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If a bacterium has motility, it will use the ability to survive and thrive. For many pathogenic species, their motilities are a crucial virulence factor. The form of motility varies among the species. Some use flagella for swimming in liquid, and others use the cell-surface machinery to move over solid surfaces. Spirochetes are distinguished from other bacterial species by their helical or flat wave morphology and periplasmic flagella (PFs). It is believed that the rotation of PFs beneath the outer membrane causes transformation or rolling of the cell body, propelling the spirochetes. Interestingly, some spirochetal species exhibit motility both in liquid and over surfaces, but it is not fully unveiled how the spirochete pathogenicity involves such amphibious motility. This review focuses on the causative agent of zoonosis leptospirosis and discusses the significance of their motility in liquid and on surfaces, called crawling, as a virulence factor.

Clostridium perfringens enterotoxin (CPE) is the main virulence factor for C. perfringens type F strains to cause human gastrointestinal diseases, which can involve lethal enterotoxemia. During type F disease, CPE encounters an adherent mucus layer overlying the intestines, so the current study evaluated if NanI potentiates CPE activity in the presence of adherent mucus. CPE alone caused more cytotoxicity transepithelial electrical resistance (TEER) and permeability to fluorescent dextran (FD) for minimal mucus-producing HT29 cells versus that in their derivative HT29-MTX-E12 cells, which produce abundant adherent mucus. However, for HT29-MTX-E12 cells, the presence of NanI significantly increased CPE binding and pore formation, which enhanced their sensitivity to CPE effects on cytotoxicity, TEER, and FD permeability. When the ability of NanI to potentiate CPE-induced enterotoxemia was then tested in a mouse small intestinal loop enterotoxemia model, a pathophysiologically relevant 50 μg/mL dose of CPE did not kill mice. However, the copresence of purified NanI resulted in significant CPE-induced lethality. More CPE was detected in the sera of mice challenged with 50 μg/mL of CPE when NanI was copresent during challenge. The copresence of NanI and CPE during challenge also significantly increased intestinal histologic damage compared to that after challenge with CPE alone, suggesting that NanI enhancement of CPE-induced intestinal damage may increase CPE absorption into blood. Overall, these results indicate that (i) mucus inhibits CPE action and (ii) NanI can potentiate CPE action in the presence of mucus, which may help explain why type F strains that produce relatively low levels of CPE are still pathogenic. IMPORTANCE NanI is a sialidase produced by some Clostridium perfringens type F strains. Here, we found that NanI can significantly increase the action of C. perfringens enterotoxin (CPE), which is the main toxin responsible for severe human enteric disease caused by type F strains. This effect likely helps to explain why even some type F strains that produce small amounts of CPE are pathogenic.

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa , uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

For many intracellular pathogens, the phagosome is the site of events and interactions that shape infection outcome. Phagosomal membrane damage, in particular, is proposed to benefit invading pathogens. To define the innate immune consequences of this damage, we profiled macrophage transcriptional responses to wild-type Mycobacterium tuberculosis (Mtb) and mutants that fail to damage the phagosomal membrane. We identified a set of genes with enhanced expression in response to the mutants. These genes represented a late component of the TLR2-dependent transcriptional response to Mtb, distinct from an earlier component that included Tnf . Expression of the later component was inherent to TLR2 activation, dependent upon endosomal uptake, and enhanced by phagosome acidification. Canonical Mtb virulence factors that contribute to phagosomal membrane damage blunted phagosome acidification and undermined the endosome-specific response. Profiling cell survival and bacterial growth in macrophages demonstrated that the attenuation of these mutants is partially dependent upon TLR2. Further, TLR2 contributed to the attenuated phenotype of one of these mutants in a murine model of infection. These results demonstrate two distinct components of the TLR2 response and identify a component dependent upon endosomal uptake as a point where pathogenic bacteria interfere with the generation of effective inflammation. This interference promotes tuberculosis (TB) pathogenesis in both macrophage and murine infection models., Competing Interests: AH, CJ, SP, WZ, NJ, AM, AB No competing interests declared, (© 2021, Hinman et al.)

Pseudomonas (P.) aeruginosa is an opportunistic pathogen that causes serious infections and hospital-acquired pneumonia in immunocompromised patients. P. aeruginosa accounts for up to 20% of all cases of hospital-acquired pneumonia, with an attributable mortality rate of ~30-40%. The poor clinical outcome of P. aeruginosa -induced pneumonia is ascribed to its ability to disrupt lung barrier integrity, leading to the development of lung edema and bacteremia. Airway epithelial and endothelial cells are important architecture blocks that protect the lung from invading pathogens. P. aeruginosa produces a number of virulence factors that can modulate barrier function, directly or indirectly, through exploiting cytoskeleton networks and intercellular junctional complexes in eukaryotic cells. This review summarizes the current knowledge on P. aeruginosa virulence factors, their effects on the regulation of the cytoskeletal network and associated components, and molecular mechanisms regulating barrier function in airway epithelial and endothelial cells. A better understanding of these processes will help to lay the foundation for new therapeutic approaches against P. aeruginosa -induced pneumonia.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI -carrying versus non -nanI -carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N -acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Virulence of the neonatal pathogen Group B Streptococcus is under the control of the master regulator CovR. Inactivation of CovR is associated with large-scale transcriptome remodeling and impairs almost every step of the interaction between the pathogen and the host. However, transcriptome analyses suggested a plasticity of the CovR signaling pathway in clinical isolates leading to phenotypic heterogeneity in the bacterial population. In this study, we characterized the CovR regulatory network in a strain representative of the CC-17 hypervirulent lineage responsible of the majority of neonatal meningitis. Transcriptome and genome-wide binding analysis reveal the architecture of the CovR network characterized by the direct repression of a large array of virulence-associated genes and the extent of co-regulation at specific loci. Comparative functional analysis of the signaling network links strain-specificities to the regulation of the pan-genome, including the two specific hypervirulent adhesins and horizontally acquired genes, to mutations in CovR-regulated promoters, and to variability in CovR activation by phosphorylation. This regulatory adaptation occurs at the level of genes, promoters, and of CovR itself, and allows to globally reshape the expression of virulence genes. Overall, our results reveal the direct, coordinated, and strain-specific regulation of virulence genes by the master regulator CovR and suggest that the intra-species evolution of the signaling network is as important as the expression of specific virulence factors in the emergence of clone associated with specific diseases., Competing Interests: The authors have declared that no competing interests exist.

Pseudomonas aeruginosa is an opportunistic pathogen, mainly affecting severe patients, such as those in intensive care units (ICUs). High levels of antibiotic resistance and a long battery of virulence factors characterise this pathogen. Among virulence factors, the T3SS (Type 3 Secretion Systems) are especially relevant, being one of the most important virulence factors in P. aeruginosa. T3SS are a complex "molecular syringe" able to inject different effectors in host cells, subverting cell machinery influencing immune responses, and increasing bacterial survival rates. While T3SS have been largely studied and the molecular structure and main effector functions have been established, a series of questions and further points remain to be clarified or established. The key role of T3SS in P. aeruginosa virulence has resulted in the search for T3SS-targeting molecules able to impair their functions and subsequently improve patient outcomes. This review aims to summarise the most relevant features of the P. aeruginosa T3SS., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The type II secretion system (T2SS) is a multi-protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long-range effectors that promote nutrient acquisition, biofilm formation, and pathogenicity. In both animals and plants, the T2SS is increasingly recognized as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focuses on the structure and mechanism of the T2SS. Advances in cryo-electron microscopy are enabling increasingly elaborate sub-complexes to be resolved. However, key questions remain regarding the mechanism of pseudopilus extension and retraction, and how this is coupled with the choreography of the substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Epithelial cells are typically connected through different types of cell junctions that are localized from the apical membrane to the basal surface. In this way, epithelium cells form the first barrier against pathogenic microorganisms and prevent their entry into internal organs and the circulatory system. Recent studies demonstrate that bacterial pathogens disrupt epithelial cell junctions through targeting junctional proteins by secreted virulence factors. In this review, we discuss the diverse strategies used by common bacterial pathogens, including Pseudomonas aeruginosa, Helicobacter pylori, and enteropathogenic Escherichia coli, to disrupt epithelial cell junctions during infection. We also discuss the potential of targeting the pathogenic mechanisms in the treatment of pathogen-associated diseases., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

Yersinia pestis is a highly virulent pathogen and the causative agent of bubonic, septicemic, and pneumonic plague. Primary pneumonic plague caused by inhalation of respiratory droplets contaminated with Y. pestis is nearly 100% lethal within 4 to 7 days without antibiotic intervention. Pneumonic plague progresses in two phases, beginning with extensive bacterial replication in the lung with minimal host responsiveness, followed by the abrupt onset of a lethal proinflammatory response. The precise mechanisms by which Y. pestis is able to colonize the lung and survive two very distinct disease phases remain largely unknown. To date, a few bacterial virulence factors, including the Ysc type 3 secretion system, are known to contribute to the pathogenesis of primary pneumonic plague. The bacterial GTPase BipA has been shown to regulate expression of virulence factors in a number of Gram-negative bacteria, including Pseudomonas aeruginosa , Escherichia coli , and Salmonella enterica serovar Typhi. However, the role of BipA in Y. pestis has yet to be investigated. Here, we show that BipA is a Y. pestis virulence factor that promotes defense against early neutrophil-mediated bacterial killing in the lung. This work identifies a novel Y. pestis virulence factor and highlights the importance of early bacterial/neutrophil interactions in the lung during primary pneumonic plague., (Copyright © 2021 Crane et al.)

Gastric cancer constitutes one of the most prevalent malignancies in both sexes; it is currently the fourth major cause of cancer-related deaths worldwide. The pathogenesis of gastric cancer is associated with the interaction between genetic and environmental factors, among which infection by Helicobacter pylori ( H. pylori ) is of major importance. The invasion, survival, colonization, and stimulation of further inflammation within the gastric mucosa are possible due to several evasive mechanisms induced by the virulence factors that are expressed by the bacterium. The knowledge concerning the mechanisms of H. pylori pathogenicity is crucial to ameliorate eradication strategies preventing the possible induction of carcinogenesis. This review highlights the current state of knowledge and the most recent findings regarding H. pylori virulence factors and their relationship with gastric premalignant lesions and further carcinogenesis.

Staphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (P cap ) upstream of the cap operon. P cap , which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the P cap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of P cap , mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases. IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus , an important human pathogen., (Copyright © 2020 American Society for Microbiology.)

Outer inflammatory protein (OipA) is an important virulence factor of Helicobacter pylori (H. pylori), but the correlation between oipA copy number and its virulence remains unknown. The study was designed to investigate whether the duplicate oipA gene loci showed more virulent than one oipA gene in vitro. H. pylori strain CCS9803 (China Chongqing Strain 9803) that carries duplicate oipA loci was used to construct one or two oipA knockout mutant strain, which was further verified by qPCR and western blot. Gastric epithelial cells AGS and GES-1 were infected with wild-type (WT) or oipA mutants for 6 or 24 h. The expression levels of IL-8, bacterial adhesion, cell apoptosis and cell cycle were performed to analyze the function of oipA. The WT and oipA mutant strains induce significantly higher mRNA and protein levels of IL-8 than the uninfected group (P < 0.05), but only oipA2 mutants induced significantly decreased expression levels than the WT-infected group (P < 0.05). Adherence to gastric cells was significantly decreased by inactivated two oipA loci (P < 0.05). The WT strain caused a significant rising proportion of early apoptosis cell, which had dropped after duplicate oipA genes were both knockout (P < 0.05). WT and oipA1 mutants failed to affect cell cycle; however, the oipA2 mutants increased M phase and reduced S phase when compared to the uninfected group. In conclusion, our study demonstrated that oipA impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its gene copy number.

Back in 1989 some studies have shown that the viral protein Vpr was dispensable for HIV-1 replication in vitro. From then the concept of accessory or auxiliary protein for Vpr has emerged and it is still used to date. However, Vpr soon appeared to be very important for in vivo virus spread and pathogenesis. Vpr has been involved in many biological functions including regulation of reverse transcriptase activity, the nuclear import of the pre-integration complex (PIC), HIV-1 transcription, gene splicing, apoptosis and in cell cycle arrest. Thus, we might rather consider Vpr as a true virulence factor instead of just an accessory factor. At present, Vpr can be regarded as a potential and promising target in different strategies aiming to fight infected cells including latently infected cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The plant pathogen Burkholderia glumae uses quorum sensing (QS) that allows bacteria to share information and alter gene expression on the basis of cell density. The wild-type strain of B. glumae produces quorum-sensing signals (autoinducers) to detect their community and upregulate QS-dependent genes across the population for performing social and group behaviors. The model organism B. glumae was selected to investigate adaptation, estimate evolutionary parameters, and test diverse evolutionary hypotheses by using experimental evolution. The wild-type B. glumae virulent strain showed genotypic changes during regular subculture due to oxygen limitation. The laboratory-evolved clones failed to produce the signaling molecule of C8-HSL/C6-HSL for activation of the quorum-sensing system. Further, the laboratory-evolved clones failed to produce catalase and oxalate for protecting themselves from the toxic environment at stationary phase and phytotoxins (toxoflavin) for infecting rice grain, respectively. The laboratory-evolved clones were completely sequenced and compared with the wild-type. Sequencing analysis of the evolved clones revealed that mutations in QS-responsible genes (iclR), sensor genes (shk, mcp), and signaling genes (luxR) were responsible for quorum-sensing activity failure. The experimental results and sequencing analysis revealed quorum-sensing process failure in the laboratory-evolved clones. In conclusion, the wild-type B. glumae strain was often exposed to oxidative stress during regular subculture and evolved as an avirulent strain (quorum-sensing mutant) by losing the phenotypic and genotypic characteristics.

Pulmonary tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), manifests with a persistent cough as both a primary symptom and mechanism of transmission. The cough reflex can be triggered by nociceptive neurons innervating the lungs, and some bacteria produce neuron-targeting molecules. However, how pulmonary Mtb infection causes cough remains undefined, and whether Mtb produces a neuron-activating, cough-inducing molecule is unknown. Here, we show that an Mtb organic extract activates nociceptive neurons in vitro and identify the Mtb glycolipid sulfolipid-1 (SL-1) as the nociceptive molecule. Mtb organic extracts from mutants lacking SL-1 synthesis cannot activate neurons in vitro or induce cough in a guinea pig model. Finally, Mtb-infected guinea pigs cough in a manner dependent on SL-1 synthesis. Thus, we demonstrate a heretofore unknown molecular mechanism for cough induction by a virulent human pathogen via its production of a complex lipid., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Kingella kingae is a gram-negative coccobacillus that is a fastidious commensal organism in the oropharynx and is being recognized increasingly as a common cause of osteoarticular infections and other invasive diseases in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, followed by translocation across the epithelial barrier, survival in the bloodstream, and dissemination to distant sites, including bones, joints, and the endocardium, among others. Characterization of the determinants of K. kingae pathogenicity has revealed a novel model of adherence that involves the interplay of type IV pili, a non-pilus adhesin, and a polysaccharide capsule and a novel model of resistance to serum killing and neutrophil killing that involves complementary functions of a polysaccharide capsule and an exopolysaccharide. These models likely apply to other bacterial pathogens as well., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Streptococcus pneumoniae is a Gram-positive pathogen with high morbidity and mortality globally but some of its pathogenesis remains unknown. Previous research has provided evidence that aminopeptidase N (PepN) is most likely a virulence factor of S. pneumoniae. However, its role in S. pneumoniae virulence and its interaction with the host remains to be confirmed. We generated a pepN gene deficient mutant strain and found that its virulence for mice was significantly attenuated as were in vitro adhesion and invasion of host cells. The PepN protein could induce a strong innate immune response in vivo and in vitro and induced secretion of IL-6 and TNF-α by primary peritoneal macrophages via the rapid phosphorylation of MAPK and PI3K/AKT signaling pathways and this was confirmed using specific pathway inhibitors. In conclusion, PepN is a novel virulence factor that is essential for the virulence of S. pneumoniae and induces host innate immunity via MAPK and PI3K/AKT signaling.

Thirty years ago Stanley Falkow formulated molecular Koch's postulates as a framework to help dissect the contribution of microbial genes to their pathogenicity (Box 1). Three years later, his advice led me to develop Mycobacterium marinum, a close genetic relative of Mycobacterium tuberculosis, as a model for tuberculosis pathogenesis. Here, I discuss insights into M. tuberculosis pathogenicity from studying M. marinum in the zebrafish, and frame them in terms of molecular Koch's postulates. The highly orchestrated life cycle of M. tuberculosis is achieved in substantial measure not by "traditional" pathogen-exclusive virulence genes acquired along its evolutionary history, but rather by genes that are shared with its environmental ancestors. Together, these genes support its tactics of subterfuge and exploitation to overcome host immunity so as to produce the transmissible disease that ensures the evolutionary survival of this obligate human pathogen., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

5'-nucleotidases (5'-NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5'-NTs have been discovered in Gram-positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5'-NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro-inflammatory immune responses. Several 5'-NTs are also able to use dAMP as substrate to generate deoxy-adenosine which is cytotoxic for macrophages. A synergy between 5'-NTs and exonucleases which are commonly expressed in Gram-positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram-positive cell wall-anchored 5'-NTs and their role as virulence factors. A potential use of 5'-NTs for vaccine development is also briefly discussed., (© 2020 John Wiley & Sons Ltd.)

The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is an important commensal resident of the human nasopharynx. Carriage is usually asymptomatic, however, S. pneumoniae can become invasive and spread from the upper respiratory tract to the lungs causing pneumonia, and to other organs to cause severe diseases such as bacteremia and meningitis. Several pneumococcal proteins important for its disease-causing capability have been described and many are expressed on the bacterial surface. The surface located pneumococcal type-1 pilus has been associated with virulence and the inflammatory response, and it is present in 20%-30% of clinical isolates. Its tip protein RrgA has been shown to be a major adhesin to human cells and to promote invasion through the blood-brain barrier. In this review we discuss recent findings of the impact of RrgA on bacterial colonization of the upper respiratory tract and on pneumococcal virulence, and use epidemiological data and genome-mining to suggest trade-off mechanisms potentially explaining the rather low prevalence of pilus-1 expressing pneumococci in humans., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Phenoloxidase (PO) is a key enzyme in the melanization process involved in elimination of pathogens in insects. The PO system is rapidly activated in response to pathogen recognition. Inhibition of PO activity can be a way to avoid immune response and increase infection effectiveness. In this study, the effects of inoculation of Galleria mellonella larvae with Aspergillus niger α-1,3-glucan and conidia on PO activity in hemolymph are analyzed in comparison with the effects of β-1,3/1,6-glucan inoculation. Our results indicate that α-1,3-glucan, a fungal cell wall polysaccharide, can play a role of a virulence factor involved in inhibition of the insect PO system., Competing Interests: Declaration of Competing Interest The authors declare no conflict interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis is primarily a respiratory pathogen. However, 15% of infections worldwide occur at extrapulmonary sites causing additional complications for diagnosis and treatment of the disease. In addition, dissemination of M. tuberculosis out of the lungs is thought to be more than just a rare event leading to extrapulmonary tuberculosis, but rather a prerequisite step that occurs during all infections, producing secondary lesions that can become latent or productive. In this review we will cover the clinical range of extrapulmonary infections and the process of dissemination including evidence from both historical medical literature and animal experiments for dissemination and subsequent reseeding of the lungs through the lymphatic and circulatory systems. While the mechanisms of M. tuberculosis dissemination are not fully understood, we will discuss the various models that have been proposed to address how this process may occur and summarize the bacterial virulence factors that facilitate M. tuberculosis dissemination., (Copyright © 2020 Moule and Cirillo.)

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions., (Copyright © 2020 American Society for Microbiology.)

Multidrug efflux systems belonging to the resistance-nodulation-division (RND) superfamily are ubiquitous in Gram-negative bacteria. RND efflux systems are often associated with multiple antimicrobial resistance and also contribute to the expression of diverse bacterial phenotypes including virulence, as documented in the intestinal pathogen Vibrio cholerae , the causative agent of the severe diarrheal disease cholera. Transcriptomic studies with RND efflux-negative V. cholerae suggested that RND-mediated efflux was required for homeostasis, as loss of RND efflux resulted in the activation of transcriptional regulators, including multiple environmental sensing systems. In this report, we investigated six RND efflux-responsive regulatory genes for contributions to V. cholerae virulence factor production. Our data showed that the V. cholerae gene VC2714, encoding a homolog of Escherichia coli OmpR, was a virulence repressor. The expression of ompR was elevated in an RND-null mutant, and ompR deletion partially restored virulence factor production in the RND-negative background. Virulence inhibitory activity in the RND-negative background resulted from OmpR repression of the key ToxR regulon virulence activator aphB , and ompR overexpression in wild-type cells also repressed virulence through aphB We further show that ompR expression was not altered by changes in osmolarity but instead was induced by membrane-intercalating agents that are prevalent in the host gastrointestinal tract and which are substrates of the V. cholerae RND efflux systems. Our collective results indicate that V. cholerae ompR is an aphB repressor and regulates the expression of the ToxR virulence regulon in response to novel environmental cues., (Copyright © 2020 American Society for Microbiology.)

The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureus S. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr -defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis -secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections. IMPORTANCE S. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections., (Copyright © 2020 American Society for Microbiology.)

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain., (© 2019 John Wiley & Sons Ltd.)

Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. The RpoE sigma factor and CpxRA two-component system are the major regulators of the extracytoplasmic stress response. In this study, we found that the CpxR has highly significant, but opposite effects on the auto-aggregation and swarming motility of S . Enteritidis. Auto-aggregation was negatively affected in the ∆ cpxR mutant, whereas the same mutant significantly out-performed its wild-type counterpart with respect to swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, biofilm-related assays showed that the CpxR is of critical importance in biofilm development under both static (microtiter plate) and dynamic (flow cell) media flow conditions. In contrast, the RpoE sigma factor showed no significant role in biofilm development under dynamic conditions. Transcriptomic analysis revealed that the cpxR mutation negatively affected the constitutive expression of the operons critical for biosynthesis of O-antigen and adherence, but positively affected the expression of virulence genes critical for Salmonella -mediated endocytosis. Conversely, CpxR induced the expression of curli csgAB and fimbrial stdAC operons only during biofilm development and flagellar motAB and fliL operons exclusively during the planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator.

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits., Competing Interests: The authors have declared that no competing interests exist.

Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island. Colibactin is a virulence factor and a putative pro-carcinogenic compound. Therefore, we aimed to decouple the antagonistic activity of EcN from its genotoxic activity. We demonstrated that the pks-encoded ClbP, the peptidase that activates colibactin, is required for the antagonistic activity of EcN. The analysis of a series of ClbP mutants revealed that this activity is linked to the transmembrane helices of ClbP and not the periplasmic peptidase domain, indicating the transmembrane domain is involved in some aspect of Mcc biosynthesis or secretion. A single amino acid substitution in ClbP inactivates the genotoxic activity but maintains the antagonistic activity. In an in vivo salmonellosis model, this point mutant reduced the clinical signs and the fecal shedding of Salmonella similarly to the wild type strain, whereas the clbP deletion mutant could neither protect nor outcompete the pathogen. The ClbP-dependent antibacterial effect was also observed in vitro with other E. coli strains that carry both a truncated form of the Mcc gene cluster and the pks island. In such strains, siderophore-Mcc synthesis also required the glucosyltransferase IroB involved in salmochelin production. This interplay between colibactin, salmochelin, and siderophore-Mcc biosynthetic pathways suggests that these genomic islands were co-selected and played a role in the evolution of E. coli from phylogroup B2. This co-evolution observed in EcN illustrates the fine margin between pathogenicity and probiotic activity, and the need to address both the effectiveness and safety of probiotics. Decoupling the antagonistic from the genotoxic activity by specifically inactivating ClbP peptidase domain opens the way to the safe use of EcN., Competing Interests: The authors have declared that no competing interests exist.

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa , the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before., (Copyright © 2019 American Society for Microbiology.)

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens., Competing Interests: The authors have declared that no competing interests exist.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV., Competing Interests: The authors have declared that no competing interests exist.

The genital pathogen Chlamydia is known to colonize the gastrointestinal tract. Orally delivered Chlamydia muridarum can reach the colon and maintain a long-lasting colonization there. However, C. muridarum with mutations in chromosomal genes tc0237 and tc0668 (designated a chromosomal mutant) or deficient in plasmid-encoded pGP3 (designated a plasmid mutant) is unable to do so. We now report that the chromosomal mutant is still able to reach the colon while the plasmid mutant fails to do so following an oral delivery, suggesting that lack of colon colonization by different mutants may involve distinct mechanisms. Consistently, a direct intracolonic delivery selectively restored the ability of the plasmid mutant, but not the chromosomal mutant, to colonize the colon. The chromosomal mutant was rescued only in the colon of mice deficient in gamma interferon (IFN-γ). Thus, the chromosomal mutant's deficiency in colonizing colonic mucosal tissue is likely due to its increased susceptibility to IFN-γ-mediated immunity. Furthermore, IFN-γ deficiency was sufficient for rescuing colon colonization of an orally delivered chromosomal mutant but not plasmid mutant while mice deficient in gastric acid production rescued the plasmid mutant but not the chromosomal mutant. Both mutants are attenuated in inducing genital tract pathology. Thus, we propose that chlamydial chromosomal-gene-encoded genital tract virulence factors may be essential for Chlamydia to maintain long-lasting colonization in the colon while the plasmid may enable Chlamydia to reach the colon by promoting evasion of gastric barriers., (Copyright © 2019 American Society for Microbiology.)

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitans A. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the β 2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent., (Copyright © 2019 American Society for Microbiology.)

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis., Competing Interests: Conflict of interest statement: V.J.T. is an inventor on patents and patent applications filed by New York University which are currently under commercial license to Janssen Biotech, Inc.

Aim: Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Aims: In this study, the association between multidrug resistance (MDR) and the expression of some virulence factors were evaluated in Escherichia coli strains isolated from infant faeces and fresh green vegetables. The effect of isolate origin on associated virulence factors was evaluated. In addition, genetic fingerprinting of a sample of these isolates (10 isolates from each group) was studied in order to detect any genetic relatedness among these isolates., Methods and Results: Escherichia coli isolates were divided into four groups based on their origin (human faeces or plant) and their antibiotic resistance (multiresistance or susceptible). PCR was used to investigate heat-labile and heat-stable enterotoxin genes, and four siderophore genes (aerobactin, enterobactin, salmochelin and yersiniabactin). Genetic fingerprinting of the isolates was performed using enterobacterial repetitive intergenic consensus PCR. Siderophore production was measured by a colorimetric method. Biofilm formation was evaluated by a crystal violet assay. The results of the study showed that the expression of MDR is not significantly associated with an increase in these virulence factors or with biofilm formation. However, the origin of isolates had a significant association with siderophore gene availability and consequently on the concentrations of siderophores released. Genetic fingerprinting indicated that human and plant isolates have the same clonal origin, suggesting their circulation among humans and plants., Conclusion: Antibiotic-susceptible strains of E. coli may be as virulent as MDR strains. Results also suggest that the environment can play a potential role in selection of strains with specific virulence factors., Significance and Impact of the Study: Antibiotic-susceptible isolates of Escherichia coli from plant or human origin can be as virulent as the multidrug resistance (MDR) ones. Genetic relatedness was detected among the isolates of plant and human origin, indicating the circulation of these bacteria among human and plants. This could imply a potential role for environmental antimicrobial resistant bacteria in human infection., (© 2019 The Society for Applied Microbiology.)

Streptococcus suis has received increasing attention for its involvement in severe infections in pigs and humans; however, their pathogenesis remains unclear. ClpX and ClpP, two subunits of the ATP-dependent caseinolytic protease Clp, play key roles in bacterial adaptation to various environmental stresses. In this study, a virulent S. suis serotype 2 strain, ZY05719, was employed to construct clpX and clpP deletion mutants (ΔclpX and ΔclpP, respectively) and their complementation strains. Both ΔclpX and ΔclpP displayed significantly reduced adaptability compared with the wild-type strain, evident through several altered phenotypes: formation of long cell chains, tendency to aggregate in culture, and reduced growth under acidic pH and H 2 O 2 -induced oxidative stress. ClpP and ClpX were required for the optimal growth during heat and cold stress, respectively. An in vitro experiment on RAW264.7 macrophage cells showed significantly increased sensitivity of ΔclpX and ΔclpP to phagocytosis compared with the wild-type strain. Mouse infection assays verified the deletion of clpX and clpP led to not only fewer clinical symptoms and lower mortality but also to a marked attenuation in bacterial colonization. These virulence-related phenotypes were restored by genetic complementation. Furthermore, the deletion of clpX or clpP caused a significant decrease in the expression of sodA, tpx, and apuA compared with the wild-type strain, suggesting that these genes may be regulated by ClpX and ClpP as downstream response factors to facilitate the bacterial tolerance against various environmental stresses. Taken together, these results suggest that ClpX and ClpP play important roles in stress tolerance for achieving the full virulence of S. suis serotype 2 during infection., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Introduction: The rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear., Methods: A retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST)., Results: The predominant virulence profile (fimH, mrkD v1 , khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of β-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p<0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 β-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways., Conclusion: This study describes an overlapping of multidrug-resistance and virulence determinants in ST-14 K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates worldwide. Monitoring data have revealed a continuing trend toward an increase in neonatal GBS infections, despite the introduction of preventive measures. We investigated this trend, by performing the first ever characterization of the prophage content for 106 GBS strains causing neonatal infections between 2002 and 2018. We determined whether the genome of each strain harbored prophages, and identified the insertion site of each of the prophages identified. We found that 71.7% of the strains carried at least one prophage, and that prophages genetically similar to livestock-associated phiD12, carrying genes potentially involved in GBS pathogenesis (e.g., genes encoding putative virulence factors and factors involved in biofilm formation, bacterial persistence, or adaptation to challenging environments) predominated. The phiD12-like prophages were (1) associated with CC17 and 1 strains ( p = 0.002), (2) more frequent among strains recovered during the 2011-2018 period than among those from 2002-2010 ( p < 0.001), and (3) located at two major insertion sites close to bacterial genes involved in host adaptation and colonization. Our data provide evidence for a recent increase in lysogeny in GBS, characterized by the acquisition, within the genome, of genetic features typical of animal-associated mobile genetic elements by GBS strains causing neonatal infection. We suggest that lysogeny and phiD12-like prophage genetic elements may have conferred an advantage on GBS strains for adaptation to or colonization of the maternal vaginal tract, or for pathogenicity, and that these factors are currently playing a key role in the increasing ability of GBS strains to infect neonates.

The acquisition of metal ions such as iron, copper and manganese is essential for the survival of microorganisms as these are constituents of metalloproteins including enzymes, storage proteins, structural elements, transcription factors and antimicrobial factors in various biological processes. However, excess of these metal ions is associated with significant toxicity due to spontaneous redox cycling of ions and obstruction of normal metabolic pathways. To overcome this, microbes have developed a variety of metal regulatory systems allowing them to adapt to the changing biotic and abiotic environments. Multi-copper oxidases (MCOs) such as ceruloplasmins, ferroxidases, laccases and nitrite reductases are such regulatory systems employed by microbes to resist the toxicity of metal ions by controlling their oxidation states under aerobic conditions. MCOs help pathogens survive during an infection by evasion of the toxic environment generated by the host immune system and thus are considered necessary determinants of virulence. This review summarizes the role of MCOs in metal homeostasis under stressful conditions and the extent to which these MCOs contribute to microbial virulence within the host that might prove as an esteemed avenue for the development of novel antimicrobial therapies., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Klebsiella pneumoniae is an important human pathogen causing hospital-acquired and community-acquired infections. Systemic K. pneumoniae infections may be preceded by gastrointestinal colonization, but the basis of this bacterium's interaction with the intestinal epithelium remains unclear. Here, we report that the K. pneumoniae Sap (sensitivity to antimicrobial peptides) transporter contributes to bacterial-host cell interactions and in vivo virulence. Gene deletion showed that sapA is required for the adherence of a K. pneumoniae blood isolate to intestinal epithelial, lung epithelial, urinary bladder epithelial, and liver cells. The ΔsapA mutant was deficient for translocation across intestinal epithelial monolayers, macrophage interactions, and induction of proinflammatory cytokines. In a mouse gastrointestinal infection model, ΔsapA yielded significantly decreased bacterial loads in liver, spleen and intestine, reduced liver abscess generation, and decreased mortality. These findings offer new insights into the pathogenic interaction of K. pneumoniae with the host gastrointestinal tract to cause systemic infection., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

Reversible switching between opaque and translucent colony formation is a novel feature of Acinetobacter baumannii that has been associated with variations in the cell morphology, surface motility, biofilm formation, antibiotic resistance and virulence. Here, we assessed a number of phenotypic alterations related to colony switching in A. baumannii clinical isolates belonging to different multi-locus sequence types. Our findings demonstrated that these phenotypic alterations were mostly strain-specific. In general, the translucent subpopulations of A. baumannii produced more dense biofilms, were more piliated, and released larger amounts of outer membrane vesicles (OMVs). In addition, the translucent subpopulations caused reduced fertility of Caenorhabditis elegans. When assessed for effects on the immune response in RAW 264.7 macrophages, the OMVs isolated from opaque subpopulations of A. baumannii appeared to be more immunogenic than the OMVs from the translucent form. However, also the OMVs from the translucent subpopulations had the potential to evoke an immune response. Therefore, we suggest that OMVs may be considered for development of new immunotherapeutic treatments against A. baumannii infections., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity., Methods: PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects., Results: PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity., Conclusions: PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.

Certain animal and plant pathogenic bacteria have developed virulence factors including effector proteins that enable them to overcome host immunity. A plant pathogen, Pseudomonas syringae pv. tomato (Pto) secretes a large repertoire of effectors via a type III secretory apparatus, thereby suppressing plant immunity. Here, we show that Pto causes sepsis in mice. Surprisingly, the effector HopQ1 disrupted animal phagocytosis by inhibiting actin rearrangement via direct interaction with the LIM domain of the animal target protein LIM kinase, a key regulator of actin polymerization. The results provide novel insight into animal host-plant pathogen interactions. In addition, the current study firstly demonstrates that certain plant pathogenic bacteria such as Pto evade phagocytosis by animal cells due to cross-kingdom suppression of host immunity., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)