Faculty Bibliography

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 alpha subunit of the alphaM/beta2 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.

Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection. Pathogen-induced bone destruction limits antimicrobial penetration to the infectious focus and compromises treatment of osteomyelitis. To investigate mechanisms of S. aureus-induced bone destruction, we developed a murine model of osteomyelitis. Microcomputed tomography of infected femurs revealed that S. aureus triggers profound alterations in bone turnover. The bacterial regulatory locus sae was found to be critical for osteomyelitis pathogenesis, as Sae-regulated factors promote pathologic bone remodeling and intraosseous bacterial survival. Exoproteome analyses revealed the Sae-regulated protease aureolysin as a major determinant of the S. aureus secretome and identified the phenol-soluble modulins as aureolysin-degraded, osteolytic peptides that trigger osteoblast cell death and bone destruction. These studies establish a murine model for pathogen-induced bone remodeling, define Sae as critical for osteomyelitis pathogenesis, and identify protease-dependent exoproteome remodeling as a major determinant of the staphylococcal virulence repertoire.

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 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 epidemic strain USA300 is a highly successful pathogen. However, the underlying basis of this success is not clear. Now, Thurlow and colleagues (2013) provide evidence linking the bacterial arginine catabolic mobile element (ACME) to the dominance of USA300 as a pathogen of the skin.

Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic towards neutrophils, but also specifically target other immune cells. Despite decades since the first description of staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins towards different immune cells remain unknown. Here we identify the human immunodeficiency virus (HIV) co-receptor CCR5 as a cellular determinant required for cytotoxic targeting of subsets of myeloid cells and T lymphocytes by the S. aureus leukotoxin ED (LukED). We further demonstrate that LukED-dependent cell killing is blocked by CCR5 receptor antagonists, including the HIV drug maraviroc. Remarkably, CCR5-deficient mice are largely resistant to lethal S. aureus infection, highlighting the importance of CCR5 targeting in S. aureus pathogenesis. Thus, depletion of CCR5(+) leukocytes by LukED suggests a new immune evasion mechanism of S. aureus that can be therapeutically targeted.

This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Deltahla) in human blood was comparable to the parental wild-type strain and polymorphonuclear leukocyte (PMN) plasma membrane permeability caused by USA300 did not require Hla. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) following infection by USA300, USA300Deltahla, and USA300Deltahla transformed with a plasmid over-expressing Hla (USA300Deltahla Comp) demonstrated this toxin plays a significant role inducing plasma membrane permeability of CD14(+), CD3(+), and CD19(+) PBMCs. Rapid plasma membrane permeability independent of Hla was observed for PMNs, CD14(+) and CD19(+) PBMCs following intoxication with USA300 supernatant while the majority of CD3(+) PBMC plasma membrane permeability induced by USA300 required Hla. Addition of recombinant Hla to USA300Deltahla supernatant rescued CD3(+) and CD19(+) PBMC plasma membrane permeability generated by USA300 supernatant. An observed delay in plasma membrane permeability caused by Hla in conjunction with Annexin V binding and ApoBrdU Tunel assays examining PBMCs intoxicated with recombinant Hla or infected with USA300, USA300Deltahla, USA300Deltahla Comp, and USA300DeltasaeR/S suggest Hla induces programmed cell death of monocytes, B cells, and T cells that results in plasma membrane permeability. Together these findings underscore the importance of Hla during S. aureus infection of human tissue and specifically demonstrate Hla activity during USA300 infection triggers programmed cell death of human monocytes, T cells and B cells that leads to plasma membrane permeability.

We investigated the antimicrobial activities of N-substituted glycine "peptoid" oligomers incorporating cationic and hydrophobic side chains. Head-to-tail macrocyclization was employed to enhance antimicrobial activity. Both linear and cyclic peptoids, ranging from six to ten residues, demonstrate potent antimicrobial activity against Gram-positive and Gram-negative bacteria. These peptoids do not cause significant lysis of human erythrocytes, indicating selective antimicrobial activity. Conformational ordering established upon macrocyclization is generally associated with an enhanced capacity to inhibit bacterial cell growth. Moreover, increased hydrophobic surface area also plays a role in improving antimicrobial activity. We demonstrate the potency of a cyclic peptoid in exerting antimicrobial activity against clinical strains of S. aureus while deterring the emergence of antimicrobial resistance.