Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes.

In order for Staphylococcus aureus to thrive inside the mammalian host, the bacterium has to overcome iron scarcity. S. aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, the most abundant iron source in mammals. Here we identify the Duffy antigen receptor for chemokines (DARC) as the receptor for the S. aureus hemolytic leukocidins LukED and HlgAB. By assessing human erythrocytes with DARC polymorphisms, we determined that HlgAB- and LukED-mediated lysis directly relates to DARC expression. DARC is required for S. aureus-mediated lysis of human erythrocytes, and DARC overexpression is sufficient to render cells susceptible to toxin-mediated lysis. HlgA and LukE bind directly to DARC through different regions, and by targeting DARC, HlgAB and LukED support S. aureus growth in a hemoglobin-acquisition-dependent manner. These findings elucidate how S. aureus targets and lyses erythrocytes to release one of the scarcest nutrients within the mammalian host.

Counter inhibition between leukotoxins attenuates Staphylococcus aureus virulence.

Staphylococcus aureus subverts host defences by producing a collection of virulence factors including bi-component pore-forming leukotoxins. Despite extensive sequence conservation, each leukotoxin has unique properties, including disparate cellular receptors and species specificities. How these toxins collectively influence S. aureus pathogenesis is unknown. Here we demonstrate that the leukotoxins LukSF-PV and LukED antagonize each other's cytolytic activities on leukocytes and erythrocytes by forming inactive hybrid complexes. Remarkably, LukSF-PV inhibition of LukED haemolytic activity on both human and murine erythrocytes prevents the release of nutrients required for in vitro bacterial growth. Using in vivo murine models of infection, we show that LukSF-PV negatively influences S. aureus virulence and colonization by inhibiting LukED. Thus, while S. aureus leukotoxins can certainly injure immune cells, the discovery of leukotoxin antagonism suggests that they may also play a role in reducing S. aureus virulence and maintaining infection without killing the host.

An rpsL-based allelic exchange vector for Staphylococcus aureus.

Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 °C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 °C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.

Single-copy vectors for integration at the SaPI1 attachment site for Staphylococcus aureus.

We have previously reported the construction of Staphylococcus aureus integration vectors based on the staphylococcal pathogenicity island 1 (SaPI1) site-specific recombination system. These are shuttle vectors that can be propagated in Escherichia coli, which allows for standard DNA manipulations. In S. aureus, these vectors are temperature-sensitive and can only be maintained at non-permissive (42 °C) temperatures by integrating into the chromosome. However, most S. aureus strains are sensitive to prolonged incubations at higher temperatures and will rapidly accumulate mutations, making the use of temperature-sensitive integration vectors impractical for single-copy applications. Here we describe improved versions of these vectors, which are maintained only in single-copy at the SaPI1 attachment site. In addition, we introduce several additional cassettes containing resistance markers, expanding the versatility of integrant selection, especially in strains that are resistant to multiple antibiotics.

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.

Staphylococcus aureus corneal infections: effect of the Panton-Valentine leukocidin (PVL) and antibody to PVL on virulence and pathology.

PURPOSE: Community-associated methicillin-resistant Staphylococcus aureus strains expressing Panton-Valentine leukocidin (PVL) are associated with severe skin and soft tissue infections, necrotizing pneumonia, and eye infections. We determined PVL's toxicity on infected mouse and cultured human corneal epithelial cells and the role of PVL and antibody to PVL in pathogenesis of murine keratitis. METHODS: Cytotoxicity on corneas and corneal epithelial cells was evaluated by LDH assays. Scratched corneas of female A/J mice were inoculated with approximately 107 CFU/eye of either WT S. aureus, isogenic ΔPVL, or strains overproducing PVL. Antibodies to PVL or control sera were topically applied to infected corneas 0, 24, and 32 hours postinfection, corneas scored for pathology and tissue levels of S. aureus were determined. RESULTS: PVL expression augmented the cytotoxicity of S. aureus on infected mouse corneas and human cultured corneal epithelial cells. Variable effects on leukocyte recruitment, pathogenesis, and immunity were obtained in the in vivo studies. Inactivation of PVL in USA300 strains caused reduced pathology and bacterial counts. Results were variable when comparing WT and ΔPVL USA400 strains, while USA400 strains overproducing PVL caused increased bacterial burdens. Topical treatment with polyclonal antibody to PVL yielded significant reductions in corneal pathology and bacterial CFU in corneas infected with USA300 strains, whereas effects were inconsistent in eyes infected with USA400 strains. CONCLUSIONS: PVL enhanced the virulence of a subset of MRSA strains in a keratitis model. Coupled with a variable effect of antibody treatment, it appears that PVL plays an inconsistent role in pathogenesis and immunity to S. aureus corneal infection.

Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.

Staphylococcus aureus causes diseases ranging from superficial wound infections to more invasive manifestations like osteomyelitis and endocarditis. The evasion of host phagocytes recruited to the site of infection is essential to the success of S. aureus as a pathogen. A single S. aureus strain can produce up to five different bicomponent pore-forming leukotoxins that lyse immune cells by forming pores in the cellular plasma membrane. Although these leukotoxins have been considered redundant due to their cytotoxic activity toward human neutrophils, each toxin displays varied species and cell-type specificities. This suggests that cellular factors may influence which cells each toxin targets. Here we describe the identification of CD11b, the α subunit of the αM/ß2 integrin (CD11b/CD18), macrophage-1 antigen, or complement receptor 3, as a cellular receptor for leukocidin A/B (LukAB), an important toxin that contributes to S. aureus killing of human neutrophils. We demonstrate that CD11b renders human neutrophils susceptible to LukAB-mediated killing by purified LukAB as well as during S. aureus infection ex vivo. LukAB directly interacts with human CD11b by binding to the I domain, a property that determines the species specificity exhibited by this toxin. Identification of a LukAB cellular target has broad implications for the use of animal models to study the role of LukAB in S. aureus pathogenesis, explains the toxin's tropism toward human neutrophils and other phagocytes, and provides a cellular therapeutic target to block the effect of LukAB toward human neutrophils.

The effects of Staphylococcus aureus leukotoxins on the host: cell lysis and beyond.

The success of Staphylococcus aureus as a leading cause of deadly hospital-acquired and community-acquired infections is attributed to its high-level resistance to most antibiotics, and the multitude of virulence factors it elaborates. Most clinical isolates produce up to four bi-component pore-forming toxins capable of lysing cells of the immune system. Subtle differences in activity and target range of each leukotoxin suggest that these toxins are not redundant, but instead may have specialized functions in attacking and/or evading host defenses. In turn, the host has developed countermeasures recognizing sublytic levels of leukotoxins as signals to activate protective immune defenses. The opposing cytotoxic and immune-activating effects of leukotoxins on host cells make for a complex dynamic between S. aureus and the host.

Staphylococcus aureus elaborates leukocidin AB to mediate escape from within human neutrophils.

Methicillin-resistant Staphylococcus aureus (MRSA) strains of the pulsed-field type USA300 are primarily responsible for the current community-associated epidemic of MRSA infections in the United States. The success of USA300 is partly attributed to the ability of the pathogen to avoid destruction by human neutrophils (polymorphonuclear leukocytes [PMNs]), which are crucial to the host immune response to S. aureus infection. In this work, we investigated the contribution of bicomponent pore-forming toxins to the ability of USA300 to withstand attack from primary human PMNs. We demonstrate that in vitro growth conditions influence the expression, production, and availability of leukotoxins by USA300, which in turn impact the cytotoxic potential of this clone toward PMNs. Interestingly, we also found that upon exposure to PMNs, USA300 preferentially activates the promoter of the lukAB operon, which encodes the recently identified leukocidin AB (LukAB). LukAB elaborated by extracellular S. aureus forms pores in the plasma membrane of PMNs, leading to PMN lysis, highlighting a contribution of LukAB to USA300 virulence. We now show that LukAB also facilitates the escape of bacteria engulfed within PMNs, in turn enabling the replication and outgrowth of S. aureus. Together, these results suggest that upon encountering PMNs S. aureus induces the production of LukAB, which serves as an extra- and intracellular weapon to protect the bacterium from destruction by human PMNs.

Poly-N-acetylglucosamine expression by wild-type Yersinia pestis is maximal at mammalian, not flea, temperatures.

UNLABELLED: Numerous bacteria, including Yersinia pestis, express the poly-N-acetylglucosamine (PNAG) surface carbohydrate, a major component of biofilms often associated with a specific appearance of colonies on Congo red agar. Biofilm formation and PNAG synthesis by Y. pestis have been reported to be maximal at 21 to 28°C or "flea temperatures," facilitating the regurgitation of Y. pestis into a mammalian host during feeding, but production is diminished at 37°C and thus presumed to be decreased during mammalian infection. Most studies of PNAG expression and biofilm formation by Y. pestis have used a low-virulence derivative of strain KIM, designated KIM6+, that lacks the pCD1 virulence plasmid, and an isogenic mutant without the pigmentation locus, which contains the hemin storage genes that encode PNAG biosynthetic proteins. Using confocal microscopy, fluorescence-activated cell sorter analysis and growth on Congo red agar, we confirmed prior findings regarding PNAG production with the KIM6+ strain. However, we found that fully virulent wild-type (WT) strains KIM and CO92 had maximal PNAG expression at 37°C, with lower PNAG production at 28°C both in broth medium and on Congo red agar plates. Notably, the typical dark colony morphology appearing on Congo red agar was maintained at 28°C, indicating that this phenotype is not associated with PNAG expression in WT Y. pestis. Extracts of WT sylvatic Y. pestis strains from the Russian Federation confirmed the maximal expression of PNAG at 37°C. PNAG production by WT Y. pestis is maximal at mammalian and not insect vector temperatures, suggesting that this factor may have a role during mammalian infection. IMPORTANCE: Yersinia pestis transitions from low-temperature residence and replication in insect vectors to higher-temperature replication in mammalian hosts. Prior findings based primarily on an avirulent derivative of WT (wild-type) KIM, named KIM6+, showed that biofilm formation associated with synthesis of poly-N-acetylglucosamine (PNAG) is maximal at 21 to 28°C and decreased at 37°C. Biofilm formation was purported to facilitate the transmission of Y. pestis from fleas to mammals while having little importance in mammalian infection. Here we found that for WT strains KIM and CO92, maximal PNAG production occurs at 37°C, indicating that temperature regulation of PNAG production in WT Y. pestis is not mimicked by strain KIM6+. Additionally, we found that Congo red binding does not always correlate with PNAG production, despite its widespread use as an indicator of biofilm production. Taken together, the findings show that a role for PNAG in WT Y. pestis infection should not be disregarded and warrants further study.

Immune-activating properties of Panton-Valentine leukocidin improve the outcome in a model of methicillin-resistant Staphylococcus aureus pneumonia.

The Panton-Valentine leukocidin (PVL) is a cytotoxin expressed by many methicillin-resistant Staphylococcus aureus (MRSA) strains that cause community-acquired infections (CA-MRSA). Its role in virulence however, is controversial, with clinical data suggesting that PVL-producing strains may cause less severe disease in humans. PVL is capable of lysing human white blood cells, but at sublytic amounts, PVL can activate protective host immunity in the absence of cell damage. The concentration-dependent reactions it elicits from host cells could be the reason for seemingly contradictory results about PVL's role in virulence. We hypothesized that a key to understanding PVL's action on host cells and, possibly, outcomes from infection is the amount of toxin present, a hypothesis previously supported in studies using a low-inoculum skin infection model, where low levels of PVL augmented innate immune resistance to infection. Here, we present additional data supporting this hypothesis using a mouse model of MRSA pneumonia, wherein we found increased virulence of isogenic Δpvl strains and further confirmed PVL's capacity to activate proinflammatory responses from mouse and human neutrophils and pulmonary cells. Activation was measured as the production of phosphorylated p38 mitogen-activated protein kinase (MAPK) and proinflammatory cytokines interleukin-8 (IL-8) and KC (from human and mouse cells, respectively), as well as the release of antibacterial factors. Conversely, PVL lowered the levels of tumor necrosis factor alpha (TNF-α) produced in active pulmonary infection, while low doses induced apoptosis, suggesting that PVL also has the capacity to regulate inflammation. Our data indicate that, independent of its cytotoxic effects, PVL also plays an important and positive immunomodulatory role during MRSA infections.

Characterization of SSR42, a novel virulence factor regulatory RNA that contributes to the pathogenesis of a Staphylococcus aureus USA300 representative.

Staphylococcus aureus is a major human pathogen that is capable of producing an expansive repertoire of cell surface-associated and extracellular virulence factors. Herein we describe an S. aureus regulatory RNA, SSR42, which modulates the expression of approximately 80 mRNA species, including several virulence factors, in S. aureus strains UAMS-1 and USA300 (LAC) during stationary-phase growth. Mutagenesis studies revealed that SSR42 codes for an 891-nucleotide RNA molecule and that the molecule's regulatory effects are mediated by the full-length transcript. Western blotting and functional assays indicated that the regulatory effects of SSR42 correlate with biologically significant changes in corresponding protein abundances. Further, in S. aureus strain LAC, SSR42 is required for wild-type levels of erythrocyte lysis, resistance to human polymorphonuclear leukocyte killing, and pathogenesis in a murine model of skin and soft tissue infection. Taken together, our results indicate that SSR42 is a novel S. aureus regulatory RNA molecule that contributes to the organism's ability to cause disease.

Enhancement of bacterial virulence by antibody neutralization of immune-activating toxins.

Bacterial pathogens release a number of toxins that are able to form pores in target host cells, which can result in their destruction. Due to this property of this subgroup of toxins, they are considered virulence factors. A lesser known ability of these toxins when present at lower concentrations that are insufficient for host cell lysis to occur, is their activation of host immune cells. The Panton-Valentine Leukocidin (PVL) secreted by an alarming percentage of Staphylococcus aureus causing community-acquired infections, is one such: toxin. Due to the low inoculum of S. aureus we used to establish skin abscesses in a murine model of infection, lower amounts of PVL are likely to be present early in infection, hence, the proinflammatory properties of PVL may be more evident. Our data not only suggested that this was indeed the case, but we also showed that antibodies directed to PVL neutralized immune activation by this toxin resulting in a less robust host innate immune response. Thus, given the high levels of antibodies to PVL present in healthy individuals, these antibodies may directly enhance the virulence of PVL-producing S. aureus by dampening the innate immune response to infection. Since many pore-forming toxins share this dual property of concentration-dependent host cell lysis and immune activation, it is interesting to speculate that antibodies raised to some bacterial toxins may have the opposite intended outcome of directly enhancing bacterial virulence instead of controlling infection.

High levels of antibody to panton-valentine leukocidin are not associated with resistance to Staphylococcus aureus-associated skin and soft-tissue infection.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) expressing Panton-Valentine leukocidin (PVL) causes severe skin and soft-tissue infection (SSTI), necrotizing pneumonia, and other invasive infections. The PVL toxin has been implicated as a virulence factor, and antibody to a component of this toxin is under investigation as a vaccine candidate. The role of PVL in pathogenesis remains controversial, and it is unknown whether human serum antibody to PVL modulates infection. METHODS: We determined antibody levels to PVL in serum samples from children aged 0-18 years presenting with polymerase chain reaction-confirmed, PVL-positive MRSA-associated SSTI (with or without prior MRSA infection or SSTI), PVL-positive MRSA invasive infection, and PVL-negative MRSA infection, as well as uninfected control subjects. We also measured antibody-mediated neutralization of PVL-induced lysis of human polymorphonuclear cells. RESULTS: Antibody to PVL was present in healthy children reaching adult levels by 4-6 years, with a nadir at 3-11 months likely due to loss of maternal antibody. Children with a primary PVL-positive MRSA infection had moderate levels of antibody to PVL that increased after infection. Children with prior MRSA infection or SSTI had high levels of antibody to PVL after the onset of PVL-positive MRSA infection. There was no increase in antibody to PVL in this population's serum samples after the onset of infection. Serum samples from children with PVL-positive MRSA-associated SSTIs, particularly those with prior MRSA infection or SSTI, and convalescent-phase serum samples from children with invasive PVL-positive MRSA infection potently inhibited PVL-induced lysis of polymorphonuclear cells. CONCLUSIONS: Neutralizing antibody to PVL does not protect children against primary or recurrent CA-MRSA-associated SSTI.

Antibody-mediated enhancement of community-acquired methicillin-resistant Staphylococcus aureus infection.

Community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA) expressing the Panton-Valentine leukocidin (PVL) are rampant, but the contribution of PVL to bacterial virulence remains controversial. While PVL is usually viewed as a cytotoxin, at sublytic amounts it activates protective innate immune responses. A leukotoxic effect might predominate in high inoculum studies, whereas protective proinflammatory properties might predominate in settings with lower bacterial inocula that more closely mimic what initially occurs in humans. However, these protective effects might possibly be neutralized by antibodies to PVL, which are found in normal human sera and at increased levels following PVL(+) S. aureus infections. In a low-inoculum murine skin abscess model including a foreign body at the infection site, strains deleted for the pvl genes replicated more efficiently within abscesses than isogenic PVL(+) strains. Coinfection of mice at separate sites with isogenic PVL(+) and PVL(-) MRSA abrogated the differences in bacterial burdens, indicating a systemic effect on host innate immunity from production of PVL. Mice given antibody to PVL and then infected with seven different PVL(+) strains also had significantly higher bacterial counts in abscesses compared with mice given nonimmune serum. Antibody to PVL had no effect on MRSA strains that did not produce PVL. In vitro, antibody to PVL incapacitated PVL-mediated activation of PMNs, indicating that virulence of PVL(+) MRSA is enhanced by the interference of PVL-activated innate immune responses. Given the high rates of primary and recurring MRSA infections in humans, it appears that antibodies to PVL might contribute to host susceptibility to infection.

PlyPH, a bacteriolytic enzyme with a broad pH range of activity and lytic action against Bacillus anthracis.

We have cloned a lytic enzyme, PlyPH, with a specific lytic effect on Bacillus anthracis strains. PlyPH remains active between pH 4 and 10.5, and a single dose rescued a significant percentage of mice infected intraperitoneally with an attenuated B. anthracis strain. We propose PlyPH as a novel therapeutic agent.

Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium.

Enterococcus faecalis and Enterococcus faecium infections are increasingly difficult to treat due to high levels of resistance to antibiotics. PlyV12, a bacteriophage lytic enzyme, was isolated and shown to effectively kill both E. faecalis and E. faecium (including vancomycin-resistant strains), as well as other human pathogens. We propose its development and use as an alternative therapeutic tool.