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Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the K D value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death., Competing Interests: V.V., S.U.L., W.P., E.B., K.S., V.M., N.N., and D.M. are employees of GSK. R.R., V.M., K.S., and D.M. report ownership of GSK shares and/or restricted GSK shares. M.M. is an employee of the University of Naples Federico II with a consultancy contract with GSK.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens., Competing Interests: The authors declare no conflict of interest.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A . marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b , mlp2 , and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security., Competing Interests: The authors declare no conflict of interest.

Bordetella species that cause respiratory infections in mammals include B. pertussis , which causes human whooping cough, and B. bronchiseptica , which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis , which causes whooping cough in humans, and B. bronchiseptica , which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis., Competing Interests: The authors declare no conflict of interest (such as defined by ASM policy).

The ability of many human pathogens to infect requires their ability to adhere to the host surfaces as a first step in the process. Porphyromonas gingivalis, a keystone oral pathogen , uses adhesins to adhere to the surface of the gingival epithelium and other members of the oral microbiome. In a previous study, we identified several proteins potentially linked to virulence whose mRNA levels are regulated by CRISPR-Cas type I-C. Among those, PGN_1547 was highly upregulated in the CRISPR-Cas 3 mutant. PGN_1547 is annotated as a hypothetical protein. Employing homology searching, our data support that PGN_1547 resembles an auto-transporter adhesin of P. gingivalis based on containing the DUF2807 domain. To begin to characterize the function of PGN_1547, we found that a deletion mutant displayed a significant decrease in virulence using a Galleria mellonela model. Furthermore, this mutant was significantly impaired in forming biofilms and attaching to the macrophage-like cell THP-1. Luminex revealed that the PGN_1547 mutant elicited a less robust cytokine and chemokine response from THP-1 cells, and TLR2 predominantly sensed that recombinant PGN_1547. Taken together, these findings broaden our understanding of the toolbox of virulence factors possessed by P. gingivalis . Importantly, PGN_1547, a hypothetical protein, has homologs in another member of the order Bacteroidales whose function is unknown, and our results could shed light on the role of this family of proteins as auto-transport adhesins in this phylogenetic group.IMPORTANCEPeriodontal diseases are among humans' most common infections, and besides their effect on the oral cavity, they have been associated with systemic inflammatory conditions. Among members of the oral microbiome implicated in the development of periodontitis, Porphyromonas gingivalis is considered a keystone pathogen. We have identified a new adhesin that acts as a virulence factor, PGN_1547, which contains the DUF2807 domain, which belongs to the putative auto-transporter adhesin, head GIN domain family. Deletion of this gene lowers the virulence of P . gingivalis and impacts the ability of P . gingivalis to form biofilm and attach to host cells. Furthermore, the broad distribution of these receptors in the order Bacteroidales suggests their importance in colonization by this important group of organisms., Competing Interests: The authors declare no conflict of interest.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation., Competing Interests: The authors declare no conflict of interest.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low- to middle-income countries. ETEC adheres to small intestinal epithelia via colonization factors (CFs) and secretes heat-stable toxin and/or heat-labile toxin, causing dysregulated ion transport and water secretion. There are over 30 CFs identified, including major CFs associated with moderate-to-severe diarrhea (MSD) and minor CFs for which a role in pathogenesis is less clear. The Global Enteric Multicenter Study identified CS14, a class 5a fimbriae, as the only minor CF significantly associated with MSD and was recommended for inclusion in ETEC vaccines. Despite detection of CS14 in ETEC isolates, the sequence conservation of the CS14 operon, its role in adherence, and functional cross-reactivity to other class 5a fimbriae like CFA/I and CS4 are not understood. Sequence analysis determined that the CS14 operon is >99.9% identical among seven geographically diverse isolates with expanded sequence analysis demonstrating SNPs exclusively in the gene encoding the tip adhesin CsuD. Western blots and electron microscopy demonstrated that CS14 expression required the growth of isolates on CFA agar with the iron chelator deferoxamine mesylate. CS14 expression resulted in significantly increased adherence to cultured intestinal cells and human enteroids. Anti-CS14 antibodies and anti-CS4 antibodies, but not anti-CFA/I antibodies, inhibited the adherence of a subset of ETEC isolates, demonstrating CS14-specific inhibition with partial cross-reactivity within the class 5a fimbrial family. These data provide support for CS14 as an important fimbrial CF and its consideration as a vaccine antigen in future strategies. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) infection causes profuse watery diarrhea in adults and children in low- to middle-income countries and is a leading cause of traveler's diarrhea. Despite increased use of rehydration therapies, young children especially can suffer long-term effects including gastrointestinal dysfunction as well as stunting and malnutrition. As there is no licensed vaccine for ETEC, there remains a need to identify and understand specific antigens for inclusion in vaccine strategies. This study investigated one adhesin named CS14. This adhesin is expressed on the bacterial surface of ETEC isolates and was recently recognized for its significant association with diarrheal disease. We demonstrated that CS14 plays a role in bacterial adhesion to human target cells, a critical first step in the disease process, and that adherence could be blocked by CS14-specific antibodies. This work will significantly impact the ETEC field by supporting inclusion of CS14 as an antigen for ETEC vaccines., Competing Interests: The authors declare no conflict of interest.

Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a Δ fleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the Δ fleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the Δ fleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens . IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens , biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens . Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins., Competing Interests: The authors declare no conflict of interest.

Considerable progress has been made toward elucidating the mechanism of Staphylococcus aureus aggregation in synovial fluid. In this study, aggregate morphology was assessed following incubation under several simulated postsurgical joint conditions. Using fluorescently labeled synovial fluid polymers, we show that aggregation occurs through two distinct mechanisms: (i) direct bridging between S. aureus cells and host fibrinogen and (ii) an entropy-driven depletion mechanism facilitated by hyaluronic acid and albumin. By screening surface adhesin-deficient mutants ( clfA , clfB , fnbB , and fnbA ), we identified the primary genetic determinant of aggregation in synovial fluid to be clumping factor A. To characterize this bridging interaction, we employed an atomic force microscopy-based approach to quantify the binding affinity of either wild-type S. aureus or the adhesin mutant to immobilized fibrinogen. Surprisingly, we found there to be cell-to-cell variability in the binding strength of the bacteria for immobilized fibrinogen. Superhigh-resolution microscopy imaging revealed that fibrinogen binding to the cell wall is heterogeneously distributed at both the single cell and population levels. Finally, we assessed the antibiotic tolerance of various aggregate morphologies arising from newly deciphered mechanisms of polymer-mediated synovial fluid-induced aggregation. The formation of macroscopic aggregates under shear was highly tolerant of gentamicin, while smaller aggregates, formed under static conditions, were susceptible. We hypothesize that aggregate formation in the joint cavity, in combination with shear, is mediated by both polymer-mediated aggregation mechanisms, with depletion forces enhancing the stability of essential bridging interactions. IMPORTANCE The formation of a bacterial biofilm in the postsurgical joint environment significantly complicates the resolution of an infection. To form a resilient biofilm, incoming bacteria must first survive the initial invasion of the joint space. We previously found that synovial fluid induces the formation of Staphylococcus aureus aggregates, which may provide rapid protection during the early stages of infection. The state of the host joint environment, including the presence of fluid flow and fluctuating abundance of synovial fluid polymers, determines the rate and size of aggregate formation. By expanding on our knowledge of the mechanism and pathogenic implications of synovial fluid-induced aggregation, we hope to contribute insights for the development of novel methods of prevention and therapeutic intervention.

Chronic obstructive pulmonary disease (COPD) is a common debilitating disorder that is the third most common cause of death globally. Chronic lower airway infection by nontypeable Haemophilus influenzae (NTHi) in adults with COPD increases airway inflammation, causes increased symptoms, and accelerates progressive loss of lung function. Little is known about the mechanisms by which NTHi survives in COPD airways. To explore this question, the present study analyzes, in detail, 14 prospectively collected, serial isolates of a strain that persisted for 543 days in a patient with COPD, including analysis of four gap-free complete genomes. The NTHi genome underwent inversion of a ~400-kb segment three times during persistence. This inversion event resulted in switching of expression of the HMW1A and HMW2A adhesins as the inversion sites are in the promoter regions of HMW1 and HMW2. Regulation of the level of expression of HMW 1 and HMW2 in the human airways was controlled by the ~400-kb inversion and by 7-bp repeats in the HMW promoters. Analysis of knockout mutants of the persistent strain demonstrated that HMW1 and HMW2 proteins both function in the adherence of NTHi to human respiratory epithelial cells during persistence and that HMW1 also facilitates invasion of epithelial cells. An inverse relationship between biofilm formation and HMW1 expression was observed during persistence. This work advances understanding of the mechanisms of persistence of NTHi in COPD airways, which can inform the development of novel interventions to treat and prevent chronic NTHi infection in COPD. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) persists in the lower airways of adults with chronic obstructive pulmonary disease (COPD) for months to years, increasing airway inflammation that accelerates the progressive loss of lung function. Understanding the mechanisms of persistence in human airways by NTHi is critical in developing novel interventions. Here, in detail, we studied longitudinally collected sequential isolates of a strain of NTHi that persisted in an adult with COPD, including analysis of four gap-free genomes and knockout mutants to elucidate how the genome adapts in human airways. The NTHi genome underwent a genome rearrangement during persistence and this inversion impacted regulation of expression of key virulence phenotypes, including adherence to respiratory epithelial cells, invasion of epithelial cells and biofilm formation. These novel observations advance our understanding of the mechanisms of persistence of NTHi in the airways of adults with COPD.

Almost all spirochetes in the genus Borrelia ( sensu lato ) naturally contain multiple variants of closely related prophages. In the Lyme disease borreliae, these prophages are maintained as circular episomes that are called c ircular p lasmid 32  kb (cp32s). The cp32s of Lyme agents are particularly unique in that they encode two distinct families of lipoproteins, namely, Erp and Rev, that are expressed on the bacterial outer surface during infection of vertebrate hosts. All identified functions of those outer surface proteins involve interactions between the spirochetes and host molecules, as follows: Erp proteins bind plasmin(ogen), laminin, glycosaminoglycans, and/or components of complement and Rev proteins bind fibronectin. Thus, cp32 prophages provide their bacterial hosts with surface proteins that can enhance infection processes, thereby facilitating their own survival. Horizontal transfer via bacteriophage particles increases the spread of beneficial alleles and creates diversity among Erp and Rev proteins.

Adhesins ( adhes ive prote ins ) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum ( Rs ) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a Δ phcA quorum-sensing mutant that cannot respond to high cell densities: radA ( R alstonia ad hesin A ), rcpA ( R alstonia c ollagen-like p rotein A ), and rcpB . Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization, Rs upregulated rcpA and rcpB , but not radA , relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with Δ rcpA/B and Δ radA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta , including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta . Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. IMPORTANCE Every microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant-pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of multiple afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three different plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to success in plants. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis.

Chronic gastric infection with Helicobacter pylori can lead to progressive tissue changes that culminate in cancer, but how H. pylori adapts to the changing tissue environment during disease development is not fully understood. In a transgenic mouse gastric metaplasia model, we found that strains from unrelated individuals differed in their ability to infect the stomach, to colonize metaplastic glands, and to alter the expression of the metaplasia-associated protein TFF3. H. pylori isolates from different stages of disease from a single individual had differential ability to colonize healthy and metaplastic gastric glands. Exposure to the metaplastic environment selected for high gastric colonization by one of these strains. Complete genome sequencing revealed a unique alteration in the frequency of a variant allele of the putative adhesin sabB , arising from a recombination event with the related sialic acid binding adhesin (SabA) gene. Mutation of sabB in multiple H. pylori strain backgrounds strongly reduced adherence to both normal and metaplastic gastric tissue, and highly attenuated stomach colonization in mice. Thus, the changing gastric environment during disease development promotes bacterial adhesin gene variation associated with enhanced gastric colonization. IMPORTANCE Chronic infection with Helicobacter pylori is the primary risk factor for developing stomach cancer. As disease progresses H. pylori must adapt to a changing host tissue environment that includes induction of new cell fates in the cells that line the stomach. We tested representative H. pylori isolates collected from the same patient during early and later stages of disease in a mouse model where we can rapidly induce disease-associated tissue changes. Only the later-stage H. pylori strains could robustly colonize the diseased stomach environment. We also found that the ability to colonize the diseased stomach was associated with genetic variation in a putative cell surface adhesin gene called sabB . Additional experiments revealed that SabB promotes binding to stomach tissue and is critical for stomach colonization by the late-stage strains. Thus, H. pylori diversifies its genome during disease progression and these genomic changes highlight critical factors for bacterial persistence.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and frequent colonizer of human skin and mucosal membranes, including the vagina, with vaginal colonization reaching nearly 25% in some pregnant populations. MRSA vaginal colonization can lead to aerobic vaginitis (AV), and during pregnancy, bacterial ascension into the upper reproductive tract can lead to adverse birth outcomes. USA300, the most prominent MRSA lineage to colonize pregnant individuals, is a robust biofilm former and causative agent of invasive infections; however, little is known about how it colonizes and ascends in the female reproductive tract (FRT). Our previous studies showed that a MRSA mutant of seven fibrinogen-binding adhesins was deficient in FRT epithelial attachment and colonization. Using both monolayer and multilayer air-liquid interface cell culture models, we determine that one class of these adhesins, the fibronectin binding proteins (FnBPA and FnBPB), are critical for association with human vaginal epithelial cells (hVECs) and hVEC invasion through interactions with α 5 β 1 integrin. We observe that both FnBPs are important for biofilm formation as single and double fnbAB mutants exhibit reduced biofilm formation on hVECs. Using heterologous expression of fnbA and fnbB in Staphylococcus carnosus, FnBPs are also found to be sufficient for hVEC cellular association, invasion, and biofilm formation. In addition, we found that an Δ fnbAB mutant displays attenuated ascension in our murine vaginal colonization model. Better understanding of MRSA FRT colonization and ascension can ultimately inform treatment strategies to limit MRSA vaginal burden or prevent ascension, especially during pregnancy and in those prone to AV.

In addition to providing a typing mechanism for group A Streptococcus (GAS) isolates (T typing), cell surface pilus production impacts GAS virulence characteristics, including adherence and immune evasion. The pilus biosynthesis genes are located in the fibronectin- and collagen-binding T-antigen (FCT) region of the genome, and nine different FCT types, encoding more than 20 different T types, have been described. GAS isolates are not uniform in their degree or pattern of pilus expression, as highlighted by pilus production being thermoregulated in isolates that harbor the FCT-type FCT-3 (e.g., M-types M3 and M49) but not in isolates that harbor FCT-2 (e.g., M-type M1). Here, we investigated the molecular basis underlying our previous finding that M3 GAS isolates produce pili in lower abundance than M1 or M49 isolates do. We discovered that, at least in part, the low pilus expression observed for M3 isolates is a consequence of the repression of pilus gene expression by the CovR/CovS two-component regulatory system and of an M3-specific mutation in the nra gene, encoding a positive regulator of pilus gene expression. We also discovered that the orthologous transcriptional regulators RofA and Nra, whose encoding genes are located within FCT-2 and FCT-3, respectively, are not functionally identical. Finally, we sequenced the genome of an M3 isolate that had naturally undergone recombinational replacement of the FCT region, changing the FCT and T types of this strain from FCT-3/T3 to FCT-2/T1. Our study furthers the understanding of strain- and type-specific variation in virulence factor production by an important human pathogen. IMPORTANCE Our ability to characterize how a pathogen infects and causes disease, and consequently our ability to devise approaches to prevent or attenuate such infections, is inhibited by the finding that isolates of a given pathogen often show phenotypic variability, for example, in their ability to adhere to host cells through modulation of cell surface adhesins. Such variability is observed between isolates of group A Streptococcus (GAS), and this study investigates the molecular basis for why some GAS isolates produce pili, cell wall-anchored adhesins, in lower abundance than other isolates do. Given that pili are being considered as potential antigens in formulations of future GAS vaccines, this study may inform vaccine design.

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA, to irreversibly adhere to surfaces. In Caulobacter crescentus, a freshwater Caulobacterales species, the holdfast is anchored at the cell pole via the h old f ast a nchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD colocalize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for the translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not being properly attached to the cell and shed into the medium. This phenotype is greater in a Δ hfaB mutant than in a Δ hfaA Δ hfaD double mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species, with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to those produced by the wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similarly to other anchor mutants. We showed that fluorescently tagged HfaE colocalizes with holdfast and that HfaE forms an SDS-resistant high-molecular-weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein and that HfaE functions to link holdfast material to the cell envelope. IMPORTANCE For surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins, and DNA. Species of the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces. In this study, we evaluate the role of a newly identified holdfast anchor protein, HfaE, in holdfast anchoring to the cell surface in two different members of the Caulobacterales with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in the Caulobacterales . Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.

Streptococcus agalactiae, otherwise known as Group B Streptococcus (GBS), is an opportunistic pathogen that vaginally colonizes approximately one third of healthy women. During pregnancy, this can lead to in utero infection, resulting in premature rupture of membranes, chorioamnionitis, and stillbirths. Furthermore, GBS causes serious infection in newborns, including sepsis, pneumonia, and meningitis. Previous studies have indicated that GBS antigen (Ag) I/II family proteins promote interaction with vaginal epithelial cells; thus, we hypothesized that the Ag I/II Group B streptococcal surface protein C (BspC) contributes to GBS colonization of the female reproductive tract (FRT). Here, we show that a Δ bspC mutant has decreased bacterial adherence to vaginal, ecto-, and endocervical cells, as well as decreased auto-aggregation and biofilm-like formation on cell monolayers. Using a murine model of vaginal colonization, we observed that the Δ bspC mutant strain exhibited a significant fitness defect compared to wild-type (WT) GBS and was less able to ascend to the cervix and uterus in vivo, resulting in reduced neutrophil chemokine signaling. Furthermore, we determined that BspC interacts directly with the host intermediate filament protein cytokeratin 19 (K19). Surface localization of K19 was increased during GBS infection, and interaction was mediated by the BspC variable (V) domain. Finally, mice treated with a drug that targets the BspC V-domain exhibited reduced bacterial loads in the vaginal lumen and reproductive tissues. These results demonstrate the importance of BspC in promoting GBS colonization of the FRT and that it may be targeted therapeutically to reduce GBS vaginal persistence and ascending infection. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract (FRT) of up to one third of women, but GBS carriage can lead to adverse pregnancy outcomes, including premature rupture of membranes, preterm labor, and chorioamnionitis. GBS colonization during pregnancy is also the largest predisposing factor for neonatal GBS disease, including pneumonia, sepsis, and meningitis. The molecular interactions between bacterial surface proteins and the host cell receptors that promote GBS colonization are vastly understudied, and a better understanding would facilitate development of novel therapeutics to prevent GBS colonization and disease. Here, we characterize the role of the GBS surface protein BspC in colonization of the FRT. We show for the first time that GBS infection induces cytokeratin 19 (K19) surface localization on vaginal epithelial cells; GBS then uses the BspC V-domain to interact with K19 to promote colonization and ascending infection. Furthermore, this interaction can be targeted therapeutically to reduce GBS carriage.

Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plf QT598 , wherein plfG was replaced by papG ; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. IMPORTANCE Fimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin-encoding plfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. A plf mutant also showed decreased colonization of the kidneys in a mouse competitive infection model. PL fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.

Early bacterial survival in the postsurgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentrations, composition, and viscosity occur. These changes, along with fluid movement resulting from postoperative joint motion, modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with the synovial fluid polymer fibrinogen. Following incubation under each simulated postoperative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes, depending on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the postsurgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with surface proteins present to aggregate under high-viscosity conditions, yielding large, globular aggregates. IMPORTANCE Infection is a major complication of knee and hip joint replacement surgery, which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint, potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.

Uropathogenic Escherichia coli (UPEC) causes the majority of uncomplicated urinary tract infections (UTI), which affect nearly half of women worldwide. Many UPEC strains carry an annotated intimin-like adhesin ( ila ) locus in their genome related to a well-characterized virulence factor in diarrheagenic E. coli pathotypes. Its role in UPEC uropathogenesis, however, remains unknown. In prototype UPEC strain CFT073, there is an ila locus that contains three predicted intimin-like genes, sinH , sinI , and ratA . We used in silico approaches to determine the phylogeny and genomic distribution of this locus among uropathogens. We found that the currently annotated intimin locus-encoded proteins in CFT073 are more closely related to invasin proteins found in Salmonella. Deletion of the individual sinH , sinI , and ratA genes did not result in measurable effects on growth, biofilm formation, or motility in vitro . On average, sinH was more highly expressed in clinical strains during active human UTI than in human urine ex vivo . Unexpectedly, we found that strains lacking this ila locus had increased adherence to bladder cells in vitro , coupled with a decrease in bladder cell invasion and death. The sinH mutant displayed a significant fitness defect in the murine model of ascending UTI, including reduced inflammation in the bladder. These data confirmed an inhibitory role in bladder cell adherence to facilitate invasion and inflammation; therefore, the ila locus should be termed invasin-like rather than intimin-like. Collectively, our data suggest that loss of this locus mediates measurable interactions with bladder cells in vitro and contributes to fitness during UTI.

Helicobacter pylori genomes encode over 60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains exhibiting differential fitness in vivo compared to in vitro , we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro . The control library proliferated as a relatively stable community in vitro , but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a nonselective bottleneck in vivo . We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro . Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro . These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

Staphylococcus aureus can colonize both the anterior nares and the gastrointestinal tract. However, colonization at these sites in the same individuals has not been studied, and the traits that facilitate colonization and persistence at these sites have not been compared. Samples from the nostrils and feces collected on 9 occasions from 3 days to 3 years of age in 65 infants were cultured; 54 samples yielded S. aureus. The numbers of nasal and fecal S. aureus strains increased rapidly during the first weeks and were similar at 1 month of age (>40% of infants colonized). Thereafter, nasal carriage declined, while fecal carriage remained high during the first year of life. Individual strains were identified, and their colonization patterns were related to their carriage of genes encoding adhesins and superantigenic toxins. Strains retrieved from both the nose and gut ( n  = 44) of an infant were 4.5 times more likely to colonize long term (≥3 weeks at both sites) than strains found only in the rectum/feces ( n  = 56) or only in the nose ( n  = 32) ( P  ≤ 0.001). Gut colonization was significantly associated with carriage of the fnbA gene, and long-term colonization at either site was associated with carriage of fnbA and fnbB. In summary, gut colonization by S. aureus was more common than nasal carriage by S. aureus in the studied infants. Gut strains may provide a reservoir for invasive disease in vulnerable individuals. Fibronectin-binding adhesins and other virulence factors may facilitate commensal colonization and confer pathogenic potential. IMPORTANCE S. aureus may cause severe infections and frequently colonizes the nose. Nasal carriage of S. aureus increases 3-fold the risk of invasive S. aureus infection. S. aureus is also commonly found in the gut microbiota of infants and young children. However, the relationships between the adhesins and other virulence factors of S. aureus strains and its abilities to colonize the nostrils and gut of infants are not well understood. Our study explores the simultaneous colonization by S. aureus of the nasal and intestinal tracts of newborn infants through 3 years of follow-up. We identify bacterial virulence traits that appear to facilitate persistent colonization of the nose and gut by S. aureus. This expands our current knowledge of the interplay between bacterial commensalism and pathogenicity. Moreover, it may contribute to the development of targeted strategies for combating S. aureus infection.

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2 hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth.

Enterotoxigenic Escherichia coli (ETEC) contributes significantly to the substantial burden of infectious diarrhea among children living in low- and middle-income countries. In the absence of a vaccine for ETEC, children succumb to acute dehydration as well as nondiarrheal sequelae related to these infections, including malnutrition. The considerable diversity of ETEC genomes has complicated canonical vaccine development approaches defined by a subset of ETEC pathovar-specific antigens known as colonization factors (CFs). To identify additional conserved immunogens unique to this pathovar, we employed an "open-aperture" approach to capture all potential conserved ETEC surface antigens, in which we mined the genomic sequences of 89 ETEC isolates, bioinformatically selected potential surface-exposed pathovar-specific antigens conserved in more than 40% of the genomes ( n  = 118), and assembled the representative proteins onto microarrays, complemented with known or putative colonization factor subunit molecules ( n  = 52) and toxin subunits. These arrays were then used to interrogate samples from individuals with acute symptomatic ETEC infections. Surprisingly, in this approach, we found that immune responses were largely constrained to a small number of antigens, including individual colonization factor antigens and EtpA, an extracellular adhesin. In a Bangladeshi cohort of naturally infected children <2 years of age, both EtpA and a second antigen, EatA, elicited significant serologic responses that were associated with protection from symptomatic illness. In addition, children infected with ETEC isolates bearing either etpA or eatA genes were significantly more likely to develop symptomatic disease. These studies support a role for antigens not presently targeted by vaccines (noncanonical) in virulence and the development of adaptive immune responses during ETEC infections. These findings may inform vaccine design efforts to complement existing approaches., (Copyright © 2021 American Society for Microbiology.)

At the intestinal host-microbe interface, the transmembrane mucin MUC1 can function as a physical barrier as well as a receptor for bacteria. MUC1 also influences epithelial cell morphology and receptor function. Various bacterial pathogens can exploit integrins to infect eukaryotic cells. It is yet unclear whether MUC1 influences the interaction of bacteria with integrins. We used Escherichia coli expressing the invasin (inv) protein of Yersinia pseudotuberculosis ( E. coli inv) to assess the effects of MUC1 on β1 integrin (ITGB1)-mediated bacterial invasion. Our results show that expression of full-length MUC1 does not yield a physical barrier but slightly enhances E. coli inv uptake. Enzymatic removal of the MUC1 extracellular domain (ED) using a secreted protease of C1 esterase inhibitor (StcE) of pathogenic Escherichia coli had no additional effect on E. coli inv invasion. In contrast, expression of a truncated MUC1 that lacks the cytoplasmic tail (CT) reduced bacterial entry substantially. Substitution of tyrosine residues in the MUC1 CT also reduced bacterial uptake, while deletion of the C-terminal half of the cytoplasmic tail only had a minor effect, pointing to a regulatory role of tyrosine phosphorylation and the N-terminal region of the MUC1 CT in integrin-mediated uptake process. Unexpectedly, StcE removal of the ED in MUC1-ΔCT cells reversed the block in bacterial invasion. Together, these findings indicate that MUC1 can facilitate β1-integrin-mediated bacterial invasion by a concerted action of the large glycosylated extracellular domain and the membrane-juxtaposed cytoplasmic tail region. IMPORTANCE Bacteria can exploit membrane receptor integrins for cellular invasion, either by direct binding of bacterial adhesins or utilizing extracellular matrix components. MUC1 is a large transmembrane glycoprotein expressed by most epithelial cells that can have direct defensive or receptor functions at the host-microbe interface and is involved in facilitating integrin clustering. We investigated the role of epithelial MUC1 on β1 integrin-mediated bacterial invasion. We discovered that MUC1 does not act as a barrier but facilitates bacterial entry through β1 integrins. This process involves a concerted action of the MUC1 O -glycosylated extracellular domain and cytoplasmic tail. Our findings add a new dimension to the complexity of bacterial invasion mechanisms and provide novel insights into the distinct functions of MUC1 domains at the host-microbe interface., (Copyright © 2021 Li et al.)

Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation. IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation., (Copyright © 2021 Gago-Córdoba et al.)

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (Δ sspA ) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene ( pig ) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σ S ). Further analysis demonstrates that the Δ sspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene ( pvd ) cluster, leading to the inhibition of siderophore production and iron uptake. The Δ sspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway. IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis., (Copyright © 2021 American Society for Microbiology.)

The pneumococcal serine-rich repeat protein (PsrP) is a high-molecular-weight, glycosylated adhesin that promotes the attachment of Streptococcus pneumoniae to host cells. PsrP, its associated glycosyltransferases (GTs), and dedicated secretion machinery are encoded in a 37-kb genomic island that is present in many invasive clinical isolates of S. pneumoniae PsrP has been implicated in establishment of lung infection in murine models, although specific roles of the PsrP glycans in disease progression or bacterial physiology have not been elucidated. Moreover, enzymatic specificities of associated glycosyltransferases are yet to be fully characterized. We hypothesized that the glycosyltransferases that modify PsrP are critical for the adhesion properties and infectivity of S. pneumoniae Here, we characterize the putative S. pneumoniae psrP locus glycosyltransferases responsible for PsrP glycosylation. We also begin to elucidate their roles in S. pneumoniae virulence. We show that four glycosyltransferases within the psrP locus are indispensable for S. pneumoniae biofilm formation, lung epithelial cell adherence, and establishment of lung infection in a mouse model of pneumococcal pneumonia. IMPORTANCE PsrP has previously been identified as a necessary virulence factor for many serotypes of S. pneumoniae and studied as a surface glycoprotein. Thus, studying the effects on virulence of each glycosyltransferase (GT) that builds the PsrP glycan is of high importance. Our work elucidates the influence of GTs in vivo We have identified at least four GTs that are required for lung infection, an indication that it is worthwhile to consider glycosylated PsrP as a candidate for serotype-independent pneumococcal vaccine design., (Copyright © 2021 American Society for Microbiology.)

Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genomewide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyperholdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor ( fss ) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD -dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion. IMPORTANCE Understanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors., (Copyright © 2021 Hershey et al.)

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced ( P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro , demonstrating the role of MbfN as an adhesin. IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis , and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence., (Copyright © 2020 American Society for Microbiology.)

Although it is normally an innocuous part of the human skin microbiota, Staphylococcus epidermidis has emerged as a major nosocomial pathogen, and implanted foreign materials are an essential risk factor for the development of an infection. The extraordinary efficiency of S. epidermidis to colonize artificial surfaces is particularly related to the ability to form biofilms. Biofilm formation itself critically depends on stable pathogen binding to extracellular host matrix components, e.g. fibronectin (Fn), covering inserted devices in vast amounts. Extracellular matrix binding protein (Embp) and its subdomains referred to as the F-repeat and the FG-repeat are critical for adherence of S. epidermidis to surface-immobilized Fn. Embp-Fn interactions preferentially occur with surface-bound, but not folded, globular Fn via binding to the F3 domain. High-resolution structure analysis of F- and FG-repeats revealed that both repeats are composed of two tightly connected triple α-helix bundles, exhibiting an elongated but rather rigid structural organization in solution. Both F- and FG-repeat possess Fn-binding capacity via interactions with type III subdomain FN12, involving residues within the C and F β-sheet. FN12 essentially supports stability of the globular Fn state, and thus these findings reasonably explain why Embp-mediated interaction of S. epidermidis necessitates Fn surface immobilization. Thus, Embp employs an uncharacterized bacterial Fn-binding mechanism to promote staphylococcal adherence. IMPORTANCE Staphylococcus epidermidis is a leading pathogen in implant-associated hospital infections. The pathogenesis critically depends on bacterial binding to ECM components, specifically fibronectin (Fn). The cell surface-localized, 1-MDa extracellular matrix binding protein (Embp) is essentially characterized by 10 F- and 40 FG-repeats. These repetitive units, each characterized by two α-helical bundles, organize themselves in a rigid, elongated form. Embp binds preferentially to surface-localized but not soluble Fn, with both F- and FG-repeats being sufficient for Fn binding and resulting bacterial adherence. Binding preferentially involves Fn type III domain, specifically residues of FN12 β-sheets C and F. Both play key role in stabilizing the globular Fn conformation, explaining the necessity of Fn surface immobilization for a subsequent interaction with Embp. In comparison to many other bacterial Fn-binding proteins using the Fn N terminus, Embp employs a previously undescribed mechanism supporting the adhesion of S. epidermidis to surface-immobilized Fn., (Copyright © 2020 Büttner et al.)

The Negativicutes are a clade of the Firmicutes that have retained the ancestral diderm character and possess an outer membrane. One of the best studied Negativicutes , Veillonella parvula , is an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial communities, including the gut and the dental plaque microbiota. Whereas the adhesion and biofilm capacities of V. parvula are expected to be crucial for its maintenance and development in these environments, studies of V. parvula adhesion have been hindered by the lack of efficient genetic tools to perform functional analyses in this bacterium. Here, we took advantage of a recently described naturally transformable V. parvula isolate, SKV38, and adapted tools developed for the closely related Clostridia spp. to perform random transposon and targeted mutagenesis to identify V. parvula genes involved in biofilm formation. We show that type V secreted autotransporters, typically found in diderm bacteria, are the main determinants of V. parvula autoaggregation and biofilm formation and compete with each other for binding either to cells or to surfaces, with strong consequences for V. parvula biofilm formation capacity. The identified trimeric autotransporters have an original structure compared to classical autotransporters identified in Proteobacteria , with an additional C-terminal domain. We also show that inactivation of the gene coding for a poorly characterized metal-dependent phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for further molecular characterization of V. parvula interactions with other bacteria and the host within complex microbiota environments. IMPORTANCE Veillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to adhere to surfaces or other bacteria and form biofilms is critical for it to inhabit complex human microbial communities such as the gut and oral microbiota. Although the adhesive capacity of V. parvula has been previously described, very little is known about the underlying molecular mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to identify surface-exposed adhesins called autotransporters as the main molecular determinants of adhesion in this bacterium. This work therefore provides new insights on an important aspect of the V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of biofilm formation to the biology of this major commensal of the oral-digestive tract., (Copyright © 2020 Béchon et al.)

Pseudomonas aeruginosa is an important pathogen that causes chronic infections that involve multicellular aggregates called biofilms. Within biofilms, bacteria are surrounded in a protective extracellular matrix of proteins, exopolysaccharides (EPS), and DNA. A key P. aeruginosa matrix protein is an extracellular adhesin called CdrA, which promotes aggregation by binding to the EPS Psl and via CdrA-CdrA interactions. We hypothesized that because of its ability to bind Psl, CdrA would be important only for strains that use Psl as the primary EPS (e.g., the laboratory strain PAO1). Thus, we predicted that cdrA might be dispensable for biofilm formation by strains that do not utilize Psl (e.g., the laboratory strain PA14). Instead, we observed that cdrA deletion strains exhibited biofilm defects, regardless of their EPS dependencies. We screened a panel of clinical and environmental P. aeruginosa isolates for the presence of the cdrA allele and production of CdrA protein. All isolates that we tested contained the cdrA allele, and these alleles had minimal sequence variation compared to the reference PAO1 cdrA gene. Additionally, all isolates except one produced detectable CdrA protein. We investigated the possible mechanisms of CdrA-promoted biofilm formation in these strains where Psl is not dominant, and we discovered that CdrA binds to Pel. Although Psl and Pel chemical structures are distinct, this appears to be a specific interaction, since previous work has shown that CdrA binds discriminately to other EPS. Our findings provide new understanding of biofilm formation across P. aeruginosa isolates and emphasize the versatility of CdrA. IMPORTANCE Depending upon the strain, Pseudomonas aeruginosa can use different exopolysaccharides (e.g., Psl, Pel, and alginate) to build its biofilm matrix. Previously, we demonstrated that the biofilm matrix protein CdrA binds to Psl, promoting biofilm formation and aggregate stability. As such, it was thought that CdrA might be important for biofilm assembly only in strains that rely upon Psl. However, past studies indicated that CdrA can interact with monosaccharides not present in Psl, including N -acetylglucosamine, a constituent of another EPS called Pel. We discovered that CdrA also binds to Pel and promotes biofilm formation by strains in which Psl is not dominant. Thus, our findings suggest that CdrA plays a common role as a biofilm matrix cross-linker across P. aeruginosa isolates with different EPS., (Copyright © 2020 Reichhardt et al.)

Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection., (Copyright © 2020 American Society for Microbiology.)

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract., (Copyright © 2020 American Society for Microbiology.)

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most factors that could respond to oxidative stress (a host cell defense mechanism). We previously found that the C terminus of Ehrlichia surface invasin, e ntry- t riggering p rotein of E hrlichia (EtpE; EtpE-C) directly binds mammalian DNase X, a glycosylphosphatidylinositol-anchored cell surface receptor and that binding is required to induce bacterial entry and simultaneously to block the generation of reactive oxygen species (ROS) by host monocytes and macrophages. However, how the EtpE-C-DNase X complex mediates the ROS blockade was unknown. A mammalian transmembrane glycoprotein CD147 (basigin) binds to the EtpE-DNase X complex and is required for Ehrlichia entry and infection of host cells. Here, we found that bone marrow-derived macrophages (BMDM) from myeloid cell lineage-selective CD147-null mice had significantly reduced Ehrlichia -induced or EtpE-C-induced blockade of ROS generation in response to phorbol myristate acetate. In BMDM from CD147-null mice, nucleofection with CD147 partially restored the Ehrlichia -mediated inhibition of ROS generation. Indeed, CD147-null mice as well as their BMDM were resistant to Ehrlichia infection. Moreover, in human monocytes, anti-CD147 partially abrogated EtpE-C-induced blockade of ROS generation. Both Ehrlichia and EtpE-C could block activation of the small GTPase Rac1 (which in turn activates phagocyte NADPH oxidase) and suppress activation of Vav1, a hematopoietic-specific Rho/Rac guanine nucleotide exchange factor by phorbol myristate acetate. Vav1 suppression by Ehrlichia was CD147 dependent. E. chaffeensis is the first example of pathogens that block Rac1 activation to colonize macrophages. Furthermore, Ehrlichia uses EtpE to hijack the unique host DNase X-CD147-Vav1 signaling to block Rac1 activation. IMPORTANCE Ehrlichia chaffeensis is an obligatory intracellular bacterium with the capability of causing an emerging infectious disease called human monocytic ehrlichiosis. E. chaffeensis preferentially infects monocytes and macrophages, professional phagocytes, equipped with an arsenal of antimicrobial mechanisms, including rapid reactive oxygen species (ROS) generation upon encountering bacteria. As Ehrlichia isolated from host cells are readily killed upon exposure to ROS, Ehrlichia must have evolved a unique mechanism to safely enter phagocytes. We discovered that binding of the Ehrlichia surface invasin to the host cell surface receptor not only triggers Ehrlichia entry but also blocks ROS generation by the host cells by mobilizing a novel intracellular signaling pathway. Knowledge of the mechanisms by which ROS production is inhibited may lead to the development of therapeutics for ehrlichiosis as well as other ROS-related pathologies., (Copyright © 2020 Teymournejad and Rikihisa.)

Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCC mec ) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB , and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR&#95;B or DR&#95;E. When inserted via DR&#95;E, the sesJ gene was encoded in the SCC element. When inserted via DR&#95;B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates. IMPORTANCE S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis , is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections., (Copyright © 2020 Arora et al.)

Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization. IMPORTANCE Staphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission., (Copyright © 2019 Deng et al.)

The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae , where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae , the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae ; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae , which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments., (Copyright © 2019 Kitts et al.)

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA., (Copyright © 2019 American Society for Microbiology.)

The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo -like conditions to study bacterial pathogenesis. IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development., (Copyright © 2019 Chanin et al.)

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium , we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium , and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease. IMPORTANCE Fusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease., (Copyright © 2019 American Society for Microbiology.)

Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments. IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa , but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment., (Copyright © 2019 Wang et al.)

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT -containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT -containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness., (Copyright © 2019 American Society for Microbiology.)

Lactobacilli play an important role in the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of Lactobacillus gasseri DSM 14869 and Lactobacillus rhamnosus DSM 14870 were previously selected to develop the probiotic EcoVag capsules and showed therapeutic effects in women with bacterial vaginosis (BV). However, the molecular mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in L. gasseri DSM 14869 that promote adhesion to vaginal epithelial cells (VEC) by constructing dedicated knockout mutants, including exopolysaccharides (EPSs), a protein containing MucBP-like domains (N506&#95;1778), and a putative novel adhesin (N506&#95;1709) with rib/alpha-like domain repeats. EPS knockout mutants revealed 20-fold and 14-fold increases in adhesion to Caco-2 and HeLa cells, respectively, compared with wild type, while the adhesion to VEC was reduced 30% by the mutation, suggesting that EPSs might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2 cells, HeLa cells, and VEC was observed in the N506&#95;1778 knockout mutant. The N506&#95;1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared with wild type (WT); in contrast, the adhesion to VEC revealed a significant decrease (42%), suggesting that N506&#95;1709 might mediate specific binding to stratified squamous epithelial cells, and this putative novel adhesin was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). Thus, we have discovered an important role for EPSs and a novel adhesin, LVEA, in the adhesive capacity of a vaginal probiotic Lactobacillus strain. IMPORTANCE Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for the prevention or treatment of urogenital diseases, such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are not fully understood. Here, we functionally identified three cell surface factors of a Lactobacillus gasseri strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPSs) and two sortase-dependent proteins (N506&#95;1778 and N506&#95;1709). We could demonstrate the tissue-specific adhesion of EPSs to vaginal cells and that N506&#95;1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal Lactobacillus spp. adhesion., (Copyright © 2019 American Society for Microbiology.)

The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis share high genome identity. Retrospective analysis of surveillance data from New Zealand indicates the potential cross-protective effect of outer membrane vesicle (OMV) meningococcal serogroup B vaccine (MeNZB) against N. gonorrhoeae A licensed OMV-based MenB vaccine, MenB-4C, consists of a recombinant FHbp, NhbA, NadA, and the MeNZB OMV. Previous work has identified several abundantly expressed outer membrane proteins (OMPs) as major components of the MenB-4C OMV with high sequence similarity between N. gonorrhoeae and N. meningitidis , suggesting a mechanism for cross-protection. To build off these findings, we performed comparative genomic analysis on 970 recent N. gonorrhoeae isolates collected through a U.S surveillance system against N. meningitidis serogroup B (NmB) reference sequences. We identified 1,525 proteins that were common to both Neisseria species, of which 57 proteins were predicted to be OMPs using in silico methods. Among the MenB-4C antigens, NhbA showed moderate sequence identity (73%) to the respective gonococcal homolog, was highly conserved within N. gonorrhoeae , and was predicted to be surface expressed. In contrast, the gonococcal FHbp was predicted not to be surface expressed, while NadA was absent in all N. gonorrhoeae isolates. Our work confirmed recent observations (E. A. Semchenko, A. Tan, R. Borrow, and K. L. Seib, Clin Infect Dis, 2018, https://doi.org/10.1093/cid/ciy1061) and describes homologous OMPs from a large panel of epidemiologically relevant N. gonorrhoeae strains in the United States against NmB reference strains. Based on our results, we report a set of OMPs that may contribute to the previously observed cross-protection and provide potential antigen targets to guide the next steps in gonorrhea vaccine development. IMPORTANCE Gonorrhea, a sexually transmitted disease, causes substantial global morbidity and economic burden. New prevention and control measures for this disease are urgently needed, as strains resistant to almost all classes of antibiotics available for treatment have emerged. Previous reports demonstrate that cross-protection from gonococcal infections may be conferred by meningococcal serogroup B (MenB) outer membrane vesicle (OMV)-based vaccines. Among 1,525 common proteins shared across the genomes of both N. gonorrhoeae and N. meningitidis , 57 proteins were predicted to be surface expressed (outer membrane proteins [OMPs]) and thus preferred targets for vaccine development. The majority of these OMPs showed high sequence identity between the 2 bacterial species. Our results provide valuable insight into the meningococcal antigens present in the current OMV-containing MenB-4C vaccine that may contribute to cross-protection against gonorrhea and may inform next steps in gonorrhea vaccine development.

Flavobacterium johnsoniae SprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTD SprB ) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTD SprB required coexpression with sprF , which lies downstream of sprB SprF is similar in sequence to Porphyromonas gingivalis PorP. Most F. johnsoniae genes encoding proteins with type B CTDs lie immediately upstream of porP / sprF -like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh&#95;3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins. IMPORTANCE The F. johnsoniae gliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylum Bacteroidetes and may have roles in adhesion, motility, and virulence., (Copyright © 2019 American Society for Microbiology.)

Bacterial adhesion to host tissues is considered the first and critical step of microbial infection. The extracellular matrix protein adhesin A (EmaA) is a collagen-binding adhesin of the periodontal pathogen Aggregatibacter actinomycetemcomitans Three 202-kDa EmaA monomers form antenna-like structures on the bacterial surface with the functional domain located at the apical end. The structure of the 30-nm functional domain has been determined by three-dimensional (3D) electron tomography and subvolume averaging. The region exhibits a complex architecture composed of three subdomains (SI to SIII) and a linker between subdomains SII and SIII. However, the molecular interaction between the adhesin receptor complexes has yet to be revealed. This study provides the first detailed 3D structure of reconstituted EmaA/collagen complexes obtained using 3D electron tomography and image processing techniques. The observed interactions of EmaA with collagen were not to whole, intact fibrils, but rather to individual collagen triple helices dissociated from the fibrils. The majority of the contacts with the EmaA functional domain encompassed subdomains SII and SIII and in some cases the tip of the apical domain, involving SI. These data suggest a multipronged mechanism for the interaction of Gram-negative bacteria with collagen. IMPORTANCE Bacterial adhesion is a crucial step for bacterial colonization and infection. In recent years, the number of antibiotic-resistant strains has dramatically increased; therefore, there is a need to search for novel antimicrobial agents. Thus, great efforts are being devoted to develop a clear understanding of the bacterial adhesion mechanism for preventing infections. In host/pathogen interactions, once repulsive forces are overcome, adhesins recognize and tightly bind to specific receptors on the host cell or tissue components. Here, we present the first 3D structure of the interaction between the collagen-binding adhesin EmaA and collagen, which is critical for the development of endocarditis in humans., (Copyright © 2019 American Society for Microbiology.)

Streptococcus pyogenes (group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization, hupYZ Here we show that HupY binds heme in vitro , affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogen Streptococcus agalactiae (group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS. IMPORTANCE Colonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogen Streptococcus pyogenes shows extensive remodeling during in vivo growth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in both S. pyogenes and the related pathogen Streptococcus agalactiae , binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization., (Copyright © 2019 Cook et al.)

Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains' antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden's population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden., (Copyright © 2019 Gullsby et al.)