Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Role of Major Toxin Virulence Factors in Pertussis Infection and Disease Pathogenesis.

Bordetella pertussis produces several toxins that affect host-pathogen interactions. Of these, the major toxins that contribute to pertussis infection and disease are pertussis toxin, adenylate cyclase toxin-hemolysin and tracheal cytotoxin. Pertussis toxin is a multi-subunit protein toxin that inhibits host G protein-coupled receptor signaling, causing a wide array of effects on the host. Adenylate cyclase toxin-hemolysin is a single polypeptide, containing an adenylate cyclase enzymatic domain coupled to a hemolysin domain, that primarily targets phagocytic cells to inhibit their antibacterial activities. Tracheal cytotoxin is a fragment of peptidoglycan released by B. pertussis that elicits damaging inflammatory responses in host cells. This chapter describes these three virulence factors of B. pertussis, summarizing background information and focusing on the role of each toxin in infection and disease pathogenesis, as well as their role in pertussis vaccination.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions.

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.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Host cell perforation by listeriolysin O (LLO) activates a Ca2+-dependent cPKC/Rac1/Arp2/3 signaling pathway that promotes Listeria monocytogenes internalization independently of membrane resealing.

Host cell invasion is an indispensable step for a successful infection by intracellular pathogens. Recent studies identified pathogen-induced host cell plasma membrane perforation as a novel mechanism used by diverse pathogens (Trypanosoma cruzi, Listeria monocytogenes, and adenovirus) to promote their internalization into target cells. It was concluded that T. cruzi and adenovirus damage the host cell plasma membrane to hijack the endocytic-dependent membrane resealing machinery, thereby invading the host cell. We studied L. monocytogenes and its secreted pore-forming toxin listeriolysin O (LLO) to identify key signaling events activated upon plasma membrane perforation that lead to bacterial internalization. Using various approaches, including fluorescence resonance energy transfer imaging, we found that the influx of extracellular Ca2+ subsequent to LLO-mediated plasma membrane perforation is required for the activation of a conventional protein kinase C (cPKC). cPKC is positioned upstream of Rac1 and the Arp2/3 complex, which activation leads to F-actin--dependent bacterial internalization. Inhibition of this pathway did not prevent membrane resealing, revealing that perforation-dependent L. monocytogenes endocytosis is distinct from the resealing machinery. These studies identified the LLO-dependent endocytic pathway of L. monocytogenes and support a novel model for pathogen uptake promoted by plasma membrane injury that is independent of membrane resealing.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina / Diversidad y distribución de genes de toxinas insecticidas Lepidoptera-específicos en cepas de Bacillus thuringiensis de Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cryl, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Se analizaron 268 cepas de Bacillus thuringiensis obtenidas de diferentes fuentes de Argentina con el objeto de determinar la diversidad y distribución de genes cryl, cry2, cry8, cry9 y vip3A, que codifican proteínas insecticidas lepidóptero-específicas. Se excluyeron las cepas gemelas. Se detectaron solo diez perfiles diferentes entre los 80 B. thuringiensis seleccionados. Dos de estos perfiles, el cry1Aa, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (35/80) y el cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (25/80), comprendieron el 75% de las cepas seleccionadas. La existencia de esta baja diversidad es una rareza, ya que en la mayor parte de las colecciones estudiadas se ha descrito una gran diversidad de perfiles de genes de toxinas insecticidas. El perfil detectado con mayor frecuencia se obtuvo principalmente de cepas procedentes de suelo (el 70% de los de esa fuente lo tenían), también fue mayoritario entre los procedentes de polvo de producto almacenado (59%) y en los que procedían de telas de arana (50%). En cambio, el perfil cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa se detectó principalmente en las cepas aisladas de hojas (40%) y de larvas de insectos muertos (50%). Seis de los perfiles identificados fueron encontrados en cepas aisladas de polvo de producto almacenado y de hojas, lo que indica una mayor diversidad de perfiles en estas fuentes que en otras. Se identificaron algunas cepas con alta actividad insecticida contra larvas de Epinotia aporema (Lepidoptera), hallazgo importante para explorar en el futuro estrategias microbianas para el control de esta plaga en la región.

Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol-dependent cytolysins.

Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum-Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore-forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore-forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol-dependent cytolysins.

Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and ß-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence.

Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial ß-hemolysin/cytolysin (ß-H/C) because an ß-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1ß, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of ß-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and ß-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder.

Nitric oxide participates in the toxicity of Bacillus thuringiensis Cry1Ab toxin to kill Manduca sexta larvae.

Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae.

The staphylococcal toxins γ-haemolysin AB and CB differentially target phagocytes by employing specific chemokine receptors.

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-haemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-haemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2(+) cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.

Bacterial delivery of Staphylococcus aureus α-hemolysin causes regression and necrosis in murine tumors.

Bacterial therapies, designed to manufacture therapeutic proteins directly within tumors, could eliminate cancers that are resistant to other therapies. To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells. To date, these properties have not been shown for a single protein. The gene for Staphylococcus aureus α-hemolysin (SAH), a pore-forming protein, was cloned into Escherichia coli. These bacteria were injected into tumor-bearing mice and volume was measured over time. The location of SAH relative to necrosis and bacterial colonies was determined by immunohistochemistry. In culture, SAH was released and killed 93% of cancer cells in 24 hours. Injection of SAH-producing bacteria reduced viable tissue to 9% of the original tumor volume. By inducing cell death, SAH moved the boundary of necrosis toward the tumor edge. SAH diffused 6.8 ± 0.3 µm into tissue, which increased the volume of affected tissue from 48.6 to 3,120 µm(3). A mathematical model of molecular transport predicted that SAH efficacy is primarily dependent on colony size and the rate of protein production. As a payload protein, SAH will enable effective bacterial therapy because of its ability to diffuse in tissue, kill cells, and expand tumor necrosis.

Factores de virulencia de Staphylococcus aureus asociados con infecciones mamarias en bovinos: relevancia y rol como agentes inmunógenos / [Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: relevance and role as immunogens].

Staphylococcus aureus is the most prevalent mastitis pathogen in Argentina and worldwide. Lack of effectiveness of traditional control measures based on milking hygiene and antibiotic therapy against this organism has led to the development of alternatives directed to prevent the disease. Among them, the manipulation of host immune mechanisms through vaccination has been explored. The identification of virulence factors able to stimulate host immune defenses is key to developing a rational vaccine. S. aureus has multiple virulence factors that interact with the host at different stages of an intramammary infection. The use of some of these factors as immunogens has been shown to elicit protective responses in the host. The structure, function, and use as immunogens of S. aureus virulence factors considered to be relevant at different stages of intrammamary infections caused by this organism are reviewed in this article.

Mecanismo de acción de la toxina alfa hemolisina de Escherichia coli / Action mechanism of Escherichia coli alpha-hemolysin toxin / Mecanismo de ação da toxina alfa-hemolisina de Escherichia coli

Escherichia coli es una de las bacterias anaerobias facultativas más predominantes en el intestino, siendo, en la mayoría de los casos, inocua para el huésped. Existen cepas que traslocan al torrente sanguíneo causando enfermedades extraintestinales como infecciones urinarias, septicemia y meningitis. Dentro de éstas se encuentran las cepas uropatogénicas (Uropathogenic Escherichia coli: UPEC), que secretan varios factores de virulencia. Estos últimos incluyen: toxinas, sistemas de adquisición de hierro, adhesinas y antígenos capsulares. Las principales toxinas secretadas son: alfa-hemolisina (HlyA) y el factor necrotizante citotóxico 1 (CNF-1). En esta revisión se presenta una descripción exhaustiva de HlyA, incluyendo su síntesis, maduración y exportación desde la bacteria. La acilación de la proteína en dos residuos internos de lisina la convierte en una toxina muy virulenta al exponer regiones intrínsecamente desordenadas que son esenciales en diferentes pasos del mecanismo de acción de la misma. Específicamente, la exposición de estas regiones está involucrada en interacciones proteína-proteína dentro del proceso de oligomerización. La formación del oligómero es responsable de la permeabilidad inducida en las células blanco. Finalmente, basado en los conocimientos acerca de las características estructurales y funcionales de HlyA, se presentan potenciales usos de HlyA en terapias basadas en toxinas.

Escherichia coli is one of the predominant species of facultative anaerobes in the human gut, and in the majority of the cases it is harmless to the host. Some strains of this species can translocate to blood and cause infection such as urinary infection, septicemia and meningitis. These are the uropathogenic E. coli strains (UPEC) that secrete a number of virulence factors. The latter include a number of secreted toxins, iron-acquisition systems, adhesins, and capsular antigens. Secreted toxins include HlyA, the cytotoxic necrotizing factor-1 (CNF-1). In this review an exhaustive description of the toxin has been delineated, including its synthesis, maturation, and export from the bacteria. The acylation of the protein at two internal lysine residues gives the toxin its virulence, by exposing intrinsic disordered regions that are essential in different steps of the toxin's mechanism of action. The further exposure of regions involved in the protein-protein interaction within the oligomerization process is responsG-ible for the permeability induced in all the target cells. Based on the already known structural and functional characteristics of HlyA, the potential use in toxin-based therapy is presented.

Escherichia coli é uma das bactérias anaérobias facultativas mais predominantes no intestino, sendo na maioria dos casos inócua para o hóspede. Há cepas que passam ao torrente sanguíneo causando doenças extraintestinais como infecção urinária, septicemia e meningite. Dentro destas se encontram as cepas uropatogênicas (Uropathogenic Escherichia coli: UPEC) que secretam varios fatores de virulência. Estos últimos incluem: toxinas, sistemas de aquisição de ferro, adesinas e antígenos capsulares. As principais toxinas secretadas são: alfa hemolisina (HlyA) e o fator necrotizante citotóxico 1 (CNF-1). Nesta revisão apresenta-se uma descrição exaustiva de HlyA incluindo sua sintese, seu amadurecimento e exportação a partir da bactéria. A acilação da proteína em dois residuos internos de lisina a transforma numa toxina muito virulenta ao expor regiões intrinsecamente desordenadas que são essenciais em diferentes passos do mecanismo de ação da mesma. Especificamente, a exposição destas regiões esta envolvida em interações proteína-proteína dentro do processo de oligomerização. A formação do oligômero é responsável pela permeabilidade induzida nas células alvo. Finalmente, com base nos conhecimentos acerca das características estruturais e funcionais de HlyA, apresentam-se potenciais usos de HlyA em terapias baseadas em toxinas.

Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis.

Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel beta-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

Listeria monocytogenes-infected human peripheral blood mononuclear cells produce IL-1beta, depending on listeriolysin O and NLRP3.

Different NOD-like receptors, including NLRP1, NLRP3, and NLRC4, as well as the recently identified HIN-200 protein, AIM2, form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of pro-IL-1beta. Listeria monocytogenes is an intracellular pathogen that is actively phagocytosed by monocytes/macrophages and subsequently escapes from the phagosome into the host cell cytosol, depending on its pore-forming toxin listeriolysin O (LLO). In this study, we demonstrate that human PBMCs produced mature IL-1beta when infected with wild-type L. monocytogenes or when treated with purified LLO. L. monocytogenes mutants lacking LLO or expressing a noncytolytic LLO as well as the avirulent Listeria innocua induced strongly impaired IL-1beta production. RNA interference and inhibitor experiments in human PBMCs as well as experiments in Nlrp3 and Rip2 knockout bone marrow-derived macrophages demonstrated that the Listeria-induced IL-1beta release was dependent on ASC, caspase-1, and NLRP3, whereas NOD2, Rip2, NLRP1, NLRP6, NLRP12, NLRC4, and AIM2 appeared to be dispensable. We found that L. monocytogenes-induced IL-1beta production was largely dependent on phagosomal acidification and cathepsin B release, whereas purified LLO activated an IL-1beta production independently of these mechanisms. Our results indicate that L. monocytogenes-infected human PBMCs produced IL-1beta, largely depending on an LLO-mediated phagosomal rupture and cathepsin B release, which is sensed by Nlrp3. In addition, an LLO-dependent but cathepsin B-independent NLRP3 activation might contribute to some extent to the IL-1beta production in L. monocytogenes-infected cells.

A critical role for hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome.

The mechanism by which bacterial pathogens activate caspase-1 via Nlrp3 remains poorly understood. In this study, we show that the ability of Staphylococcus aureus, a leading cause of infection in humans, to activate caspase-1 and induce IL-1beta secretion resides in culture supernatants of growing bacteria. Caspase-1 activation induced by S. aureus required alpha-, beta-, and gamma-hemolysins and the host Nlrp3 inflammasome. Mechanistically, alpha- and beta-hemolysins alone did not trigger caspase-1 activation, but they did so in the presence of bacterial lipoproteins released by S. aureus. Notably, caspase-1 activation induced by S. aureus supernatant was independent of the P2X7 receptor and the essential TLR adaptors MyD88 and TIR domain-containing adapter-inducing IFN-beta, but was inhibited by extracellular K(+). These results indicate that S. aureus hemolysins circumvent the requirement of ATP and the P2X7 receptor to induce caspase-1 activation via Nlrp3. Furthermore, these studies revealed that hemolysins promote in the presence of lipoproteins the activation of the Nlrp3 inflammasome.

Staphylococcal alpha-toxin is not sufficient to mediate escape from phagolysosomes in upper-airway epithelial cells.

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.

Relevance of fatty acid covalently bound to Escherichia coli alpha-hemolysin and membrane microdomains in the oligomerization process.

alpha-Hemolysin (HlyA) is an exotoxin secreted by some pathogenic strains of Escherichia coli that causes lysis of several mammalian cells, including erythrocytes of different species. HlyA is synthesized as a protoxin, pro-HlyA, which is activated by acylation at two internal lysines Lys-563 and Lys-689. It has been proposed that pore formation is the mechanism of cytolytic activity for this toxin, as shown in experiments with whole cells, planar lipid membranes, and liposomes, but these experiments have yielded conflicting results about the structure of the pore. In this study, HlyA cysteine replacement mutant proteins of amino acids have been labeled with Alexa-488 and Alexa-546. Fluorescence resonance energy transfer measurements, employing labeled toxin bound to sheep ghost erythrocytes, have demonstrated that HlyA oligomerizes on erythrocyte membranes. As the cytotoxic activity is absolutely dependent on acylation, we have studied the role of acylation in the oligomerization, demonstrating that fatty acids are essential in this process. On the other hand, fluorescence resonance energy transfer and the hemolytic activity decrease when the erythrocyte ghosts are cholesterol-depleted, hence indicating the role of membrane microdomains in the clustering of HlyA. Simultaneously, HlyA was found in detergent-resistant membranes. Pro-HlyA has also been found in detergent-resistant membranes, thus demonstrating that the importance of acyl chains in toxin oligomerization is the promotion of protein-protein interaction. These results change the concept of the main role assigned to acyl chain in the targeting of proteins to membrane microdomains.

Real-time observation of listeria monocytogenes-phagocyte interactions in living zebrafish larvae.

The zebrafish, Danio rerio, has become a popular vertebrate model for the study of infections, mainly because of its excellent optical accessibility at the embryonic and larval stages, when the innate immune system is already effective. We have thus tested the susceptibility of zebrafish larvae to the human pathogen Listeria monocytogenes, a gram-positive, facultative, intracellular bacterium that is known to survive and multiply in professional phagocytes and that causes fatal meningitis and abortions. Intravenous injection of early zebrafish larvae resulted in a progressive and ultimately fatal infection. Blood-borne L. monocytogenes bacteria were quickly trapped and engulfed by macrophages, an event that, for the first time, could be captured in vivo and in real time. Granulocytes also participated in the innate immune response. As in mammals, bacteria could escape the macrophage phagosome in a listeriolysin-dependent manner and accessed the cytosol; this event was critical for bacterial virulence, as listeriolysin-deficient bacteria were completely avirulent. Actin comet tails and protrusions were observed, suggesting cell-to-cell spread; these phenomena also played a role in virulence in zebrafish larvae, as actA-deficient bacteria were attenuated. These results demonstrate the relevance of the genetically tractable and optically accessible zebrafish model for the study of L. monocytogenes pathogenesis and particularly for the dissection of its interactions with phagocytes in vivo, a key factor of L. monocytogenes virulence.

NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation.

Staphylococcus aureus is a major cause of community-acquired and nosocomial infections including the life-threatening conditions endocarditis, necrotizing pneumonia, necrotizing fasciitis, and septicemia. Toll-like receptor (TLR)-2, a membrane-bound microbial sensor, detects staphylococcal components, but macrophages lacking TLR2 or both TLR2 and TLR4 remain S. aureus responsive, suggesting that an alternative microbial recognition receptor might be involved. The cytoplasmic sensor nucleotide-binding oligomerization domain containing (NOD) 2/caspase recruitment domain (CARD) 15 detects muramyl dipeptide from bacterial peptidoglycans and mediates cytokine responses to S. aureus in vitro, but the physiological significance of these observations is not well defined. Here we show that NOD2-deficient mice exhibit a delayed but ultimately exacerbated ulcerative response and impaired bacterial clearance after s.c. infection with S. aureus. NOD2-dependent recognition of S. aureus and muramyl dipeptide is facilitated by alpha-toxin (alpha-hemolysin), a pore-forming toxin and virulence factor of the pathogen. The action of NOD2 is dependent on IL-1beta-amplified production of IL-6, which promotes rapid bacterial killing by neutrophils. These results significantly broaden the physiological importance of NOD2 in innate immunity from the recognition of bacteria that primarily enter the cytoplasm to the detection of bacteria that typically reside extracellularly and demonstrate that this microbial sensor contributes to the discrimination between commensal bacteria and bacterial pathogens that elaborate pore-forming toxins.