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The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion. [ABSTRACT FROM AUTHOR]

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

ABSTRACT Stenotrophomonas maltophilia is an emerging opportunistic respiratory pathogen in people with cystic fibrosis (CF). S. maltophilia is frequently observed in polymicrobial infections, and we have previously shown that Pseudomonas aeruginosa promotes colonization and persistence of S. maltophilia in mouse respiratory infections. In this study, we used host and bacterial RNA sequencing to further understand the molecular underpinnings of this interaction. To evaluate S. maltophilia transcript profiles, we used a recently described method for selective capture of bacterial mRNA transcripts with strain-specific RNA probes. We found that factors associated with the type IV pilus, including the histidine kinase subunit of a chemotactic two-component signaling system (chpA), had increased transcript levels during dual-species infection. Using immortalized CF respiratory epithelial cells, we found that infection with P. aeruginosa increases adherence of S. maltophilia, at least in part due to disruption of epithelial tight junctions. In contrast, an isogenic S. maltophilia chpA mutant strain lacked cooperative adherence to CF epithelia and decreased bacterial burden in vivo in dual-species infections with P. aeruginosa. Similarly, P. aeruginosa lacking elastase (lasB) failed to promote S. maltophilia adherence or bacterial colonization and persistence in vivo. Based on these results, we propose that disruption of lung tissue integrity by P. aeruginosa facilitates adherence of S. maltophilia to the lung epithelia, likely in a type IV pilus-dependent manner. These data lend insight into S. maltophilia colonization and persistence in people in later stages of CF disease and may have implications for interactions with other bacterial opportunists. IMPORTANCE Despite advances in treatment options for people with CF, complications of bacterial infections remain the greatest driver of morbidity and mortality in this patient population. These infections often involve more than one bacterial pathogen, and our understanding of how interspecies interactions impact disease progression is lacking. Previous work in our lab found that two CF pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, can work together in the lung to cause more severe infection. In the present study, we found that infection with P. aeruginosa promotes persistence of S. maltophilia by interfering with epithelial barrier integrity. Depolarization of the epithelial cell layer by P. aeruginosa-secreted elastase increased S. maltophilia adherence, likely in a type IV pilus-dependent manner. Ultimately, this work sheds light on the molecular mechanisms governing an important multispecies interaction seen in pulmonary diseases such as CF.

ABSTRACT Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA, pilB, and pilQ, which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

ABSTRACT Hydrocephalus, the leading indication for childhood neurosurgery worldwide, is particularly prevalent in low- and middle-income countries. Hydrocephalus preceded by an infection, or postinfectious hydrocephalus, accounts for up to 60% of hydrocephalus in these areas. Since many children with hydrocephalus suffer poor long-term outcomes despite surgical intervention, prevention of hydrocephalus remains paramount. Our previous studies implicated a novel bacterial pathogen, Paenibacillus thiaminolyticus, as a causal agent of neonatal sepsis and postinfectious hydrocephalus in Uganda. Here, we report the isolation of three P. thiaminolyticus strains, Mbale, Mbale2, and Mbale3, from patients with postinfectious hydrocephalus. We constructed complete genome assemblies of the clinical isolates as well as the nonpathogenic P. thiaminolyticus reference strain and performed comparative genomic and proteomic analyses to identify potential virulence factors. All three isolates carry a unique beta-lactamase gene, and two of the three isolates exhibit resistance in culture to the beta-lactam antibiotics penicillin and ampicillin. In addition, a cluster of genes carried on a mobile genetic element that encodes a putative type IV pilus operon is present in all three clinical isolates but absent in the reference strain. CRISPR-mediated deletion of the gene cluster substantially reduced the virulence of the Mbale strain in mice. Comparative proteogenomic analysis identified various additional potential virulence factors likely acquired on mobile genetic elements in the virulent strains. These results provide insight into the emergence of virulence in P. thiaminolyticus and suggest avenues for the diagnosis and treatment of this novel bacterial pathogen. IMPORTANCE Postinfectious hydrocephalus, a devastating sequela of neonatal infection, is associated with increased childhood mortality and morbidity. A novel bacterial pathogen, Paenibacillus thiaminolyticus, is highly associated with postinfectious hydrocephalus in an African cohort. Whole-genome sequencing, RNA sequencing, and proteomics of clinical isolates and a reference strain in combination with CRISPR editing identified type IV pili as a critical virulence factor for P. thiaminolyticus infection. Acquisition of a type IV pilus-encoding mobile genetic element critically contributed to converting a nonpathogenic strain of P. thiaminolyticus into a pathogen capable of causing devastating diseases. Given the widespread presence of type IV pilus in pathogens, the presence of the type IV pilus operon could serve as a diagnostic and therapeutic target in P. thiaminolyticus and related bacteria.

ABSTRACT Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.

ABSTRACT With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson’s disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.

Type IV pili (Tfp) are known to mediate several biological activities, including surface-dependent twitching motility. Although a pil gene cluster for Tfp biosynthesis is found in all sequenced Streptococcus sanguinis strains, Tfp-mediated twitching motility is less commonly detected. Upon examining 81 clinical strains, 39 strains generated twitching zones on blood agar plates (BAP), while 27 strains displayed twitching on Todd-Hewitt (TH) agar. Although BAP appears to be more suitable for the development of twitching zones, 5 strains exhibited twitching motility only on TH agar, indicating that twitching motility is not only strain specific but also sensitive to growth media. Furthermore, different twitching phenotypes were observed in strains expressing comparable levels of pilT, encoding the retraction ATPase, suggesting that the twitching phenotype on agar plates is regulated by multiple factors. By using a PilT-null and a pilin protein-null derivative (CHW02) of twitching-active S. sanguinis CGMH010, we found that Tfp retraction was essential for biofilm stability. Further, biofilm growth was amplified in CHW02 in the absence of shearing force, indicating that S. sanguinis may utilize other ligands for biofilm formation in the absence of Tfp. Similar to SK36, Tfp from CGMH010 were required for colonization of host cells, but PilT only marginally affected adherence and only in the twitching-active strain. Taken together, the results suggest that Tfp participates in host cell adherence and that Tfp retraction facilitates biofilm stability. [ABSTRACT FROM AUTHOR]

ABSTRACT Type IV pili (T4P) are common bacterial surface appendages involved in different biological processes such as adherence, motility, competence, pathogenesis, and conjugation. In this work, we describe the T4P of TP114, an IncI2 enterobacterial conjugative plasmid recently shown to disseminate at high rates in the mouse intestinal tract. This pilus is composed of the major PilS and minor PilV pilins that are both important for conjugation in broth and in the gut microbiota but not on a solid support. The PilV-coding sequence is part of a shufflon and can bear different C-terminal domains. The shufflon is a multiple DNA inversion system containing many DNA cassettes flanked by recombination sites that are recognized by a shufflon-specific tyrosine recombinase (shufflase) promoting the recombination between DNA segments. The different PilV variants act as adhesins that can modify the affinity for different recipient bacteria. Eight PilV variants were identified in TP114, including one that has not been described in other shufflons. All PilV variants allowed conjugative transfer with different recipient Escherichia coli strains. We conclude that the T4P carried by TP114 plays a major role in mating pair stabilization in broth as well as in the gut microbiota and that the shufflon acts as a biological switch modifying the conjugative host range specificity. IMPORTANCE Conjugative plasmids are involved in horizontal gene transfer in the gut microbiota, which constitutes an important antibiotic resistance gene reservoir. However, the molecular mechanisms used by conjugative plasmids to select recipient bacteria and transfer at high rates in the mouse gut microbiota remain poorly characterized. We studied the type IV pilus carried by TP114 and demonstrated that the minor pilin PilV acts as an adhesin that can efficiently select target cells for conjugative transfer. Moreover, the pilV gene can be rapidly modified by a shufflon, hence modulating the nature of the recipient bacteria during conjugation. Our study highlights the role of mating pair stabilization for conjugation in broth as well as in the gut microbiome and explains how the host spectrum of a plasmid can be expanded simply by remodeling the PilV adhesin.

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which has largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection have been identified and characterized in detail. Here, we isolate a phage, vB_Pae_QDWS, capable of infecting Pseudomonas aeruginosa PAO1 and report that its encoded Gp21 protein protects PAO1 against phage infection. Expression of Gp21 regulates bacterial quorum sensing with an inhibitory effect in low cell density and an activation effect in high cell density. By testing the type IV pilus (TFP)-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found to decrease the pilus synthesis. Further, by constructing the TFP synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated that the Gp21 protein could block phage infection via decreasing the TFP-mediated phage adsorption. Gp21 is a novel protein that inhibits phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. IMPORTANCE The majority of the annotated phage genes are currently deposited as “hypothetical protein” with unknown function. Research has revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Conversely, we report a phage encoded protein Gp21 that protects the host against phage infection. The pathways that Gp21 involved in antiphage defense in Pseudomonas aeruginosa PAO1 interfere with quorum sensing and decrease type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage-derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into phage-host evolutionary dynamics. [ABSTRACT FROM AUTHOR]

ABSTRACT Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be important determinants of community-level properties. Spatial intermixing emerging from microbial interaction is one of the best-studied characteristics of spatial patterns. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two mutualistic Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (here referred to as a ‘bubble-burst’ pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments. IMPORTANCE When growing on surfaces, multispecies microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, enabling otherwise impermissible metabolic functions to occur as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells, and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganisms, but its role on pattern formation within microbial communities is unclear. Here, we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy for engineering high-performance synthetic microbial communities.

Exopolysaccharide (EPS) layers on the bacterial cell surface are key determinants of biofilm establishment and maintenance, leading to the formation of higher‐order 3D structures that confer numerous survival benefits to a cell community. In addition to a specific cell‐associated EPS glycocalyx, we recently revealed that the social δ‐proteobacterium Myxococcus xanthus secretes a novel biosurfactant polysaccharide (BPS) to the extracellular milieu. Together, secretion of the two polymers (EPS and BPS) is required for type IV pilus (T4P)‐dependent swarm expansion via spatio‐specific biofilm expression profiles. Thus the synergy between EPS and BPS secretion somehow modulates the multicellular lifecycle of M. xanthus. Herein, we demonstrate that BPS secretion functionally alters the EPS glycocalyx via destabilization of the latter, fundamentally changing the characteristics of the cell surface. This impacts motility behaviors at the single‐cell level and the aggregative capacity of cells in groups via cell‐surface EPS fibril formation as well as T4P production, stability, and positioning. These changes modulate the structure of swarm biofilms via cell layering, likely contributing to the formation of internal swarm polysaccharide architecture. Together, these data reveal the manner by which the combined secretion of two distinct polymers induces single‐cell changes that modulate swarm biofilm communities. [ABSTRACT FROM AUTHOR]

Abstract Lysobacter enzymogenes OH11 is a non-flagellated, ubiquitous soil bacterium with broad-spectrum antifungal activities. Although lacking flagella, it employs another type of motile behavior, known as twitching motility that is powered by type IV pilus (T4P) to move towards neighboring crop fungal pathogens to kill them as food. At present, little is known about how this non-flagellated bacterium controls twitching motility that is crucial for its predatory lifestyle. Herein, we present a report on how a non-canonical PilZ domain, PilZLe3639, controls such motility in the non-flagellated L. enzymogenes; it failed to bind with c-di-GMP but seemed to be required for twitching motility. Using bacterial two-hybrid and pull-down approaches, we identified PilBLe0708, one of the PilZLe3639-binding proteins that are essential for the bacterial twitching motility, could serve as an ATPase to supply energy for T4P extension. Through site-mutagenesis approaches, we identified one essential residue of PilZLe3639 that is required for its binding affinity with PilBLe0708 and its regulatory function. Besides, two critical residues within the ATPase catalytic domains of PilBLe0708 were detected to be essential for regulating twitching behavior but not involved in binding with PilZLe3639. Overall, we illustrated that the PilZ-PilB complex formation is indispensable for twitching motility in a non-flagellated bacterium.

Akkermansia muciniphila is a kind of beneficial microorganism colonized in the human gut. A. muciniphila is closely related to human intestinal health and has a good effect on diseases related to intestinal metabolism. The proteins encoded by the Amuc_1098-Amuc_1102 gene cluster, which are related to the formation and assembly of the pilus, are highly expressed in the membrane protein components of A. muciniphila. In this paper, we report the crystal structure of Amuc_1102 at a resolution of 1.75 Å, which adopts an immunoglobulin (Ig)-like fold. Amuc_1102 shares a similar fold to three archaeal proteins related to type IV pilus (T4P)-like structure, Pilin, FlaF, and FlaG, indicating a similar function. Amuc_1102 exists as a trimer both in the crystal structure and in solution, which differs from the assemblies of Pilin, FlaF, and FlaG. This study provides a structural basis for the elucidation of the T4P formation of A. muciniphila. • We solved the crystal structure of Amuc_1102 from Akkermansia muciniphila. • Amuc_1102 adopts an immunoglobulin-like fold related to archaeal type IV pilus, indicating a similar function. • Amuc_1102 exists as a trimer both in the crystal structure and in solution. [ABSTRACT FROM AUTHOR]

Mastitis remains a major infection of dairy cows and an important issue for the dairy farmers, and Escherichia coli (E. coli) bovine mastitis is a disease of significant economic importance in the dairy industry. Our study identified six isolates belong to phylogroup B2 from 69 bovine mastitis E. coli strains. Except for one serotype O1 strain, all group B2 isolates were identified into serotype O2 and showed significantly higher mortality in the mouse infection than other phylogroups' strains. Genomic analyses and further tests were performed to examine the role of secretion systems, fimbriae, and toxins during the systemic infection of O2:K1 strain BCE049. Two integral T6SS loci and three predicted effectors clusters were found to assemble the functional T6SS complex and deliver diverse toxic effectors to modulate bacterial virulence in the mouse infection model. A total of four T4SS loci were harbored in the BCE049 genome, three of them are encoded in different plasmids, respectively, whereas the last one locates within the bacterial chromosome at FQU84_16715 to FQU84_16760, and was significantly involved in the bacterial pathogenicity. Numerous predicted pilus biosynthesis gene loci were found in the BCE049 genome, whereas most of them lost long fragments encoding key genes for the pili assembly. Unexpectedly, a type IV pilus gene locus locating at FQU84_01405 to FQU84_01335 in the plasmid 2, was found to be required for the full virulence of mastitis strain BCE049. It should be noted that a genetic neighborhood inserted with diverse genes is encoded by the plasmid 1, which harbors three prominent toxins including β-hemolysin, cytotoxic necrotizing factor 2 and cytolethal distending toxin type III. Consequent studies verified that these toxins significantly contributed to the bacterial pathogenicity. These findings provide a molecular blueprint for understanding the underlying mechanisms employed by the bovine mastitis E. coli to colonize in host and cause systemic infection.

Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellularmovements, a conserved feature in twitching bacteria. [ABSTRACT FROM AUTHOR]

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ54 factor RpoN2 regulated bacterial virulence on rice in a flagellum‐independent manner in Xoo. In this study, both yeast two‐hybrid and pull‐down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two‐component system PilS‐PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT‐PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2‐binding sequence GGN10GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT‐PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2‐PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo. [ABSTRACT FROM AUTHOR]

The gram‐negative plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) is able to infect the host rice and effectively colonize in vascular tissues. The type IV pilus (T4P) is one of the major virulence factors playing an important role in migration of Xoo through host vascular tissues. Here, we identified PilN, a T4P alignment subcomplex protein, which is involved in regulation of swimming motility, and analysed its contribution to bacterial surface‐associated behaviours and virulence. We found that the pilN deletion mutant exhibited dramatically reduced twitching motility and scarcely detectable levels of T4P major pili PilA, as well as enhanced biofilm formation and exopolysaccharide (EPS) production. In addition, deletion of the pilN gene in Xoo resulted in impaired virulence in host rice and attenuated type III secretion system (T3SS) genes expression, which is independent of PilA assembly. Expression of the relevant pilN gene in trans was capable of restoring twitching motility and biofilm formation to the wild‐type levels in the pilN mutant but partially recovering EPS production and virulence. Moreover, the expression of trh and xrvA genes, which encode the HrpG positive regulators, was decreased in the pilN mutant. Our results suggest that PilN executes versatile functions in bacterial virulence and cell surface‐associated behaviours. [ABSTRACT FROM AUTHOR]

Biological hydrolysis of cellulose above 70°C involves microorganisms that secrete free enzymes, and deploy separate protein systems to adhere to their substrate. Strongly cellulolytic Caldicellulosiruptor bescii is one such extreme thermophile, which deploys modular, multi36 functional carbohydrate acting enzymes to deconstruct plant biomass. Additionally, C. bescii also encodes for non-catalytic carbohydrate binding proteins, which likely evolved as a mechanism to compete against other heterotrophs in carbon limited biotopes that these bacteria inhabit. Analysis of the Caldicellulosiruptor pangenome identified a type IV pilus (T4P) locus encoded upstream of the tāpirins, that is encoded for by all Caldicellulosiruptor species. In this study, we sought to determine if the C. bescii T4P plays a role in attachment to plant polysaccharides. The major C. bescii pilin (CbPilA) was identified by the presence of pilin-like protein domains, paired with transcriptomics and proteomics data. Using immuno-dot blots, we determined that the plant polysaccharide, xylan, induced production of CbPilA 10 to 14-fold higher than glucomannan or xylose. Furthermore, we are able to demonstrate that recombinant CbPilA directly interacts with xylan, and cellulose at elevated temperatures. Localization of CbPilA at the cell surface was confirmed by immunofluorescence microscopy. Lastly, a direct role for CbPilA in cell adhesion was demonstrated using recombinant CbPilA or anti-CbPilA antibodies to reduce C. bescii cell adhesion to xylan and crystalline cellulose up to 4.5 and 2- fold, respectively. Based on these observations, we propose that CbPilA and by extension, the T4P, play a role in Caldicellulosiruptor cell attachment to plant biomass. [ABSTRACT FROM AUTHOR]

• Transcutaneous immunization via bandaid can induce protective immune responses. • We've designed a two-adhesin directed immunogen, 'chimV4'. • TCI with chimV4 resolved nontypeable Haemophilus influenzae -induced otitis media. • Bandaid immunization also induced durable and boostable immunity. Otitis media (OM) is a very common pediatric disease and nontypeable Haemophilus influenzae (NTHI) is the predominant causative agent. We've developed a chimeric immunogen, chimV4, that simultaneously targets two NTHI adhesins, OMP P5 and the type IV pilus. Transcutaneous immunization (TCI) via bandaid with chimV4 plus the adjuvant dmLT provides significant protection against experimental NTHI-induced OM in chinchilla models. Herein, we now examined the durability and boostability of the induced immune response. Bandaid immunization with chimV4+dmLT followed by two sequential middle ear challenges with NTHI resulted in rapid bacterial clearance and significantly accelerated disease resolution. Moreover, TCI with chimV4+dmLT significantly increased mature B-cell phenotypes and antibody-secreting cells within nasal-associated lymphoid tissues, a response that was further augmented upon TCI two months later. Thus, bandaid immunization induced durable and boostable immunity. The simplicity and non-invasive nature of TCI with chimV4+dmLT supports its utility as a highly effective additional immunization strategy for NTHI-induced OM. [ABSTRACT FROM AUTHOR]

Despite the availability of antibiotics and vaccines, Neisseriameningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections. [ABSTRACT FROM AUTHOR]

Otitis media (OM) is one of the most common diseases of childhood, and nontypeable Haemophilus influenzae (NTHI) is the predominant causative agent of chronic and recurrent OM, as well as OM for which treatment has failed. Moreover, NTHI is now as important a causative agent of acute OM as the pneumococcus. NTHI colonizes the human nasopharynx asymptomatically. However, upon perturbation of the innate and physical defenses of the airway by upper respiratory tract viral infection, NTHI can replicate, ascend the Eustachian tube, gain access to the normally sterile middle ear space, and cause disease. Bacterial biofilms within the middle ear, including those formed by NTHI, contribute to the chronic and recurrent nature of this disease. These multicomponent structures are highly resistant to clearance by host defenses and elimination by traditional antimicrobial therapies. Herein, we review several strategies utilized by NTHI to persist within the human host and interventions currently under investigation to prevent and/or resolve NTHI-induced diseases of the middle ear and uppermost airway. [ABSTRACT FROM AUTHOR]

Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain, S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5' to the cluster, and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5' promoter, which is located more than 800 bases 5' to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early-stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hair-like surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313-2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes down-regulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation. [ABSTRACT FROM AUTHOR]

Highlights • Not only the apfABCD (apf) operon but also the pilMNOPQ (pil) operon is necessary for Tfp biogenesis in A. pleuropneumoniae. • The apf and pil operons are present and highly conserved among 15 serotypes of A. pleuropneumoniae. • The transcription of the pil operon was greatly upregulated upon bacterial contact with host cells. • Each gene product encoded by the apf and pil operons is essential for bacterial adherence and biofilm formation in vitro. Abstract Actinobacillus pleuropneumoniae is a species of Gram-negative bacteria that colonizes the respiratory tract, causing porcine pleuropneumoniae. Given the high agricultural impact of this disease, a detailed understanding of the pathogenesis of A. pleuropneumoniae is crucial. Previous work found that only the apfABCD (apf) operon encodes type IV pili (Tfp), which promotes bacterial adherence to host cells, in A. pleuropneumoniae. However, the detailed biogenesis and function of Tfp in A. pleuropneumoniae remained unclear. Here, we found that the apfABCD and pilMNOPQ (pil) gene clusters are operons, and the products of each gene within the apf and pil loci are required for Tfp biogenesis and function. Sequencing analysis revealed that the apf and pil operons are highly conserved among different serotypes of A. pleuropneumoniae. Our data demonstrate that the transcription of the pil operon was greatly upregulated upon bacterial contact with host cells. Mutants with single deletions of each gene in the pil and apf operons had defective adherence when they were in contact with host cells and, additionally, these mutants were also deficient in biofilm formation in vitro. In conclusion, these results indicate that the products of each gene within the apf and pil operons are necessary for the normal biogenesis and function of Tfp in A. pleuropneumoniae. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is one of the best-studied surface-attached appendages in bacteria. Flagellar assembly in vivo is promoted by its own protein export apparatus, a type III secretion system (T3SS) in pathogenic bacteria. Lysobacter enzymogenes OH11 is a non-flagellated soil bacterium that utilizes type IV pilus (T4P)-driven twitching motility to prey upon nearby fungi for food. Interestingly, the strain OH11 encodes components homologous to the flagellar type III protein apparatus (FT3SS) on its genome, but it remains unknown whether this FT3SS-like system is functional. Here, we report that, despite the absence of flagella, the FT3SS homologous genes are responsible not only for the export of the heterologous flagellin in strain OH11 but also for twitching motility. Blocking the FT3SS-like system by in-frame deletion mutations in either flhB or fliI abolished the secretion of heterologous flagellin molecules into the culture medium, indicating that the FT3SS is functional in strain OH11. A deletion of flhA, flhB, fliI, or fliR inhibited T4P-driven twitching motility, whereas neither that of fliP nor fliQ did, suggesting that FlhA, FlhB, FliI, and FliR may obtain a novel function to modulate the twitching motility. The flagellar FliI ATPase was required for the secretion of the major pilus subunit, PilA, suggesting that FliI would have evolved to act as a PilB-like pilus ATPase. These observations lead to a plausible hypothesis that the non-flagellated L. enzymogenes OH11 could preserve FT3SS-like genes for acquiring a distinct function to regulate twitching motility associated with its predatory behavior.

Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect filamentous fungal pathogens, through secretion of antifungal antibiotic HSAF (heat-stable antifungal factor), formation of T4P (type IV pilus)-mediated twitching motility, and production of extracellular chitinase. Interestingly, all such key antifungal factors seem to be controlled by Clp, a master regulator in L. enzymogenes; however, the underlying mechanisms are poorly understood. Here, employing strain OH11 as a working model, we show that Clp plays a dual role in controlling OH11 twitching motility. It controls transcription of pilA, a major T4P structure pilin gene, via directly binding to its promoter region, as well as regulates the gene transcription of pilMONOPQ operon, whose products were essential for T4P assembly, by directly binding to a similar promoter sequence. We also truncated the Clp-binding region of the pilA promoter fragment down to 41 bp to identify the potential Clp-binding sequence. In addition, the Clp-recognized pilM promoter motif of the L. enzymogenes strains is similarly conserved as the pilA promoter, both with a conserved 5′-GTG and a conserved CAC-3′, spaced by ten highly variable nucleotides. Thus, this study identified two direct and previously uncharacterized gene targets of Clp contributing to its regulation in the L. enzymogenes twitching motility. Overall, our findings further elucidate the molecular genetics of Clp-dependent twitching motility in Lysobacter. [ABSTRACT FROM AUTHOR]

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting coordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the coordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies. [ABSTRACT FROM AUTHOR]

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4Pmediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoNOH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11. [ABSTRACT FROM AUTHOR]

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce's disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce's Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines. [ABSTRACT FROM AUTHOR]

Type IV pilus (T4P) is widespread in bacteria, yet its biogenesis mechanism and functionality is only partially elucidated in a limited number of bacterial species. Here, by using strain OH11 as the model organism, we reported the identification of 26 T4P structural or functional component (SFC) proteins in the Gram-negative Lysobacter enzymogenes, which is a biocontrol agent potentially exploiting T4P-mediated twitching motility for antifungal activity. Twenty such SFC coding genes were individually knocked-out in-frame to create a T4P SFC deletion library. By using combined phenotypic and genetic approaches, we found that 14 such SFCs, which were expressed from four operons, were essential for twitching motility. These SFCs included the minor pilins (PilE, PilX, PilV, and FimT), the anti-retraction protein PilY1, the platform protein PilC, the extension/extraction ATPases (PilB, PilT, and PilU), and the PilMNOPQ complex. Among these, mutation of pilT or pilU caused a hyper piliation, while the remaining 12 SFCs were indispensable for pilus formation. Ten (FimT, PilY1, PilB, PilT, PilU, and the PilMNOPQ complex) of the 14 SFC proteins, as well as PilA, were further shown to play a key role in L. enzymogenes biofilm formation. Overall, our results provide the first report to dissect the genetic basis of T4P biogenesis and its role in biofilm formation in L. enzymogenes in detail, which can serve as an alternative platform for studying T4P biogenesis and its antifungal function. [ABSTRACT FROM AUTHOR]

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type II secretion. [ABSTRACT FROM AUTHOR]

Type IV pili are long filamentous structures on the surface of bacteria, which can be rapidly assembled or disassembled with pilin subunits by molecular motors. They can generate force during retraction and are involved in many bacterial functions. Steered molecular dynamics simulations with coarse-grained MARTINI models are carried out to investigate the mechanical behaviors of pili under tension. Our study is the first to report a Young's modulus of 0.80 ± 0.07 GPa and a spring constant of 1294.6 ± 116.5 kJ mol −1 nm −2 for pilus. Our results show the mechanical responses of pili are different from those described by the worm-like chain model and the van der Waal's interactions play a critical role in the mechanical responses. Moreover, the effects of pulling rates and virtual spring constants of pilus on Young's modulus are studied and two distinct morphological stages with the conformational changes appear during the extension of pilus are observed. This work provide insight into the mechanics and the deformation mechanism of pilus assembly. [ABSTRACT FROM AUTHOR]

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella--structures required for motility--share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air--liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876--also critical for both motility and adhesion--will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects. [ABSTRACT FROM AUTHOR]

Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tnseq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization. Comparison of Tn-seq results from laboratory cultures and from monoinoculated worker bees reveal that 519 of 2,226 protein-coding genes in S. alvi are essential in culture, whereas 399 are not essential but are beneficial for gut colonization. Genes facilitating colonization fall into three broad functional categories: extracellular interactions, metabolism, and stress responses. Extracellular components with strong fitness benefits in vivo include trimeric autotransporter adhesins, O antigens, and type IV pili (T4P). Experiments with T4P mutants establish that T4P in S. alvi likely function in attachment and biofilm formation, with knockouts experiencing a competitive disadvantage in vivo.Metabolic processes promoting colonization include essential amino acid biosynthesis and iron acquisition pathways, implying nutrient scarcity within the hindgut environment. Mechanisms to deal with various stressors, such as for the repair of double-stranded DNA breaks and protein quality control, are also critical in vivo. This genome- wide study identifies numerous genetic networks underlying colonization by a gut commensal in its native host environment, including some known from more targeted studies in other host--microbe symbioses. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologues. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy imagens to show that the LngB protein could be localized at the tip of CS21, and probably helps to control CS21 length. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.

Bacterial cells can display different types of motility, due to the presence of external appendages such as flagella and type IV pili. To date, little information on the mechanisms involved in the motility of the Lysobacter species has been available. Recently, L. capsici AZ78, a biocontrol agent of phytopathogenic oomycetes, showed the ability to move on jellified pea broth. Pea broth medium improved also the biocontrol activity of L. capsici AZ78 against Plasmopara viticola under greenhouse conditions. Noteworthy, the quantity of pea residues remaining on grapevine leaves fostered cell motility in L. capsici AZ78. Based on these results, this unusual motility related to the composition of the growth medium was investigated in bacterial strains belonging to several Lysobacter species. The six L. capsici strains tested developed dendrite-like colonies when grown on jellified pea broth, while the development of dendrite-like colonies was not recorded in the media commonly used in motility assays. To determine the presence of genes responsible for biogenesis of the flagellum and type IV pili, the genome of L. capsici AZ78 was mined. Genes encoding structural components an d regulatory factors of type IV pili were upregulated in L. capsici AZ78 cells grown on the above-mentioned medium, as compared with the other tested media. These results provide new insight into the motility mechanism of L. capsici members and the role of type IV pili and pea compounds on the epiphytic fitness and biocontrol features of L. capsici AZ78.

Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA , pilB , and pilQ , which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

Stenotrophomonas maltophilia is an emerging opportunistic respiratory pathogen in people with cystic fibrosis (CF). S. maltophilia is frequently observed in polymicrobial infections, and we have previously shown that Pseudomonas aeruginosa promotes colonization and persistence of S. maltophilia in mouse respiratory infections. In this study, we used host and bacterial RNA sequencing to further understand the molecular underpinnings of this interaction. To evaluate S. maltophilia transcript profiles, we used a recently described method for selective capture of bacterial mRNA transcripts with strain-specific RNA probes. We found that factors associated with the type IV pilus, including the histidine kinase subunit of a chemotactic two-component signaling system ( chpA ), had increased transcript levels during dual-species infection. Using immortalized CF respiratory epithelial cells, we found that infection with P. aeruginosa increases adherence of S. maltophilia, at least in part due to disruption of epithelial tight junctions. In contrast, an isogenic S. maltophilia chpA mutant strain lacked cooperative adherence to CF epithelia and decreased bacterial burden in vivo in dual-species infections with P. aeruginosa. Similarly, P. aeruginosa lacking elastase ( lasB ) failed to promote S. maltophilia adherence or bacterial colonization and persistence in vivo . Based on these results, we propose that disruption of lung tissue integrity by P. aeruginosa facilitates adherence of S. maltophilia to the lung epithelia, likely in a type IV pilus-dependent manner. These data lend insight into S. maltophilia colonization and persistence in people in later stages of CF disease and may have implications for interactions with other bacterial opportunists. IMPORTANCE Despite advances in treatment options for people with CF, complications of bacterial infections remain the greatest driver of morbidity and mortality in this patient population. These infections often involve more than one bacterial pathogen, and our understanding of how interspecies interactions impact disease progression is lacking. Previous work in our lab found that two CF pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, can work together in the lung to cause more severe infection. In the present study, we found that infection with P. aeruginosa promotes persistence of S. maltophilia by interfering with epithelial barrier integrity. Depolarization of the epithelial cell layer by P. aeruginosa-secreted elastase increased S. maltophilia adherence, likely in a type IV pilus-dependent manner. Ultimately, this work sheds light on the molecular mechanisms governing an important multispecies interaction seen in pulmonary diseases such as CF.

Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and the adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologs. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy images to show that the LngB protein could be localized at the tip of CS21. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells. [ABSTRACT FROM AUTHOR]

Bacterial cells can display different types of motility, due to the presence of external appendages such as flagella and type IV pili. To date, little information on the mechanisms involved in the motility of the Lysobacter species has been available. Recently, L. capsici AZ78, a biocontrol agent of phytopathogenic oomycetes, showed the ability to move on jellified pea broth. Pea broth medium improved also the biocontrol activity of L. capsici AZ78 against Plasmopara viticola under greenhouse conditions. Noteworthy, the quantity of pea residues remaining on grapevine leaves fostered cell motility in L. capsici AZ78. Based on these results, this unusual motility related to the composition of the growth medium was investigated in bacterial strains belonging to several Lysobacter species. The six L. capsici strains tested developed dendrite-like colonies when grown on jellified pea broth, while the development of dendrite-like colonies was not recorded in the media commonly used in motility assays. To determine the presence of genes responsible for biogenesis of the flagellum and type IV pili, the genome of L. capsici AZ78 was mined. Genes encoding structural components and regulatory factors of type IV pili were upregulated in L. capsici AZ78 cells grown on the above-mentioned medium, as compared with the other tested media. These results provide new insight into the motility mechanism of L. capsici members and the role of type IV pili and pea compounds on the epiphytic fitness and biocontrol features of L. capsici AZ78. [ABSTRACT FROM AUTHOR]

The sophisticated DNA-uptake machinery used during natural transformation is still poorly characterized, especially in Gram-negative bacteria where the transforming DNA has to cross two membranes as well as the peptidoglycan layer before entering the cytoplasm. The DNA-uptake machinery was hypothesized to take the form of a pseudopilus, which, upon repeated cycles of extension and retraction, would pull external DNA towards the cell surface or into the periplasmic space, followed by translocation across the cytoplasmic membrane. In this review, we summarize recent advances on the DNA-uptake machinery of V. cholerae , highlighting the presence of an extended competence-induced pilus and the contribution of a conserved DNA-binding protein that acts as a ratchet and reels DNA into the periplasm. [ABSTRACT FROM AUTHOR]

Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.