Genetic variation of staphylococcal LukAB toxin determines receptor tropism.

Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.

All major cholesterol-dependent cytolysins use glycans as cellular receptors.

Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P1 antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.

The Staphylococcus aureus superantigen SElX is a bifunctional toxin that inhibits neutrophil function.

Bacterial superantigens (SAgs) cause Vß-dependent T-cell proliferation leading to immune dysregulation associated with the pathogenesis of life-threatening infections such as toxic shock syndrome, and necrotizing pneumonia. Previously, we demonstrated that staphylococcal enterotoxin-like toxin X (SElX) from Staphylococcus aureus is a classical superantigen that exhibits T-cell activation in a Vß-specific manner, and contributes to the pathogenesis of necrotizing pneumonia. Here, we discovered that SElX can also bind to neutrophils from human and other mammalian species and disrupt IgG-mediated phagocytosis. Site-directed mutagenesis of the conserved sialic acid-binding motif of SElX abolished neutrophil binding and phagocytic killing, and revealed multiple glycosylated neutrophil receptors for SElX binding. Furthermore, the neutrophil binding-deficient mutant of SElX retained its capacity for T-cell activation demonstrating that SElX exhibits mechanistically independent activities on distinct cell populations associated with acquired and innate immunity, respectively. Finally, we demonstrated that the neutrophil-binding activity rather than superantigenicity is responsible for the SElX-dependent virulence observed in a necrotizing pneumonia rabbit model of infection. Taken together, we report the first example of a SAg, that can manipulate both the innate and adaptive arms of the human immune system during S. aureus pathogenesis.

Monoclonal Antibodies Against the Staphylococcus aureus Bicomponent Leukotoxin AB Isolated Following Invasive Human Infection Reveal Diverse Binding and Modes of Action.

The 2-component leukotoxin LukAB is critical for Staphylococcus aureus targeting and killing of human neutrophils ex vivo and is produced in the setting of human infection. We report 3 LukAB-specific human monoclonal antibodies (mAbs) with distinct mechanisms of toxin neutralization and in vivo efficacy. Three hybridomas secreting mAbs with anti-LukAB activity (designated SA-13, -15, and -17) were generated from B cells obtained from a 12-year-old boy with S. aureus osteomyelitis. Each of the 3 mAbs neutralized LukAB-mediated neutrophil toxicity, exhibited differing levels of potency, recognized different antigenic sites on the toxin, and displayed at least 2 distinct mechanisms for cytotoxic inhibition. SA-15 bound exclusively to the dimeric form of the toxin, suggesting that human B cells recognize epitopes on the dimerized form of LukAB during natural infection. Both SA-13 and SA-17 bound the LukA monomer and the LukAB dimer. Although all 3 mAbs potently neutralized cytotoxicity, only SA-15 and SA-17 significantly inhibited toxin association with the cell surface. Treatment with a 1:1 mixture of mAbs SA-15 and SA-17 resulted in significantly lower bacterial colony counts in heart, liver, and kidneys in a murine model of S. aureus sepsis. These data describe the isolation of diverse and efficacious antitoxin mAbs.

Staphylococcus aureus Biofilms Induce Macrophage Dysfunction Through Leukocidin AB and Alpha-Toxin.

UNLABELLED: The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. IMPORTANCE: Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans.

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.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

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.

Biochemical activities of Streptococcus pneumoniae serotype 2 capsular glycosyltransferases and significance of suppressor mutations affecting the initiating glycosyltransferase Cps2E.

The capsular polysaccharide (CPS) is essential for Streptococcus pneumoniae virulence. Its synthesis requires multiple enzymes, and defects that block completion of the pathway can be lethal in the absence of secondary suppressor mutations. In this study, we examined the functions of three capsular glycosyltransferases (Cps2F, Cps2G, and Cps2I) involved in serotype 2 CPS synthesis, whose deletions select for secondary mutations. We demonstrate that Cps2F is a rhamnosyltransferase that catalyzes addition of the third and fourth sugars in the capsule repeat unit, while Cps2G adds the fifth sugar (glucose). Addition of the terminal residue (glucuronic acid) could not be detected; however, activities of the other glycosyltransferases together with bioinformatic analyses suggest that this step is mediated by Cps2I. Most of the secondary suppressor mutations resulting from loss of these enzymes occur in cps2E, the gene encoding the initiating glycosyltransferase. Examination of the 69 S. pneumoniae serotypes containing Cps2E homologues yielded a consensus amino acid sequence for this protein and demonstrated that there is a highly significant association between the residues that are 100% conserved and those altered by suppressor mutations. Cps2E contains an extracytoplasmic loop whose function is unknown. Among our collection of mutants, six contained missense mutations affecting amino acids in the extracytoplasmic loop. These residues are highly conserved among S. pneumoniae Cps2E homologues, and mutations therein severely reduced CPS synthesis and Cps2E activity. The critical functions of these amino acids suggest a role for the Cps2E extracytoplasmic loop in initiation, and possibly regulation, of capsule synthesis.

Genetic and biochemical characterizations of enzymes involved in Streptococcus pneumoniae serotype 2 capsule synthesis demonstrate that Cps2T (WchF) catalyzes the committed step by addition of ß1-4 rhamnose, the second sugar residue in the repeat unit.

Five genes (cps2E, cps2T, cps2F, cps2G, and cps2I) are predicted to encode the glycosyltransferases responsible for synthesis of the Streptococcus pneumoniae serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant S. pneumoniae enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In in vitro assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose. cps2T, cps2F, and cps2I deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2G mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2I mutants contained different secondary suppressor mutations in cps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2T mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the ß1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.