Host–pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin–receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin–host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10–15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

Staphylococcus aureus can produce up to five different bi-component cytotoxins: two gamma-hemolysins HlgAB and HlgCB, and leukocidins SF-PV (Panton Valentine leukocidin), ED (LukED) and GH (LukGH, also called LukAB). Their major function in S. aureus pathogenesis is to evade innate immunity by attacking phagocytic cells and to support bacterial growth by lysing red blood cells. The five cytotoxins display different levels of amino acid sequence conservation (30-82%), but all form a remarkably similar beta-barrel type pore structure (greatly resembling the mono-component toxin alpha-hemolysin) that inserts into the target cell membrane leading to necrotic cell death. This review provides an overview of the culmination of decades of research on the structure of these toxins, their unique sequence and structural features that helps to explain the observed functional differences, such as toxin potency towards different cell types and species, receptor specificity and formation of functional non-cognate toxin pairs. The vast knowledge accumulated in this field supports novel approaches and the design of therapeutics targeting these cytotoxins to tame virulence and fight S. aureus infections.

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