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Ziarenkowce z rodzaju Enterococcus spp. są składnikiem mikrobioty ludzi i zwierząt oraz powszechnie występują w środowisku naturalnym. Należą do patogenów warunkowo chorobotwórczych, mogących wywoływać zakażenia o różnej lokalizacji. Bakterie te rzadko wywołują zakażenia pozaszpitalne. Mimo, że do niedawna były uważane za drobnoustroje o niskim potencjale chorobotwórczym, stały się jednym z istotnych patogenów szpitalnych. Ich powszechne występowanie i zdolność do przeżycia w środowisku szpitalnym przyczyniają się do notowanej od kilku lat i nadal wzrastającej częstości ich izolacji, również z zakażeń inwazyjnych. Gatunki najczęściej izolowane z przypadków zakażeń to E. faecalis i E. faecium, stwarzające problemy terapeutyczne w związku z ich narastającą wielolekoopornością. Z powodu rosnącego znaczenia klinicznego, mechanizmów naturalnej i nabytej oporności na antybiotyki oraz potencjalnych czynników wirulencji, ziarenkowce z rodzaju Enterococcus stały się przedmiotem wielu badań. Celem pracy jest przedstawienie aktualnej wiedzy dotyczącej najistotniejszych czynników wirulencji mogących występować u bakterii z rodzaju Enterococcus, do których należą – antygen wydzielniczy SagA, białko EfaA, białko powierzchniowe Esp, białko wiążące kolagen Ace, cytolizyna, hialuronidaza, hemaglutynina, lipaza, proteaza serynowa, substancja agregująca, zewnątrzkomórkowe nadtlenki oraz żelatynaza.

Bacterial vaginosis (BV) is a dysbiotic condition of the vaginal microbiome associated with higher risk of infection by Neisseria gonorrhoeae— the cause of gonorrhea. Here we test if one known facet of BV—the presence of bacterial cytolysins—leads to mobilization of intracellular contents that enhance gonococcal virulence. We cloned and expressed recombinant vaginolysin (VLY), a cytolysin produced by the BV-associated bacterium Gardnerella , verifying that it liberates contents of cervical epithelial (HeLa) cells, while vector control preparations did not. We tested if VLY mediates a well-known gonococcal virulence mechanism—the molecular mimicry of host glycans. To evade host immunity, N. gonorrhoeae caps its lipooligosaccharide (LOS) with α2-3-linked sialic acid. For this, gonococci must scavenge a metabolite made inside host cells. Flow cytometry-based lectin-binding assays showed that gonococci exposed to vaginolysin-liberated contents of HeLa cells displayed greater sialic acid capping of their LOS. This higher level of bacterial sialylation was accompanied by increased binding of the complement regulatory protein factor H, and greater resistance to complement attack. Together these results suggest that cytolytic activities present during BV may enhance the ability of N. gonorrhoeae to capture intracellular metabolites and evade host immunity via glycan molecular mimicry. [ABSTRACT FROM AUTHOR]

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials. [ABSTRACT FROM AUTHOR]

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Sea anemones are well-spread everywhere in the World Ocean and represent the most ancient active poisonous organisms. Their main instrument of attack on other animals are the nematocysts – stinging organelles with the curtailed hollow thread with poisonous edge on the end. In order to attract their potential victims, they use fluorescent proteins. These proteins became a separate object of research as genetically coded markers for the observation of activity of promotors of genes. The poisonous secret of sea anemones is characterized by the presence of maximum number of peptides of various structural classes and spatial structures among the studied land and marine organisms (bees, spiders, scorpions, snakes ect.). This fact complicates the identification of sea anemones' secret and its differentiation from poisons of animals of other taxons, if the concrete source of its origin is unknown. The toxicity of some biologically active sea anemone peptides (RpI, RpIII) at intravenous administration to experimental animals is comparable with that of the most well-known and dangerous representatives of natural toxins with the similar mechanism of action (an alpha-hemolysine and tetrodotoxin), or chemical warfare agents, such as sarin and hydrogen cyanide. Based on their toxic effect, the biologically active sea anemone peptides generally can be classified as neurotoxins due to their impact on the functioning of sodium channels in the cells of the nervous system of animals. Cardiotoxic effect of sea anemone secret is caused by the specificity of interaction between its separate neurotoxins and one of the sub-types of sodium channels of muscle cells, characteristic for heart tissues. The main ways of identification of sea anemone neurotoxins in samples (for example, during the investigation of biological crimes) can be sequence by Edman`s method or tandem mass spectrometry (the analysis of fragments of toxin molecule for the establishment of its structure). Further study on the mechanisms of interaction between the sea anemone neurotoxins and the ion channels of the cells of nervous and muscular systems may result in the creation of medicines for treatment of channelopathy, as well as pluripotential antidotes, blocking the toxins, that influence on sodium channels.

SUMMARY: Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis‐and‐ethylene‐inducing peptide 1 (Nep1)‐like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant‐specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP‐producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome‐localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity‐related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over‐accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP‐producing microbial pathogens. Significance Statement: NLP‐producing phytopathogens are a big threat to agriculture worldwide. There is no report on R proteins/enzymes that counteract the toxicity of NLPs in plants. This study reports that cotton uses a peroxisome‐localized enzyme, lysophospholipase, to neutralize NLP toxin secreted by Verticillium dahliae, a soil‐borne fungal pathogen that causes vascular disease in a broad range host, thus enhancing plant resistance to wilt disease caused by this pathogen. [ABSTRACT FROM AUTHOR]

Intracellular pathogens are responsible for an enormous amount of worldwide morbidity and mortality, and each has evolved specialized strategies to establish and maintain their replicative niche. Listeria monocytogenes is a facultative intracellular pathogen that secretes a pore-forming cytolysin called listeriolysin O (LLO), which disrupts the phagosomal membrane and, thereby, allows the bacteria access to their replicative niche in the cytosol. Nonsynonymous and synonymous mutations in a PEST-like domain near the LLO N terminus cause enhanced LLO translation during intracellular growth, leading to host cell death and loss of virulence. Here, we explore the mechanism of translational control and show that there is extensive codon restriction within the PEST-encoding region of the LLO messenger RNA (mRNA) (hly). This region has considerable complementarity with the 5' UTR and is predicted to form an extensive secondary structure that overlaps the ribosome binding site. Analysis of both 5' UTR and synonymous mutations in the PEST-like domain that are predicted to disrupt the secondary structure resulted in up to a 10,000-fold drop in virulence during mouse infection, while compensatory double mutants restored virulence to WT levels. We showed by dynamic protein radiolabeling that LLO synthesis was growth phase-dependent. These data provide a mechanism to explain how the bacteria regulate translation of LLO to promote translation during starvation in a phagosome while repressing it during growth in the cytosol. These studies also provide a molecular explanation for codon bias at the 5' end of this essential determinant of pathogenesis.

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials.

Group B Streptococcus (GBS) emerged as a leading cause of invasive infectious disease in neonates in the 1970s, but has recently been identified as an escalating public health threat in non-pregnant adults, particularly those of advanced aged or underlying medical conditions. GBS infection can rapidly develop into life-threatening disease despite prompt administration of effective antibiotics and initiation of state-of-the-art intensive care protocols and technologies due to deleterious bacterial virulence factors, such as the GBS pore-forming toxin β-hemolysin/cytolysin (β-H/C). β-H/C is known to have noxious effects on a wide range of host cells and tissues, including lung epithelial cell injury, blood brain barrier weakening, and immune cell apoptosis. Neonatal and adult survivors of GBS infection are at a high risk for substantial long-term health issues and neurologic disabilities due to perturbations in organ systems caused by bacterial- and host- mediated inflammatory stressors. Previously engineered anti-virulence inhibitors, such as monoclonal antibodies and small molecular inhibitors, generally require customized design for each different pathogenic toxin and do not target deleterious host pro-inflammatory responses that may cause organ injury, septic shock, or death. By simply wrapping donor red blood cells (RBCs) around polymeric cores, we have created biomimetic "nanosponges." Because nanoparticles retain the same repertoire of cell membrane receptors as their host cell, they offer non-specific and all-purpose toxin decoy strategies with a broad ability to sequester and neutralize various bacterial toxins and host pro-inflammatory chemokines and cytokines to attenuate the course of infectious disease. This proof-of-concept study successfully demonstrated that intervention with nanosponges reduced the hemolytic activity of live GBS and stabilized β-H/C in a dose-dependent manner. Nanosponge treatment also decreased lung epithelial and macrophage cell death following exposure to live GBS bacteria and stabilized β-H/C, improved neutrophil killing of GBS, and diminished GBS-induced macrophage IL-1β production. Our results, therefore, suggest biomimetic nanosponges provide a titratable detoxification therapy that may provide a first-in-class treatment option for GBS infection by sequestering and inhibiting β-H/C activity.

Cholesterol-dependent cytolysin (CDC) is a bacterial toxin that binds to eukaryotic cholesterol-containing membranes, forms oligomeric complexes, and is inserted into the bilayer to create large aqueous pores. Recently, we reported a species-specific duplication of the hemolysin gene in group III Clostridium botulinum. The duplicated genes (bly1 and bly2) encoded two separate CDC proteins (botulinolysins; BLY1 and BLY2). Here, we aimed to investigate whether BLY1 and BLY2 exert differential cytotoxicity. We isolated two bly genes from C. botulinum and evaluated the cytotoxicity of two recombinant BLY proteins (rBLY1 and rBLY2) in HeLa, IEC-6, and NRK cells. rBLYs were cytotoxic to equine erythrocytes. rBLY1 showed higher hemolytic activity than rBLY2. rBLY2 showed no or very weak cytotoxicity to the HeLa, IEC-6, and NRK cells, whereas rBLY1 showed high cytotoxicity to these cells. The comparison of the amino acid sequence of BLYs with those of other CDCs revealed that the already-known amino acid residues involved in cholesterol-containing membrane recognition, oligomerization, and insertion into membranes are well conserved in both BLYs. However, several amino acid substitutions were observed in the conserved regions, particularly in L2 and L3 regions involved in cell binding. These findings suggest that gene duplication in group III C. botulinum evolved distinct functional specializations, and differential cytotoxicity of BLY1 and BLY2 could be due to the amino acid substitution in the conserved regions. However, the structural and functional comparisons of the two BLYs are essential to gain insights into the function of the CDCs. • Group III Clostridium botulinum strains possess two hemolysin genes, bly1 and bly2. • BLY1 exert stronger hemolytic activity than BLY2. • Cytotoxicity of BLY1 to HeLa, IEC-6, and NRK cells stronger than that of BLY2. • BLY1 and BLY2 revealed amino acid substitutions in the conserved L2 and L3 regions. [ABSTRACT FROM AUTHOR]

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenolsoluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter a- and one of the longer b-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMa of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus d-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of d-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host. [ABSTRACT FROM AUTHOR]

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenol-soluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter α- and one of the longer β-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMα of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus δ-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of δ-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host.

ABSTRACT Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT β-hemolysin/cytolysin (βhc). Little is understood about the cellular factors involved in βhc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to βhc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for βhc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of βhc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on βhc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that βhc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to βhc, a toxin required for GBS virulence. These data suggest that βhc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.

Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.

The β-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Introduction: Enterococci are one of the most important factors in nosocomial infections. Enterococcus genus has 38 species, the most important of which are Enterococcus faecalis and Enterococcus faecium. Enterococci have a medical significance due to bacteremia, urinary tract infections, endocarditis, meningitis and wound infections. Methods: 280 specimens collected from medical diagnostic laboratories in Tehran were used. In order to isolate and identify the bacteria, blood agar culture medium and the specific medium of Kanamycin esculin azide agar were used and then the colonies of bacteria were identified by biochemical methods. The molecular confirmation of bacteria was also carried out by a specific molecular kit of enterococci. Finally, the presence of the cytolysin gene in isolates was measured by PCR method. Results: Of 280 samples examined, 50 samples (17.8%) were identified for Enterococcus faecalis species. Presence of cytolysin gene (cyt) in 34% of isolates was confirmed by PCR. Conclusion: The results show that dissemination of enterococci containing pathogenic genes among patients with urinary tract infection can be dangerous for their health. These results show more attention to individual health.

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 (EtOH)‐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 EtOH administration and the chronic‐binge EtOH 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 EtOH‐induced liver injury, steatosis, and inflammation without affecting EtOH metabolism. Dietary supplementation of the α1‐2‐fucosylated glycan 2′‐fucosyllactose (2′‐FL) ameliorates EtOH‐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 EtOH‐fed mice. Conclusions: Intestinal α1‐2‐fucosylation acts as a host‐protective mechanism against EtOH‐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. [ABSTRACT FROM AUTHOR]

Background: The cellular process of autophagy is essential for maintaining the health of ocular tissue. Dysregulation of autophagy is associated with several ocular diseases including keratoconus and macular degeneration. It is known that autophagy can be used to respond to microbial infections and that certain microbes can exploit the autophagic process to their benefit. In this study, a genetic approach was used to identify surface-associated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy.Methods: A recombinant human corneal limbal epithelial cell line expressing a LC3-GFP fusion protein was challenged with normalized secretomes from wild-type and mutant S. marcescens derivatives. LC3-GFP fluorescence patterns were used to assess the ability of wild-type and mutant bacteria to influence autophagy. Purified prodigiosin was obtained from stationary phase bacteria and used to challenge ocular cells.Results: Mutations in the global regulators eepR and gumB genes highly reduced the ability of the bacteria to activate autophagy in corneal cells. This effect was further narrowed down to the secreted cytolysin ShlA and the biologically active pigment prodigiosin. Purified prodigiosin and ShlA from Escherichia coli further supported the role of these factors in activating autophagy in human corneal cells. Additional genetic data indicate a role for flagellin and type I pili, but not the nuclease, S-layer protein, or serratamolide biosurfactant in activation of autophagy.Conclusions: This work identifies specific bacterial components that activate autophagy and give insight into potential host-pathogen interactions or compounds that can be used to therapeutically manipulate autophagy. [ABSTRACT FROM AUTHOR]

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.

Group B Streptococcus (GBS) is the leading cause of severe bacterial infections in human newborn infants. Advances in streptococcal molecular genetics and refinement of in vitro and in vivo model systems of GBS disease have led to the discovery and characterization of several extracellular virulence factors elaborated by this pathogen. This review summarizes our current understanding of GBS extracellular virulence factors including the beta-hemolysin/cytolysin, C5a-peptidase, hyaluronate lyase, CAMP factor, oligopeptidase, and carbohydrate exotoxin CM101. The molecular basis and potential pathogenic role(s) of each factor are considered in the context of neonatal infection.

Background: Sea anemone is ocean dwelling and typically organisms sessile. These tentacles produce a variety of peptides and proteins that act as, neurotoxins and cytolysins toxins. Cytolysins due to their effect on specific tissue and lysing properties are known as antiparasitic or antitumor compounds. Materials and Methods: Sea anemone Stichodactyla haddoni were collected from the coast of Hormuz Island in November 2012 and tentacles extraction was performed by using of saline PBS solvents. Peptides above 10kDa and peptides below 10kDa were separated by amicon® Ultra-15 10K device. The hemolytic activity was assessed by the micro hemolytic method for human, rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio erythrocytes. Results: Uniform red color suspension in the wells considered as positive hemolysis. The crude extracts, peptides above 10kDa and peptides below 10kDa at various concentrations were showed hemolytic effect on human, common carp Cyprinus carpioand rainbow trout Oncorhynchus mykiss erythrocytes. Conclusion: The crude extract, peptides above 10kDa and peptides below 10kDa have a hemolytic effect on human erythrocytes and two species of fish. The venoms showed the most hemolytic activities on human blood among others.

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.

ABSTRACT 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.

Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

Gardnerella vaginalis produces cytolysin vaginolysin (VLY), which has been suggested to be a contributor to bacterial vaginosis pathogenesis. VLY along with intermedilysin (ILY) from Streptococcus intermedius have been attributed to a group of cholesterol-dependent cytolysins (CDCs) whose pore-forming activity depends on human CD59 (hCD59). Here, we show that different types of cells lacking hCD59 are susceptible to VLY-mediated lysis, albeit to different extents. We analyze the effects of both hCD59 and cholesterol on VLY cytolytic activity. We show that VLY binds to cholesterol-rich membranes of non-human cells, while VLY with an impaired cholesterol recognition site retains binding to the hCD59-containing cells. We further demonstrate that cholesterol binding by VLY is sufficient to trigger the formation of oligomeric complexes on cholesterol rich-liposomes lacking hCD59. Thus, VLY may induce cell lysis following two alternative pathways. One requires only cholesterol and does not depend on hCD59. The second pathway involves hCD59 contribution similarly to ILY. Apparently, under physiological conditions VLY acts in the most effective way by accepting the assistance of hCD59.

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.

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. Study design: Retrospective. Methods: 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). Results: 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 < 0.05), and retinal destruction was related to poor visual outcome ( P < 0.05). Conclusions: Our data suggest that enterococcal secreted proteases were correlated to retinal destruction, thereby fostering poor visual outcomes in eyes with enterococcal endophthalmitis. [ABSTRACT FROM AUTHOR]

Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata . HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions. [ABSTRACT FROM AUTHOR]

Quorum-sensing systems control major virulence determinants in Enterococcus faecalis, which causes nosocomial infections. The E. faecalis quorum-sensing systems include several virulence factors that are regulated by the cytolysin operon, which encodes the cytolysin toxin. In addition, the E. faecalis Fsr regulator system controls the expression of gelatinase, serine protease, and enterocin O16. The cytolysin and Fsr virulence factor systems are linked to enterococcal diseases that affect the health of humans and other host models. Therefore, there is substantial interest in understanding and targeting these regulatory pathways to develop novel therapies for enterococcal infection control. Quorum-sensing inhibitors could be potential therapeutic agents for attenuating the pathogenic effects of E. faecalis. Here, we discuss the regulation of cytolysin, the LuxS system, and the Fsr system, their role in E. faecalis-mediated infections, and possible therapeutic approaches to prevent E. faecalis infection. [ABSTRACT FROM AUTHOR]

Cholesterol-dependent cytolysins (CDCs) are proteinaceous toxins widely distributed in gram-positive pathogenic bacteria. CDCs can be classified into three groups (I–III) based on the mode of receptor recognition. Group I CDCs recognize cholesterol as their receptor. Group II CDC specifically recognizes human CD59 as the primary receptor on the cell membrane. Only intermedilysin from Streptococcus intermedius has been reported as a group II CDC. Group III CDCs recognize both human CD59 and cholesterol as receptors. CD59 contains five disulfide bridges in its tertiary structure. Therefore, we treated human erythrocytes with dithiothreitol (DTT) to inactivate CD59 on membranes. Our data showed that DTT treatment caused a complete loss of recognition of intermedilysin and an anti-human CD59 monoclonal antibody. In contrast, this treatment did not affect the recognition of group I CDCs, judging from the fact that DTT-treated erythrocytes were lysed with the same efficiency as mock-treated human erythrocytes. The recognition of group III CDCs toward DTT-treated erythrocytes was partially reduced, and these results are likely due to the loss of human CD59 recognition. Therefore, the degree of human CD59 and cholesterol requirements of uncharacterized group III CDCs frequently found in Mitis group streptococci can be easily estimated by comparing the amounts of hemolysis between DTT-treated and mock-treated erythrocytes. • Group III CDCs recognize hCD59 and cholesterol as receptors. • DTT treatment of human erythrocytes abolish the receptor function of hCD59. • DTT treatment did not alter cholesterol receptor function. • Group III CDCs only recognize cholesterol in DTT-treated human erythrocytes. • Receptor requirements of Group III CDCs can be determined using above properties. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed.

Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.