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The Enterococcus faecalis (E. Faecalis) virulence factor plays an essential role in the persistence of root canal infection. Virulence factors of Enterococcus faecalis such as lipoteichoic acid, extracellular superoxide, gelatinase, hyaluronidase, and cytolysin are known to increase the ability of Enterococcus faecalis to induce inflammatory processes, colonization formation, and increase resistance. The virulence factor of E. faecalis is mediated by LTA, which has pattern recognition receptors for cytokine release, bone resorption and triggers apoptosis of osteoblasts, osteoclasts, periodontal connective tissue, macrophages, and neutrophils, which have implications for the occurrence of periradicular lesions. Lipoteichoic acid is also involved in producing D-alanine, which stimulates signals to other bacteria to form biofilms. The E. faecalis will change the balance of oxygen radical production in the periapical lesion, fragment collagen. The fight host's defense mechanisms that cause periapical damage and worsening bone loss. Furthermore, cytolysin will respond to changes in oxygen conditions in the depleting root canals for the dominance of E. faecalis against other bacteria. The energy needs of E. faecalis that assisted by hyaluronidase, which degrades hyaluronan dentin. İt has to produce disaccharide degradation products that can be transported and metabolized intracellularly. These materials hydrolyzing the substrate to obtain essential carbon for its growth. This article aims to describe the molecular aspect of E. faecalis virulence that is involved in root canal infections.

To evaluate strains of Enterococcus faecalis isolated from endodontic failures cases for (a) presence of virulence genes, namely, gelatinase production (gelE), surface protein (esp), collagen-binding adhesin (ace), cytolysin activator (cylA), E. faecalis antigen A (efaA) and aggregation substance (asa), all by using PCR; (b) biofilm formation capacity; and (c) activity of gelatinase and β-lactamase. Twenty-five strains of E. faecalis were tested. The DNA extracted from these strains was used for identification of virulence genes by PCR and 1% agarose gel. Biofilm formation was performed on polystyrene microplates by using the violet crystal staining method. For assessment of the gelatinase activity, inoculum of pure cultures was deposited in tubes containing gelatin and a nutrient broth, whereas nitrocefin disks were used to assess the β-lactamase action. The virulence genes efaA and cylA were detected in 100% of the strains, whereas gelE was present in 84%, ace in 68%, esp in 56% and asa in 48%. Four strains had no biofilm formation, 17 had poor formation and four had moderate formation. Gelatinase production was observed in three strains and β-lactamase resistance in five strains of E. faecalis. Diverse patterns of virulence gene detection were observed among the E. faecalis strains, with predominance of those capable of forming biofilm. A few strains have been found to hydrolyze gelatin proteins, whereas β-lactamase resistance was detected in different isolates. To understand the influence of virulence factors in E. faecalis on the host heath status.

Elevated levels of iron are strongly correlated with the growth and virulence of highly lethal human-pathogen Vibrio vulnificus. The present study examined the expression level of hemolysin/cytolysin-encoding gene (vvhA) and the growth rate of four clinical and four environmental strains of V. vulnificus under different iron concentrations (7, 10, 15, 30, and 50 μM). The expression levels of vvhA were determined according to the 2-ΔΔCT method. vvhA transcription was down-regulated as iron concentration increased. A significant difference was observed at the level of 30 and 50 μM ferric chloride concentrations compared to the lower concentrations in all the strains tested (p

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and for gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and to other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted ten genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE:Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding ten secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

Biofilm formation is closely related to the pathogenicity of E. faecalis in persistent root canal infections. Limited information is available about the ability and characteristics of E. faecalis biofilm-forming in the persistent pathogenicity of root canal infections. Based on these problems, the purpose of this paper is to improve the molecular understanding of E. faecalis on virulence factors associated with biofilm formation against persistent root canal infections to support laboratory diagnosis and therapy of oral E. faecalis. In conclusion, the release of cytokines triggers the dlt gene of LTA to improving: homeostasis, autolytic activity, and bacterial envelope properties. The role of cytolysin activated by the cylLL and cylLS genes improving the survival ability of E. Faecalis. Hyaluronidase will facilitate other bacteria to migrate from the root canal to periapical lesions resulting in the triggering of toxin production, which will increase damage and inflammation in the host. Enterococcus faecalis, through dentine matrix formation, hydrolyze collagen and induce apatite deposition in developing biofilms. Besides, these bacteria can also increase their tolerance to antimicrobials by blocking the inflammatory response's acid reaction. Alkaline conditions will neutralize the lactic acid secreted by osteoclasts to absorb hard tissue

Several studies show associations between gut bacterial dysbiosis and chronic liver diseases, but causative mechanisms are largely unclear. We recently identified cytolysin, a bacterial exotoxin expressed and secreted by Enterococcus faecalis to cause liver damage in the setting of alcohol-related liver disease. Cytolysin was increased and highly correlated with liver disease severity and mortality in alcoholic hepatitis patients. In this study, we investigated if faecal cytolysin-positivity can be linked to non-alcoholic fatty liver disease, a highly prevalent disease where new biomarkers and treatment targets are urgently needed. In contrast to what we observed in alcoholic hepatitis, only seven out of 96 non-alcoholic fatty liver disease patients were cytolysin-positive, and these patients did not have increased liver disease activity compared with cytolysin-negative patients. These results indicate that the association of cytolysin carriage with worse clinical outcome might be specific for alcoholic hepatitis.

Bacillus thuringiensis (Bt) produces two families of insecticidal crystal proteins, i.e., crystalline (Cry) and cytolytic (Cyt) toxins. Cyt3Aa1, the newest Cyt family member, is produced by Bt TD516. Bioassay results have shown that Cyt3Aa1 has weak hemolytic activity against human red blood cells and is not toxic to A. aegypti larvae, but causing a teratogenic effect. The three-dimensional structure of Cyt3Aa1 has a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers, resembling the previously reported Cyt1Aa and Cyt2Aa structures, which indicated that Cyt3Aa1 might be a membrane-perforation toxin and could induce synergism with Cry protein. This study provides a new source of insecticidal crystal proteins, and presents a foundation for understanding the biological characterization of it, which will aid in the development of strategies to cope with the potential problem of insect resistance.

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore-forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore-forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein-protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins., his study was supported by grants from the Spanish Ministerio de Economía y Competitividad BFU2017-82758-P (H.O.) and of Basque Government (Grupos Consolidados IT1264-19). D.G.B was recipients of a fellowship from the Bizkaia Biophysics Foundation, and JA was recipient of a fellowship from the Basque Government.

IntroductionPatients with alcoholic hepatitis have an increase in cytolysin-producing Enterococcus faecalis that correlates with disease severity and mortality.AimTo determine whether patients with chronic alcoholic pancreatitis have an elevated abundance of cytolysin-producing E. faecalis.MethodsQuantification by qPCR of cytolysin-producing E. faecalis in controls and patients with alcoholic hepatitis or pancreatitis.ResultsPatients with alcoholic pancreatitis had a higher proportion of intestinal cytolysin-positive E. faecalis than healthy controls and patients with alcoholic hepatitis.ConclusionCytolytic E. faecalis may also be involved in this other alcohol-related complication and benefit from targeted microbiota editing strategies.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Enterococcal cytolysin is a hemolytic virulence factor linked to human disease and increased patient mortality. Produced by pathogenic strains of Enterococcus faecalis, cytolysin is made up of two small, post-translationally modified peptides called CylL(L)” and CylL(S)”. They exhibit a unique toxicity profile where lytic activity is observed for both mammalian cells and gram-positive bacteria that is dependent on the presence of both peptides. In this study, we performed alanine substitution of all residues in CylL(L)” and CylL(S)” and determined the effect on both activities. We identified key residues involved in overall activity and residues that dictate cell type specificity. All (methyl)lanthionines as well as a Gly-rich hinge region were critical for both activities. In addition, we investigated the binding of the two subunits to bacterial cells suggesting that the large subunit CylL(L)” has stronger affinity for the membrane or a target molecule therein. Genome mining identified other potential two-component lanthipeptides and provides insights into potential evolutionary origins.

By leveraging the ability of bacteria to express therapeutic protein cytolysin A (ClyA) through plasmid transformation, a thermally-activated biohybrid (TAB@Au) is constructed by biomineralizing gold nanoparticles (AuNPs) on the E. coli surface. Due to the feature of anaerobic bacteria homing to tumor microenvironments, the bacteria-based antitumor vehicles can be efficaciously accumulated at tumor sites. Under NIR laser irradiation, the biomineralized AuNPs harvest transdermal photons and convert them into local heat for photothermal therapy. After that, the produced heat elicits the expression of ClyA for killing tumor cells. In vitro and in vivo experiments verify the conception that the current therapeutic modality greatly inhibits the proliferation of tumor cells. In terms of the spatial specificity and non-invasiveness of NIR laser, the bacteria-based phototherapy represents an appealing way for tumor therapy.

Sepsis is an acute inflammatory response that leads to life-threatening complications if not quickly and adequately treated. Cytolysin, hemolysin, and pneumolysin are toxins produced by gram-positive bacteria and are responsible for resistance to antimicrobial drugs, cause virulence and lead to sepsis. This work assessed the effects of aloe-emodin (AE) and photodynamic therapy (PDT) on sepsis-associated gram-positive bacterial toxins. Standard and antibiotic-resistant Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia bacterial strains were cultured in the dark with varying AE concentrations and later irradiated with 72 J/cm-2 light. Colony and biofilm formation was determined. CCK-8, Griess reagent reaction, and ELISA assays were done on bacteria-infected RAW264.7 cells to determine the cell viability, NO, and IL-1β and IL-6 pro-inflammatory cytokines responses, respectively. Hemolysis and western blot assays were done to determine the effect of treatment on hemolysis activity and sepsis-associated toxins expressions. AE-mediated PDT reduced bacterial survival in a dose-dependent manner with 32 μg/ml of AE almost eliminating their survival. Cell proliferation, NO, IL-1β, and IL-6 cytokines production were also significantly downregulated. Further, the hemolytic activities and expressions of cytolysin, hemolysin, and pneumolysin were significantly reduced following AE-mediated PDT. In conclusion, combined use of AE and light (435 ± 10 nm) inactivates MRSA, S. aureus (ATCC 29213), S. pneumoniae (ATCC 49619), MDR-S. pneumoniae, E. faecalis (ATCC 29212), and VRE (ATCC 51299) in an AE-dose dependent manner. AE and light are also effective in reducing biofilm formations, suppressing pro-inflammatory cytokines, hemolytic activities, and inhibiting the expressions of toxins that cause sepsis.

Streptococcus suis, a globally distributed bacterial pathogen, is an important zoonotic agent for humans and animals that can lead to multiple deaths and cause major economic losses. Suilysin (SLY), secreted by most pathogenic S. suis strains, is a cytotoxic toxin that belongs to the cholesterol-dependent cytolysin family; this toxin plays a key role in a mouse meningitis model, suggesting that effective interference with the biological activity of SLY may be a potential treatment for S. suis infection. In addition, the inflammatory response induced by S. suis is an important manifestation in infections and is associated with multiple fatal diseases. In this study, we found that the natural compound quercetin can directly inhibit the pore-forming activity of SLY without affecting bacterial growth and SLY secretion at the concentrations tested in our assay. In addition, quercetin treatment significantly alleviated cytotoxicity caused by S. suis infection and effectively reduced the release of the pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) stimulated by bacteria. Significantly decreased mortality was observed for the S. suis-infected mice that received quercetin. Our results suggested that quercetin may represent a promising therapeutic candidate for S. suis infection by targeting SLY and the subsequent inflammation. The present study provides a new strategy and leading compound for S. suis infection.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.

Introduction. Serratia marcescens is a bacterial pathogen that causes ventilator-associated pneumonia and ocular infections. The FlhD and FlhC proteins complex to form a heteromeric transcription factor whose regulon, in S. marcescens , regulates genes for the production of flagellum, phospholipase A and the cytolysin ShlA. The previously identified mutation, scrp-31, resulted in highly elevated expression of the flhDC operon. The scrp-31 mutant was observed to be more cytotoxic to human airway and ocular surface epithelial cells than the wild-type bacteria and the present study sought to identify the mechanism underlying the increased cytotoxicity phenotype. Hypothesis/Gap Statement. Although FlhC and FlhD have been implicated as virulence determinants, the mechanisms by which these proteins regulate bacterial cytotoxicity to different cell types remains unclear. Aim. This study aimed to evaluate the mechanisms of FlhDC-mediated cytotoxicity to human epithelial cells by S. marcescens . Methodology. Wild-type and mutant bacteria and bacterial secretomes were used to challenge airway and ocular surface cell lines as evaluated by resazurin and calcein AM staining. Pathogenesis was further tested using a Galleria mellonella infection model. Results. The increased cytotoxicity of scrp-31 bacteria and secretomes to both cell lines was eliminated by mutation of flhD and shlA. Mutation of the flagellin gene had no impact on cytotoxicity under any tested condition. Elimination of the phospholipase gene, phlA, had no effect on bacteria-induced cytotoxicity to either cell line, but reduced cytotoxicity caused by secretomes to airway epithelial cells. Mutation of flhD and shlA, but not phlA, reduced bacterial killing of G. mellonella larvae. Conclusion. This study indicates that the S. marcescens FlhDC-regulated secreted proteins PhlA and ShlA, but not flagellin, are cytotoxic to airway and ocular surface cells and demonstrates differences in human epithelial cell susceptibility to PhlA.

Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.

International audience; Here, we report a novel approach to improve the anti-Clostridium perfringens activity of the leaderless two-peptide enterocin 14 (EntDD14), produced by Enterococcus faecalis 14. This strategy consists of loading EntDD14 onto alginate nanoparticles (Alg NPs), which are made of a safe polymer. The resulting formulation (EntDD14/Alg NPs) was able to reduce up to four times the minimum inhibitory concentration (MIC) of EntDD14 against C. perfringens pathogenic strains isolated from a chicken affected by necrotic enteritis (NE). Interestingly, this formulation remained active under conditions mimicking the human and chicken gastric tract. Assays conducted to establish the impact of this formulation on the intestinal epithelial cell line Caco-2 and the human colorectal adenocarcinoma cell line HT29 revealed the absence of cytotoxicity of both free-EntDD14 and EntDD14 loaded onto the alginate nanoparticles (EntDD14/Alg NPs) against the aforementioned eukaryotic cells, after 24 h of contact. Notably, EntDD14 and EntDD14/Alg NPs, both at a sub-inhibitory concentration, affected the expression of genes coding for clostridial toxins such as toxin α, enteritis B-like toxin, collagen adhesion protein and thiol-activated cytolysin. Further, expression of these genes was significantly down-regulated following the addition of EntDD14/Alg NPs, but not affected upon addition of EntDD14 alone. This study revealed that adsorption of EntDD14 onto Alg NPs leads to a safe and active formulation (EntDD14/Alg NPs) capable of affecting the pathogenicity of C. perfringens. This formulation could therefore be used in the poultry industry as a novel approach to tackle NE.

The Vibrio vulnificus (V. vulnificus) hemolysin (VVH) is a pore-forming cholesterol-dependent cytolysin (CDC). Although there has been some debate surrounding the in vivo virulence effects of the VVH, it is becoming increasingly clear that it drives different cellular outcomes and is involved in the pathogenesis of V. vulnificus. This minireview outlines recent advances in our understanding of the regulation of vvhA gene expression, the biological activity of the VVH and its role in pathogenesis. An in-depth examination of the role of the VVH in V. vulnificus pathogenesis will help reveal the potential targets for therapeutic and preventive interventions to treat fatal V. vulnificus septicemia in humans. Future directions in VVH research will also be discussed.

In this study, we tested the hypothesis that the conserved bacterial Rcs stress response system mediates corneal pathogenesis associated withSerratia marcescensocular infections. This was accomplished by modifying Rcs activity using mutant strains. These include a mutant that has a hyper-active Rcs system due to deletion of the IgaA family gene,gumB, and agumB rcsCdouble mutant that is defective for Rcs signaling. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known. Here we observed that the Rcs-activatedgumBmutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h, and demonstrated a notable reduction in inflammation based on inflammatory signs and proinflammatory markers measured at the RNA and protein levels. ThegumBmutant phenotypes could be complemented by wild-typegumBon a plasmid and partially complemented by restoration ofshlAcytolysin expression and elimination of capsular polysaccharide production. We observed that inactivation of the Rcs stress response system completely restored corneal virulence to thegumBmutant. NanoString transcriptional analysis of bacterial genes expressed during microbial keratitis demonstrated expression ofgumB, rcsB, shlA, and three metalloprotease genes. Data suggest that the bacterial capsular polysaccharide is not necessary for infection, but capsule overexpression reduces inflammation. Together, these data indicate that GumB regulates virulence factor production through the Rcs system and this overall stress response system is a key mediator of a bacterium’s ability to induce vision-threatening keratitis.

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of L. monocytogenes from phagosomes and enables the bacteria to grow within the host. LLO is a versatile tool allowing Listeria to trigger several cellular responses. In this study, rapid phosphorylation of ERK1/2 on Caco-2 cells caused by Listeria infection was demonstrated to be highly dependent on LLO activity. The effect could be strongly induced by adding purified recombinant LLO alone and could be inhibited by exogenous cholesterol. Lack of the PEST sequence, known to tightly control cytotoxicity of LLO, did not affect ERK1/2 activation. However, the recombinant non-cytolytic LLOT515AL516A, with mutations in the cholesterol-binding motif, was unable to trigger this response. Recombinant LLON478AV479A, which lacks most of the cytolytic activity, also failed to activate ERK1/2 phosphorylation, and this effect could be rescued when the protein concentration reached a cytolytic level. Infection with an LLO-deficient mutant (Δhly) or the mutant complementing LLOT515AL516A abrogated the capacity of the bacteria to activate ERK1/2. However, infection with the Δhly mutant complementing LLON478AV479A, which retained partial pore-forming ability and could grow intracellularly, was capable of triggering ERK1/2 phosphorylation. Collectively, these data suggest that ERK1/2 activation by L. monocytogenes depends on the permeabilization activity of LLO and more importantly correlates with the cholesterol-binding motif of LLO.

BackgroundGroup B streptococci (GBS) are β-hemolytic, Gram-positive bacteria associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A key factor promoting GBS virulence is the β-hemolysin/cytolysin, a pigmented ornithine rhamnolipid (also known as granadaene) associated with the bacterial surface.MethodsA previous study indicated that GBS produce small structures known as membrane vesicles (MVs), which contain virulence-associated proteins. In this study, we show that GBS MVs are pigmented and hemolytic, indicating that granadaene is functionally active in MVs.ResultsIn addition, MVs from hyperhemolytic GBS induced greater cell death of neutrophils, T cells, and B cells compared with MVs from isogenic nonhemolytic GBS, implicating MVs as a potential mechanism for granadaene-mediated virulence. Finally, hemolytic MVs reduced oxidative killing of GBS and aggravated morbidity and mortality of neonatal mice infected with GBS.ConclusionsThese studies, taken together, reveal a novel mechanism by which GBS deploy a crucial virulence factor to promote bacterial dissemination and pathogenesis.

As a serious public health concern, alcohol-related liver disease is associated with dysregulations in the intestinal barrier function and the gut microbiota. The liver and gut communicate via the gut-liver axis, through which microbial products and metabolites translocate to the liver. Here, the current knowledge of various microbial products and metabolites which contribute to the alcohol-related liver diseases, including bile acids, indole-3-acetic acid, butyrate, long-chain fatty acids, endotoxin, cytolysin, β-glucan, and candidalysin is reviewed. Some of these might serve as therapeutic targets for alcohol-related liver disease.

Examination of genes encoding for the virulence factors, hemolysin/cytolysin (vvhA) and capsular polysaccharide (CPS allele 1), during biofilm formation revealed that their expression was influenced by the maturity of the biofilm as well as by temperature. At 24 °C, expression of vvhA during biofilm formation was low between 4 and 12 h but increased 10-fold by 24 h to (5.1 × 104 ± 6.3 × 103mRNA copies/ml) as the biofilm matured. Compared to planktonic cells, expression of vvhA during biofilm formation at 24 °C was initially up-regulated at 4 h (1.07 ± 0.00-fold) but then was down-regulated almost four-fold during the intermediate and mature stages of biofilm formation. In contrast, vvhA expression at 37 °C was up-regulated almost four-fold in the early stages (4 and 6 h) of biofilm formation and remained two-fold up-regulated by 24 h even as the biofilm was deteriorating. CPS allele 1 expression at 24 °C during biofilm formation was up-regulated (1.50 ± 0.18-fold) during the initial attachment phase of the cells but was strongly down-regulated during the intermediate phases at 8 and 10 h (74.42 ± 42.16-fold and 453.76 ± 193.32-fold, respectively), indicating that capsular polysaccharide (CPS) is not important to intermediate biofilm architecture. Interestingly, as the biofilm matured by 24 h, expression of CPS allele 1 was again up-regulated (1.88 ± 1.07), showing that CPS plays a role in mature biofilm. At 37 °C, CPS allele 1 expression was significantly up-regulated (up to 105) during biofilm formation, indicating that the biofilm form of V. vulnificus may be preferred over the planktonic form in the human host.

Group B Streptococcus (GBS) is a b-hemolytic, Gram-positive bacterium that commonly colonizes the female lower genital tract and is associated with fetal injury, preterm birth, spontaneous abortion, and neonatal infections. A major factor promoting GBS virulence is the b-hemolysin/cytolysin, which is cytotoxic to several host cells. We recently showed that the ornithine rhamnolipid pigment, Granadaene, produced by the gene products of the cyl operon, is hemolytic. Here, we demonstrate that heterologous expression of the GBS cyl operon conferred hemolysis, pigmentation, and cytoxicity to Lactococcus lactis, a model non-hemolytic Gram-positive bacterium. Similarly, pigment purified from L. lactis is hemolytic, cytolytic, and identical in structure to Granadaene extracted from GBS, indicating the cyl operon is sufficient for Granadaene production in a heterologous host. Using a systematic survey of phyletic patterns and contextual associations of the cyl genes, we identify homologs of the cyl operon in physiologically diverse Gram-positive bacteria and propose undescribed functions of cyl gene products. Together, these findings bring greater understanding to the biosynthesis and evolutionary foundations of a key GBS virulence factor and suggest that such potentially toxic lipids may be encoded by other bacteria., This work was supported by funding from the National Institutes of Health grants R01AI112619, R01AI33976, R01AI00989, and R21AI125907 to LR. The NIH training grant T32AI07509 (PI: Lee Ann Campbell) supported CW. LI and LA were supported by intramural funds from NLM, NIH.

BACKGROUND: Fucosyltransferase 2 (Fut2)-mediated intestinal α1-2-fucosylation is important in maintaining a symbiotic host-microbiota relationship and can protect against several pathogens. Intestinal dysbiosis is an important factor for the progression of experimental ethanol-induced liver disease, but the role of Fut2 in modulating the intestinal glycocalyx during alcohol-associated liver disease is unknown. We investigated the role of Fut2-mediated intestinal α1-2-fucosylation for the development of alcohol-associated liver disease. METHODS: Immunohistochemistry staining was applied to evaluate α1-2-fucosylation in duodenal biopsies from patients with alcohol use disorder. Wild type (WT) and Fut2 deficient littermate mice were subjected to Lieber DeCarli models of chronic ethanol administration and the chronic-binge ethanol diet (NIAAA model). RESULTS: Intestinal α1-2-fucosylation was down-regulated in patients with alcohol use disorder. Lack of α1-2-fucosylation in Fut2 deficient mice exacerbates chronic ethanol-induced liver injury, steatosis and inflammation without affecting ethanol metabolism. Dietary supplementation of the α1-2-fucosylated glycan 2’-fucosyllactose ameliorates ethanol-induced liver disease in Fut2 deficient mice in the NIAAA model. Despite no direct effects on growth of Enterococcus faecalis in vitro, intestinal α1-2-fucosylation reduces colonization of cytolysin-positive E. faecalis in the intestine of ethanol-fed mice. CONCLUSIONS: Intestinal α1-2-fucosylation acts as a host protective mechanism against ethanol-induced liver disease. 2’-FL is an oligosaccharide naturally present in human milk that could be considered as therapeutic agent for alcohol-associated liver disease.

Spreading of multidrug-resistant bacteria has become a growing and significant threat to environmental and public health. Our understanding of environmental variables such as nutrients which contribute to the dissemination of antibiotic resistance and pathogenesis is still limited. In this study, we operated outdoor mesocosm experiments to evaluate how nitrate and phosphate ions with different concentrations influence on an adaptation of Enterococcus faecalis to aquatic environments. E. faecalis were isolated from the mesocosms at 8 sampling events for 96 days to evaluate biofilm production, quorum-sensing-related genes expression, and sensitivity to oxytetracycline, erythromycin, ciprofloxacin, ampicillin, vancomycin, and chloramphenicol. Quantitative real-time PCR was used to compare mRNA levels of E. faecalis quorum-sensing-related genes. E. faecalis isolates exhibited resistance to oxytetracycline, ampicillin, and ciprofloxacin, respectively. We observed that the biofilm production of E. faecalis isolates on day 60 and 96 was significantly increased (p

Purpose. This study explored the prevalence and characteristics of Enterococcus faecalis biofilm formation by urinary tract infection (UTI) isolates in order to identify virulence factors associated with biofilm formation.Methodology. A total of 113 E. faecalis isolates were collected from UTI patients in Shenzhen, China. The isolates were subjected to multilocus sequence typing based on housekeeping genes. Biofilms were detected by crystal violet staining and the expression levels of the E. faecalis genes were detected by quantitative real-time PCR.Results/Key findings. The main sequence types (STs) were ST16 and ST179 with the ST16 isolates more likely to form strong biofilms than the ST179 isolates (P=0.008). Strong biofilm formation was more frequently detected in aggregation substance (agg)-positive (+) isolates than in negative (-) isolates (P=0.033). Biofilm formation was also more common in isolates containing enterococcal surface protein (esp), or cytolysin A (cylA)-positive (+) isolates than in isolates negative (-) for these virulence factors. Multivariate regression analysis indicated that cylA [odds ratio (OR), 7.143, P=0.012] was associated with weak biofilm formation, and that agg (OR, 4.471, P=0.004) was associated with strong biofilm formation. The expression of cylA was increased (8.75- to 23.05-fold) in weak biofilm, and the expression of agg was greatly elevated (11.99- to 439.10-fold) in strong biofilm isolates when compared to biofilm-negative isolates.Conclusion. ST16 classification was positively associated with strong biofilm formation in E. faecalis as was agg, while cylA was associated with weak biofilm formation.

Elizabethkingia meningoseptica is an emerging, healthcare-associated pathogen causing a high mortality rate in immunocompromised patients. We report the draft genome sequence of E. meningoseptica Em3, isolated from sputum from a patient with multiple underlying diseases. The genome has a length of 4,037,922 bp, a GC-content 36.4%, and 3673 predicted protein-coding sequences. Average nucleotide identity analysis (>95%) assigned the bacterium to the species E. meningoseptica. Genome analysis showed presence of the curli formation and assembly operon and a gene encoding hemagglutinins, indicating ability to form biofilm. In vitro biofilm assays demonstrated that E. meningoseptica Em3 formed more biofilm than E. anophelis Ag1 and E. miricola Emi3, both lacking the curli operon. A gene encoding thiol-activated cholesterol-dependent cytolysin in E. meningoseptica Em3 (potentially involved in lysing host immune cells) was also absent in E. anophelis Ag1 and E. miricola Emi3. Strain Em3 showed α-hemolysin activity on blood agar medium, congruent with presence of hemolysin and cytolysin genes. Furthermore, presence of heme uptake and utilization genes demonstrated adaptations for bloodstream infections. Strain Em3 contained 12 genes conferring resistance to β-lactams, including β-lactamases class A, class B, and metallo-β-lactamases. Results of comparative genomic analysis here provide insights into the evolution of E. meningoseptica Em3 as a pathogen. Electronic supplementary material The online version of this article (10.1186/s40793-017-0269-8) contains supplementary material, which is available to authorized users.

Purpose Postoperative endophthalmitis caused by Enterococcus faecalis often results in poor visual outcome due to retinal destruction. We evaluated the virulence factors of Enterococcus faecalis clinical isolates and analyzed their relationship with retinal destruction and poor visual outcomes in postoperative bacterial endophthalmitis (PBE) following cataract surgery. Retrospective. E. faecalis isolates from nine acute-onset PBE cases were examined using pulsed-field gel electrophoresis of genomic DNA, plasmid isolation and pheromone response experiments. Virulence determinant genes, including cytolysin (cylA), aggregation substance (asa1), enterococcal surface protein (esp), gelatinase (gelE) and adhesin of collagen from enterococci (ace), were examined by polymerase chain reaction. Expression of cytolysin and secreted protease were also tested. Retinal findings observed during vitrectomy surgery and final visual acuities were compared to obtained microbiological data. Poor visual outcome was defined as final visual acuity of less than 0.1 (decimal). Although nine isolates were collected, two were identical. Out of the eight different isolates, three were positive for both expressions of cytolysin and secreted proteases. Plasmid DNAs were isolated from six of eight isolates, while pheromone response was observed in five isolates. The asa1, esp and ace genes were positive in six, five and eight of the isolates, respectively. Patients with retinal destruction exhibited significantly higher prevalence of secreted protease expression versus those without (P

Enterokoklar insan ve hayvanlarda kommensal olarak bulunan ve özel koşullarda hastalığa neden olan bakterilerdir. Hastalığın oluşumunda birçok virülens faktörü rol oynamaktadır. Bu çalışmada balıklardan enterokok izolasyonu ve izole edilen enterokoklarda virülens faktörlerinin belirlenmesi amaçlanmıştır. Çalışma kapsamında Ege bölgesinde bulunan balık işletmelerinden toplanan balıklardan 26 (%13) adet Enterococcus faecalis izole edilmiştir. İzolatlarda sitolizin ve jelatinaz aktivitesi ile birlikte agregasyon substans üretimi incelenmiştir. İzolatların hiçbirisinde sitolizin aktivitesi tespit edilememiştir. İzolatların %27'sinde agregasyon substans üretimi ve %11,5'inde ise jelatinaz aktivitesi saptanmıştır. Sonuç olarak bu çalışmada; balık kökenli enterokoklarda önemli virülens faktörlerinin varlığı belirlenmiş ve bu suşların insan ve hayvanlarda hastalık oluşturma potansiyeli olduğu kanısına varılmıştır Enterococci are commensal organisms of human and animals, and may cause diseases in particular conditions. Several virulence factors are responsible in the production of diseases. The aim of this study was to isolate enterococci from fish and to determine virulence factors of the isolates. A total of 26 (13%) Enterococcus faecalis strains were isolated from live, moribund and dead fish collected from fish farms in Aegian Region. Cytolysin and gelatinase activities and aggregation substance production of these strains were examined.Cytolysin production was not detected in any of E. faecalis strains. Of 26 strains tested, 27% was found to produce aggregation substance. Gelatinase activity was found in 11.5% of strains. The presence of strains with important virulence factors in enterococci from fish was established. It was suggested that these strains have the potential of producing disease in human and animals

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylumFirmicutes. In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the speciesEnterococcus faeciumwith highly probable species identification andE. faecalisEEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesinE. faecium(efaAfm, in 22E. faeciumstrains and in oneE. faecalis). AdhesinefaAfsgene was detected inE. faecalisEEV20 and in twoE. faecium.GelEgene was present in three strains.E. faeciumEF/EC31 was absent of virulence factor genes.

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

We developed an engineered bacterium based, RNA interference mediated therapeutic method to significantly reduce the symptoms in the most frequently used animal model of inflammatory bowel disease. This transkingdom RNA interference strategy was based on the non-pathogenic E. coli MDS42 strain, which was engineered to constitutively produce invasin and histeriolysin cytolysin. These proteins enabled the bacteria first to invade the colon epithelium and then degrade in the phagosome. This allowed the delivery of a plasmid encoding shRNA targeting TNF alpha into the cytoplasm of the target cells. The expression levels of TNF alpha and other cytokines significantly decreased and the inflammatory symptoms were significantly reduced. With further safety modifications this method could serve as a safe and side-effect free alternative to biologicals targeting TNF-alpha.

Streptococcus suis is an emerging swine and human pathogen causing severe infections and sudden death. During infection, complement C3a and C5a were reported to induce immune cells towards infection and injury sites via their corresponding receptors C3aR and C5aR. However, how S. suis evade immune surveillance mediated by C3aR and C5aR remains unclear. In this study, we analyze and construct an S. suis bacterial two-hybrid prey library containing 39 LPXTG motif anchored proteins and 18 secreted proteins. Two highly possible C3aR-binding proteins: thiol-activated toxin Suilysin, putative RTX family exoprotein A gene and three highly possible C5aR-binding proteins: thiol-activated toxin Suilysin, putative 5'-nucleotidase and subtilisin-like serine protease are identified through bacterial two-hybrid assay. Far-western blot assay confirms that a cholesterol-binding cytolysin Suilysin can interact with both C3aR and C5aR. Chemotaxis assays demonstrate that recombinant and natural Suilysin can inhibit monocyte chemotaxis mediated by C3a and C5a. These findings enlarge our knowledge of suilysin biological significance and provide a new perspective on S. suis complement evasion.

Helicobacter pylori is a human-specific Gram-negative pathogenic bacterium which colonizes the gastric mucosal layer in the stomach causing diseases such as peptic ulcer, adenocarcinoma, and gastric lymphoma. It is estimated that approximately half of the world's population is infected with H. pylori making it the most intensively characterized microbial pathogen up to now. Hemolysis has been suggested to significantly contribute to colonization of the stomach and disease progression by H. pylori. In a number of earlier studies, TlyA was characterized as a putative pore-forming cytolysin. Although a few observations in the literature suggest a role for TlyA as significant virulence factor of H. pylori, the molecular and structural characterization of this protein is much curtailed at present. Given the intensive characterization of numerous H. pylori virulence factors over the past decade, surprisingly little information exists for the TlyA toxin and its significance for pathogenesis. This review provides a brief overview on microbial hemolysis and its role for pathogenesis and discusses recent research efforts aimed at an improved understanding of the role of the 'non-conventional' hemolysin and its associated RNA methyltransferase TlyA from H. pylori.

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 μg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 μM) and the β-adrenergic antagonist propranolol (10 μM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut-and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests. © 2015, National Research Council of Canada. All Rights Reserved.

Photobacterium damselae subsp. damselae , an important pathogen of marine animals, may also cause septicemia or hyperaggressive necrotizing fasciitis in humans. We previously showed that hemolysin genes are critical for virulence of this organism in mice and fish. In the present study, we characterized the hlyA gene product, a putative small β-pore-forming toxin, and termed it phobalysin P (PhlyP), for “photobacterial lysin encoded on a plasmid.” PhlyP formed stable oligomers and small membrane pores, causing efflux of K + , with no significant leakage of lactate dehydrogenase but entry of vital dyes. The latter feature distinguished PhlyP from the related Vibrio cholerae cytolysin. Attack by PhlyP provoked a loss of cellular ATP, attenuated translation, and caused profound morphological changes in epithelial cells. In coculture experiments with epithelial cells, Photobacterium damselae subsp. damselae led to rapid hemolysin-dependent membrane permeabilization. Unexpectedly, hemolysins also promoted the association of P. damselae subsp. damselae with epithelial cells. The collective observations of this study suggest that membrane-damaging toxins commonly enhance bacterial adherence.

After invading a host, bacterial pathogens secrete diverse protein toxins to disrupt host defense systems. To ensure successful infection, however, pathogens must precisely regulate the expression of those exotoxins because uncontrolled toxin production squanders energy. Furthermore, inappropriate toxin secretion can trigger host immune responses that are detrimental to the invading pathogens. Therefore, bacterial pathogens use diverse transcriptional regulators to accurately regulate multiple exotoxin genes based on spatiotemporal conditions. This review covers three major exotoxins in pathogenic Vibrio species and their transcriptional regulation systems. When Vibrio encounters a host, genes encoding cytolysin/hemolysin, multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin, and secreted phospholipases are coordinately regulated by the transcriptional regulator HlyU. At the same time, however, they are distinctly controlled by a variety of other transcriptional regulators. How this coordinated but distinct regulation of exotoxins makes Vibrio species successful pathogens? In addition, anti-virulence strategies that target the coordinating master regulator HlyU and related future research directions are discussed.

Streptococcus suis is a bacterial pathogen that mainly colonizes the upper respiratory tract of pigs. It is known to cause severe infections such as septicemia, meningitis, arthritis, and endocarditis in pigs and to be responsible for major economic losses in the swine industry worldwide. To better understand the interactions between S. suis and the porcine respiratory epithelium, we investigated the ability of this pathogen to cause damage to the tracheal epithelial barrier. We showed that S. suis compromises the integrity of a tracheal epithelial barrier model as determined by measuring transepithelial electrical resistance and paracellular flux of FITC-dextran. As a consequence of this breakdown, S. suis translocates across the epithelial cell monolayer. On the other hand, a S. suis mutant deficient in the production of suilysin, a cholesterol-dependent cytolysin, was significantly impaired in its ability to cause damage to the epithelial barrier. In addition, a recombinant suilysin disrupted the integrity of the tracheal epithelial barrier. Immunofluorescence staining suggested that suilysin affects two major tight junction proteins (occludin and zonula occludens-1). In summary, S. suis is able to compromise the function of the porcine respiratory epithelial barrier through the action of suilysin. This better knowledge of the interactions between S. suis and tracheal epithelial cells may help in the development of novel strategies to prevent the invasion of the epithelium by this and other swine respiratory pathogens.

Vibrio vulnificus is a food-borne marine pathogen that causes both life-threatening primary septicemia and necrotizing wound infections which accompany severe inflammation. Cytolysin is a very powerful virulence factor of V. vulnificus and is one of the likely candidates in the pathogenesis of V. vulnificus infections. However, the pathogenetic roles of cytolysin in V. vulnificus-induced inflammation are not well understood. In this study, we used the recombinant protein Vibrio vulnificus cytolysin (VVC) to demonstrate that VVC can induce inflammatory responses in RAW264.7 macrophages. Low dose (5 μg/ml) VVC had no impact on cell viability and induced pro-inflammatory cytokines production in RAW264.7 macrophages such as IL-6 and TNF-α. Moreover, VVC induced p65, p38, ERK1/2, and AKT phosphorylation in RAW264.7 macrophages. We further demonstrated that BAPTA-AM, a specific intracellular calcium chelator, inhibited VVC-induced inflammatory responses including pro-inflammatory cytokines production and inflammatory signaling activation in RAW264.7 macrophages. In addition, VVC primed rather than actived NLRP3 inflammasome in RAW264.7 macrophages. To determine whether VVC have a direct inflammatory effect on the host, we examined the effects of VVC injected into the skin of mice. VVC stimulated a significant induction of mRNAs for the pro-inflammatory cytokine IL-6 and inflammatory chemokines such as MCP-1 and IP-10. Histology data also showed that VVC caused inflammatory responses in the skin of mice. Collectively, our findings indicated that VVC induced inflammatory responses in RAW264.7 macrophages and in vivo and suggested the possibility of targeting VVC as a strategy for the clinical management of V. vulnificus-induced inflammatory responses.

Vibrio cholerae is an aquatic gram-negative microbe responsible for cholera, a pandemic disease causing life-threatening diarrheal outbreaks in populations with limited access to health care. Like most pathogenic bacteria, V. cholerae secretes virulence factors to assist colonization of human hosts, several of which bind carbohydrate receptors found on cell-surfaces. Understanding how pathogenic virulence proteins specifically target host cells is important for the development of treatment strategies to fight bacterial infections. Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin with a carboxy-terminal β-prism domain that targets complex N-glycans found on mammalian cell-surface proteins. To investigate glycan selectivity, we studied the VCC β-prism domain and two additional β-prism domains found within the V. cholerae biofilm matrix protein RbmC. We show that the two RbmC β-prism domains target a similar repertoire of complex N-glycan receptors as VCC and find through binding and modeling studies that a branched pentasaccharide core (GlcNAc2-Man3) represents the likely footprint interacting with these domains. To understand the structural basis of V. cholerae β-prism selectivity, we solved high-resolution crystal structures of fragments of the pentasaccharide core bound to one RbmC β-prism domain and conducted mutagenesis experiments on the VCC toxin. Our results highlight a common strategy for cell-targeting utilized by both toxin and biofilm matrix proteins in Vibrio cholerae and provide a structural framework for understanding the specificity for individual receptors. Our results suggest that a common strategy for disrupting carbohydrate interactions could affect multiple virulence factors produced by V. cholerae, as well as similar β-prism domains found in other vibrio pathogens., Author summary Bacterial pathogens secrete multiple virulence factors to aid in infection including adhesion molecules, effector proteins, enzymes, toxins and biofilm proteins. To increase the potency and specificity of these molecules, many factors contain binding sites for host cell-surface receptors. This study involves two such factors from the human pathogen Vibrio cholerae: a toxin that forms cytotoxic pores in the cell-membranes of target cells (most likely immune cells like neutrophils) and biofilm matrix proteins that help form a protective sheath around growing bacterial colonies. We show that both factors utilize similar carbohydrate receptors to recognize cell surfaces. Uncovering the structural basis for how host cells are targeted is important in understanding how V. cholerae and similar organisms cause disease.

Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E. faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E. faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections., Intraocular infections are a potentially blinding complication of common ocular surgeries and traumatic eye injuries. Bacterial toxins synthesized in the eye can damage intraocular tissue, often resulting in poor visual outcomes. Enteroccocus faecalis causes blinding infections and is responsible for 8 to 17% of postoperative endophthalmitis cases. These infections are increasingly difficult to treat due to the emergence of multidrug-resistant strains. Virulent E. faecalis isolates secrete a pore-forming bicomponent cytolysin that contributes to retinal tissue damage during endophthalmitis. We hypothesized that a biomimetic nanosponge, which mimics erythrocytes, might adsorb subunits of the cytolysin and reduce retinal damage, protecting vision. To test the efficacy of nanosponges in neutralizing the cytolysin in vitro, hemoglobin release assays were performed on culture supernatants from cytolysin-producing E. faecalis with and without preincubation with nanosponges. Treatment with nanosponges for 30 min reduced hemolytic activity by ~70%. To determine whether nanosponges could neutralize the cytolysin in vivo, electroretinography was performed on mice 24 h after intravitreal injection with cytolysin-containing supernatants treated with nanosponges. Pretreatment of cytolysin-containing supernatants with nanosponges increased the A-wave retention from 12.2% to 65.5% and increased the B-wave retention from 21.0% to 77.0%. Histology revealed that in nanosponge-treated eyes, retinas remained intact and attached, with little to no damage. Rabbit nanosponges were also nontoxic and noninflammatory when injected into mouse eyes. In an experimental murine model of E. faecalis endophthalmitis, injection of nanosponges into the vitreous 6 h after infection with a wild-type cytolysin-producing strain increased A-wave retention from 5.9% to 31% and increased B-wave retention from 12.6% to 27.8%. Together, these results demonstrated that biomimetic nanosponges neutralized cytolysin activity and protected the retinas from damage. These results suggest that this novel strategy might also protect eyes from the activities of pore-forming toxins of other virulent ocular bacterial pathogens. IMPORTANCE Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E. faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E. faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections.

Enterococcus faecalis biofilm traits and distribution characteristics in China have not been clarified. This study aimed to determine the prevalence and characteristics of E. faecalis biofilm formation in a sample of clinical isolates and to explore the virulence factors associated with biofilm formation in those isolates. A total of 265 E. faecalis isolates were collected from patients in Shenzhen, China. Virulence genes were detected within the genomes of the microbes by polymerase chain reaction. The isolates were subjected to multilocus sequence typing (MLST) based on housekeeping genes. Biofilms were detected by crystal violet staining. The expression levels of the clinical E. faecalis isolates’ genes were determined by quantitative real-time polymerase chain reaction. The prevalence of biofilm formation among E. faecalis clinical isolates was 47.2%. MLST yielded 44 different sequence types (STs). The main STs were ST16 and ST179; the ST16 isolates were more likely to form strong or medium biofilm than the ST179 isolates (p < 0.001). Strong or medium biofilm formation was more common in linezolid-resistant isolates than in linezolid-sensitive isolates (p = 0.001). Biofilm formation was more frequently detected in enterococcal surface protein (esp+), surface aggregating protein (asa1+), cytolysin A (cylA+), or aggregation substance (agg+) positive isolates than in isolates that were negative (-) for these virulence factors. Multivariate regression analysis indicated that cylA [odds ratio (OR) 4.083, p < 0.001] was a risk factor for weak biofilm formation, and that esp (OR 8.207, p < 0.001) was a risk factor for strong or medium biofilm formation. The expression of cylA was raised (4.02 to 6.00-fold) in weak biofilm isolates compared to the biofilm-negative isolates, and the expression of esp was greatly elevated (11.39 to 134.08-fold) in strong biofilm isolates compared to biofilm-negative isolates. In conclusion, the ST16 classification and linezolid resistance were positively associated with strong/medium biofilm formation in clinical E. faecalis isolates. cylA was associated with weak biofilm formation, and esp was only associated with strong or medium biofilm formation of the clinical E. faecalis isolates.

Serratia marcescens , a member of the carbapenem-resistant Enterobacteriaceae , is an important emerging pathogen that causes a wide variety of nosocomial infections, spreads rapidly within hospitals, and has a systemic mortality rate of ≤41%. Despite multiple clinical descriptions of S. marcescens nosocomial pneumonia, little is known regarding the mechanisms of bacterial pathogenesis and the host immune response. To address this gap, we developed an oropharyngeal aspiration model of lethal and sublethal S. marcescens pneumonia in BALB/c mice and extensively characterized the latter. Lethal challenge (>4.0 × 10 6 CFU) was characterized by fulminate hemorrhagic pneumonia with rapid loss of lung function and death. Mice challenged with a sublethal dose (6 CFU) rapidly lost weight, had diminished lung compliance, experienced lung hemorrhage, and responded to the infection with extensive neutrophil infiltration and histopathological changes in tissue architecture. Neutrophil extracellular trap formation and the expression of inflammatory cytokines occurred early after infection. Mice depleted of neutrophils were exquisitely susceptible to an otherwise nonlethal inoculum, thereby demonstrating the requirement for neutrophils in host protection. Mutation of the genes encoding the cytolysin ShlA and its transporter ShlB resulted in attenuated S. marcescens strains that failed to cause profound weight loss, extended illness, hemorrhage, and prolonged lung pathology in mice. This study describes a model of S. marcescens pneumonia that mimics known clinical features of human illness, identifies neutrophils and the toxin ShlA as a key factors important for defense and infection, respectively, and provides a solid foundation for future studies of novel therapeutics for this important opportunistic pathogen.

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faecium BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto-aggregation phenotypes. Among them, En. faecium BGGO9-28 showed the strongest auto-aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen-binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, agg, gelE, esp, hylN, ace, efaAfs , and efaAfm ) showed that BGGO9-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature.

Streptococcus intermedius is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when S. intermedius PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants nanA -null and msgA -null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM N -acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of S. intermedius toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of S. intermedius .