Pauline Yoong

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/β2 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.

Staphylococcus aureus Elaborates Leukocidin AB To Mediate Escape from within Human Neutrophils

ABSTRACT 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.

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.

Identification of a Crucial Residue Required for Staphylococcus aureus LukAB Cytotoxicity and Receptor Recognition

ABSTRACT The bicomponent leukotoxins produced by Staphylococcus aureus kill host immune cells through osmotic lysis by forming β-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.

Children with Invasive Staphylococcus aureus Disease Exhibit a Potently Neutralizing Antibody Response to the Cytotoxin LukAB

ABSTRACT 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.

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 molecules 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 organisms ability to cause disease.

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 others 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.

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.

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.

Animal models and imaging technologies: paving the way towards insights into Staphylococcus aureus-induced osteomyelitis

1515 Staphylococcus aureus osteomyelitis Staphylococcus aureus is a highly successful pathogen capable of causing infections in every human organ. Importantly, a large number of clinical strains are largely refractory to treatment with conventional antibiotics [1]. As such, S. aureus is a leading cause of severe illness and death, both in nosocomial and community settings. This bacterium is responsible for more cases of osteomyelitis (infection of the bone) than any other bacteria [2,3]. This article will highlight the obstacles to treating osteomyelitis and explore the technologies used in monitoring infections in the clinical and research settings. Research on S. aureus osteomyelitis will also be summarized, with a focus on recent developments.