Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Quorum-sensing agr system of Staphylococcus aureus primes gene expression for protection from lethal oxidative stress.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H2O2, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr increased both respiration and fermentation but decreased ATP levels and growth, suggesting that Δagr cells assume a hyperactive metabolic state in response to reduced metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H2O2 doses. Increased survival of wild-type agr cells during H2O2 exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H2O2. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived "memory" of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Nox2-/-) mice. These results demonstrate the importance of protection that anticipates impending ROS-mediated immune attack. The ubiquity of quorum sensing suggests that it protects many bacterial species from oxidative damage.

Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections.

Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Prospective Randomized, Double-Blind, Placebo-Controlled Study of a Standardized Oral Pomegranate Extract on the Gut Microbiome and Short-Chain Fatty Acids.

Punica granatum L., commonly known as the pomegranate, is an abundant source of polyphenols, including hydrolyzable ellagitannins, ellagic acid, anthocyanins, and other bioactive phytochemicals shown to be effective in defending against oxidative stress, and has immunomodulatory activities. Ellagitannins, and their hydrolyzed product ellagic acid, interact with the gut microbiota to yield secondary metabolites known as urolithins that may have health benefits. The objective of this study was to determine the effects of supplementation with a standardized punicalagin-enriched pomegranate extract, Pomella® (250 mg), on the gut microbiome, circulating short-chain fatty acids, and gut microbial-derived ellagitannin metabolite urolithins. A randomized, double-blind, placebo-controlled study was conducted over 4 weeks on healthy volunteers aged 25-55 years. Subjects were randomly assigned to receive either an oral supplement containing 75 mg of punicalagin or an oral placebo. Stool sample collection and venipuncture were performed to analyze the gut microbiome, SCFAs, and urolithin. There was no significant change in the gut microbial diversity in both cohorts after 4 weeks of intervention, but there was a significantly increased relative abundance of Coprococcus eutectus, Roseburia faecis, Roseburia inullnivorans, Ruminococcus bicirculans, Ruminococcus calidus, and Faecalibacterium prausnitzii. Pomegranate extract (PE) supplementation led to the augmentation of circulating propionate levels (p = 0.02) and an increasing trend for acetate levels (p = 0.12). The pomegranate extract (PE) supplementation group had an increased level of circulating urolithins compared to the placebo group (6.6% vs. 1.1%, p = 0.13). PE supplementation correlated with shifts in the gut microbiome and with higher circulating levels of propionate and acetate. Further studies should explore the implications in larger cohorts and over a longer duration.

Prospective Randomized Double-Blind Placebo-Controlled Study of Oral Pomegranate Extract on Skin Wrinkles, Biophysical Features, and the Gut-Skin Axis.

(1) Background: The pomegranate fruit (Punica granatum L.) has been widely used in traditional medicine and has increasingly gained popularity among consumers in order to manage different facets of health. The objective of this study was to evaluate the effects of the fruit extract of P. granatum L. on different parameters of skin health. (2) Methods: A prospective, double-blind placebo-controlled study was conducted on both healthy males and females aged 25−55 years. Subjects were supplemented with a standardized punicalagin enriched oral pomegranate extract [Pomella® (Verdure Science, Noblesville, IN, USA), PE group] or a placebo (control group) daily for four weeks. Changes in wrinkle severity, facial biophysical properties, skin microbiome, and the gut microbiome were assessed. (3) Results: The PE group had significant reductions in wrinkle severity (p < 0.01) and a decreasing trend in the forehead sebum excretion rate (p = 0.14). The participants in the PE group with a higher relative abundance of Eggerthellaceae in the gut had a decrease in their facial TEWL (p < 0.05) and wrinkle severity (p = 0.058). PE supplementation led to an increase in the Staphylococcus epidermidis species and the Bacillus genus on the skin. (4) Conclusions: Overall, the study demonstrated improvements in several biophysical properties, wrinkles, and shifts in the skin microbiome with oral PE supplementation in healthy subjects.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Vaccination With Detoxified Leukocidin AB Reduces Bacterial Load in a Staphylococcus aureus Minipig Deep Surgical Wound Infection Model.

Vaccines against Staphylococcus aureus have eluded researchers for >3 decades while the burden of staphylococcal diseases has increased. Early vaccine attempts mainly used rodents to characterize preclinical efficacy, and all subsequently failed in human clinical efficacy trials. More recently, leukocidin AB (LukAB) has gained interest as a vaccine antigen. We developed a minipig deep surgical wound infection model offering 3 independent efficacy readouts: bacterial load at the superficial and at the deep-seated surgical site, and dissemination of bacteria. Due to similarities with humans, minipigs are an attractive option to study novel vaccine candidates. With this model, we characterized the efficacy of a LukAB toxoid as vaccine candidate. Compared to control animals, a 3-log reduction of bacteria at the deep-seated surgical site was observed in LukAB-treated minipigs and dissemination of bacteria was dramatically reduced. Therefore, LukAB toxoids may be a useful addition to S. aureus vaccines and warrant further study.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Exome sequencing in schizophrenia-affected parent-offspring trios reveals risk conferred by protein-coding de novo mutations.

Protein-coding de novo mutations (DNMs) are significant risk factors in many neurodevelopmental disorders, whereas schizophrenia (SCZ) risk associated with DNMs has thus far been shown to be modest. We analyzed DNMs from 1,695 SCZ-affected trios and 1,077 published SCZ-affected trios to better understand the contribution to SCZ risk. Among 2,772 SCZ probands, exome-wide DNM burden remained modest. Gene set analyses revealed that SCZ DNMs were significantly concentrated in genes that were highly expressed in the brain, that were under strong evolutionary constraint and/or overlapped with genes identified in other neurodevelopmental disorders. No single gene surpassed exome-wide significance; however, 16 genes were recurrently hit by protein-truncating DNMs, corresponding to a 3.15-fold higher rate than the mutation model expectation (permuted 95% confidence interval: 1-10 genes; permuted P = 3 × 10-5). Overall, DNMs explain a small fraction of SCZ risk, and larger samples are needed to identify individual risk genes, as coding variation across many genes confers risk for SCZ in the population.

Contributions of common genetic variants to risk of schizophrenia among individuals of African and Latino ancestry.

Schizophrenia is a common, chronic and debilitating neuropsychiatric syndrome affecting tens of millions of individuals worldwide. While rare genetic variants play a role in the etiology of schizophrenia, most of the currently explained liability is within common variation, suggesting that variation predating the human diaspora out of Africa harbors a large fraction of the common variant attributable heritability. However, common variant association studies in schizophrenia have concentrated mainly on cohorts of European descent. We describe genome-wide association studies of 6152 cases and 3918 controls of admixed African ancestry, and of 1234 cases and 3090 controls of Latino ancestry, representing the largest such study in these populations to date. Combining results from the samples with African ancestry with summary statistics from the Psychiatric Genomics Consortium (PGC) study of schizophrenia yielded seven newly genome-wide significant loci, and we identified an additional eight loci by incorporating the results from samples with Latino ancestry. Leveraging population differences in patterns of linkage disequilibrium, we achieve improved fine-mapping resolution at 22 previously reported and 4 newly significant loci. Polygenic risk score profiling revealed improved prediction based on trans-ancestry meta-analysis results for admixed African (Nagelkerke's R2 = 0.032; liability R2 = 0.017; P < 10-52), Latino (Nagelkerke's R2 = 0.089; liability R2 = 0.021; P < 10-58), and European individuals (Nagelkerke's R2 = 0.089; liability R2 = 0.037; P < 10-113), further highlighting the advantages of incorporating data from diverse human populations.

Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa.

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

Identification of common genetic risk variants for autism spectrum disorder.

Autism spectrum disorder (ASD) is a highly heritable and heterogeneous group of neurodevelopmental phenotypes diagnosed in more than 1% of children. Common genetic variants contribute substantially to ASD susceptibility, but to date no individual variants have been robustly associated with ASD. With a marked sample-size increase from a unique Danish population resource, we report a genome-wide association meta-analysis of 18,381 individuals with ASD and 27,969 controls that identified five genome-wide-significant loci. Leveraging GWAS results from three phenotypes with significantly overlapping genetic architectures (schizophrenia, major depression, and educational attainment), we identified seven additional loci shared with other traits at equally strict significance levels. Dissecting the polygenic architecture, we found both quantitative and qualitative polygenic heterogeneity across ASD subtypes. These results highlight biological insights, particularly relating to neuronal function and corticogenesis, and establish that GWAS performed at scale will be much more productive in the near term in ASD.

Discovery of the first genome-wide significant risk loci for attention deficit/hyperactivity disorder.

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable childhood behavioral disorder affecting 5% of children and 2.5% of adults. Common genetic variants contribute substantially to ADHD susceptibility, but no variants have been robustly associated with ADHD. We report a genome-wide association meta-analysis of 20,183 individuals diagnosed with ADHD and 35,191 controls that identifies variants surpassing genome-wide significance in 12 independent loci, finding important new information about the underlying biology of ADHD. Associations are enriched in evolutionarily constrained genomic regions and loss-of-function intolerant genes and around brain-expressed regulatory marks. Analyses of three replication studies: a cohort of individuals diagnosed with ADHD, a self-reported ADHD sample and a meta-analysis of quantitative measures of ADHD symptoms in the population, support these findings while highlighting study-specific differences on genetic overlap with educational attainment. Strong concordance with GWAS of quantitative population measures of ADHD symptoms supports that clinical diagnosis of ADHD is an extreme expression of continuous heritable traits.

Stenotrophomonas maltophilia Serine Protease StmPr1 Induces Matrilysis, Anoikis, and Protease-Activated Receptor 2 Activation in Human Lung Epithelial Cells.

Stenotrophomonas maltophilia is an emerging, opportunistic nosocomial pathogen that can cause severe disease in immunocompromised individuals. We recently identified the StmPr1 and StmPr2 serine proteases to be the substrates of the Xps type II secretion system in S. maltophilia strain K279a. Here, we report that a third serine protease, StmPr3, is also secreted in an Xps-dependent manner. By constructing a panel of protease mutants in strain K279a, we were able to determine that StmPr3 contributes to the previously described Xps-mediated rounding and detachment of cells of the A549 human lung epithelial cell line as well as the Xps-mediated degradation of fibronectin, fibrinogen, and the cytokine interleukin-8 (IL-8). We also determined that StmPr1, StmPr2, and StmPr3 account for all Xps-mediated effects toward A549 cells and that StmPr1 contributes the most to Xps-mediated activities. Thus, we purified StmPr1 from the S. maltophilia strain K279a culture supernatant and evaluated the protease's activity toward A549 cells. Our analyses revealed that purified StmPr1 behaves more similarly to subtilisin than to trypsin. We also determined that purified StmPr1 likely induces cell rounding and detachment of A549 cells by targeting cell integrin-extracellular matrix connections (matrilysis) as well as adherence and tight junction proteins for degradation. In this study, we also identified anoikis as the mechanism by which StmPr1 induces the death of A549 cells and found that StmPr1 induces A549 IL-8 secretion via activation of protease-activated receptor 2. Altogether, these results suggest that the degradative and cytotoxic activities exhibited by StmPr1 may contribute to S. maltophilia pathogenesis in the lung by inducing tissue damage and inflammation.

Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported that S. maltophilia strain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps- and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of the S. maltophilia Xps type II secretion system and its StmPr1 and StmPr2 substrates.

Staphylococcus aureus Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular, but Not Intracellular.

Staphylococcus aureus infections are a growing health burden worldwide, and paramount to this bacterium's pathogenesis is the production of virulence factors, including pore-forming leukotoxins. Leukocidin A/B (LukAB) is a recently discovered toxin that kills primary human phagocytes, though the underlying mechanism of cell death is not understood. We demonstrate here that LukAB is a major contributor to the death of human monocytes. Using a variety of in vitro and ex vivo intoxication and infection models, we found that LukAB activates Caspase 1, promotes IL-1ß secretion and induces necrosis in human monocytes. Using THP1 cells as a model for human monocytes, we found that the inflammasome components NLRP3 and ASC are required for LukAB-mediated IL-1ß secretion and necrotic cell death. S. aureus was shown to kill human monocytes in a LukAB dependent manner under both extracellular and intracellular ex vivo infection models. Although LukAB-mediated killing of THP1 monocytes from extracellular S. aureus requires ASC, NLRP3 and the LukAB-receptor CD11b, LukAB-mediated killing from phagocytosed S. aureus is independent of ASC or NLRP3, but dependent on CD11b. Altogether, this study provides insight into the nature of LukAB-mediated killing of human monocytes. The discovery that S. aureus LukAB provokes differential host responses in a manner dependent on the cellular contact site is critical for the development of anti-infective/anti-inflammatory therapies that target the NLRP3 inflammasome.

Children with invasive Staphylococcus aureus disease exhibit a potently neutralizing antibody response to the cytotoxin LukAB.

Despite the importance of Staphylococcus aureus as a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity of S. aureus toward human neutrophils. Children with culture-proven S. aureus infection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers against S. aureus exotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently described S. aureus bicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicity in vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies to S. aureus antigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is produced in vivo and that it elicits a functional humoral response.

Identification of a crucial residue required for Staphylococcus aureus LukAB cytotoxicity and receptor recognition.

The bicomponent leukotoxins produced by Staphylococcus aureus kill host immune cells through osmotic lysis by forming ß-barrel pores in the host plasma membrane. The current model for bicomponent pore formation proposes that octameric pores, comprised of two separate secreted polypeptides (S and F subunits), are assembled from water-soluble monomers in the extracellular milieu and multimerize on target cell membranes. However, it has yet to be determined if all staphylococcal bicomponent leukotoxin family members exhibit these properties. In this study, we report that leukocidin A/B (LukAB), the most divergent member of the leukotoxin family, exists as a heterodimer in solution rather than two separate monomeric subunits. Notably, this property was found to be associated with enhanced toxin activity. LukAB also differs from the other bicomponent leukotoxins in that the S subunit (LukA) contains 33- and 10-amino-acid extensions at the N and C termini, respectively. Truncation mutagenesis revealed that deletion of the N terminus resulted in a modest increase in LukAB cytotoxicity, whereas the deletion of the C terminus rendered the toxin inactive. Within the C terminus of LukA, we identified a glutamic acid at position 323 that is critical for LukAB cytotoxicity. Furthermore, we discovered that this residue is conserved and required for the interaction between LukAB and its cellular target CD11b. Altogether, these findings provide an in-depth analysis of how LukAB targets neutrophils and identify novel targets suitable for the rational design of anti-LukAB inhibitors.

Cell targeting by the Staphylococcus aureus pore-forming toxins: it's not just about lipids.

Staphylococcus aureus employs numerous pore-forming cytotoxins to injure host immune cells and promote infection. Until recently, it was unclear how these cytotoxins targeted specific cell types for lysis. Membrane lipids were initially postulated to be cytotoxin receptor candidates. However, the cell-type specificity and species-dependent targeting of these toxins did not support lipids as sole receptors. The recent identification of proteinaceous receptors for several S. aureus cytotoxins now provides an explanation for the observed tropism. These findings also have important implications for the implementation of animal models to study S. aureus pathogenesis, and for the development of novel therapeutics.