Protection from the acquisition of Staphylococcus aureus nasal carriage by cross-reactive antibody to a pneumococcal dehydrogenase Rebeccah S. Lijeka, Santiago L. Luquea, Qian Liub, Dane Parkerc, Taeok Baeb, and Jeffrey N. Weisera,1 aDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; bDepartment of Microbiology and Immunology, Indiana University School of Medicine-Northwest, Gary, IN 46408; and cDepartment of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032 Edited* by Porter W. Anderson, Harvard Medical School, Boston, MA, and approved July 16, 2012 (received for review May 16, 2012) Nasal colonization by Staphylococcus aureus is the major risk factor (22). Instead, we and others (21) have hypothesized that an im- for disease and transmission. Epidemiological studies have reported munological mechanism may be involved, because the antagonistic a reduced risk of S. aureus carriage in immunocompetent but not in effect of pneumococcal colonization on S. aureus carriage is ob- immunocompromised children colonized by Streptococcus pneu- served in HIV-negative but not immunocompromised HIV-positive moniae. We investigate the hypothesis that the immune response individuals within the same cohort (8, 9, 23). To date, the only study to pneumococcal colonization affects S. aureus colonization. We that has addressed the role of the immune system measured anti- demonstrate that pneumococcal colonization in mice inhibits sub- body titers to 17 predetermined pneumococcal proteins and found sequent S. aureus acquisition in an antibody-dependent manner no correlation with S. aureus carriage in 57 infants (24). Therefore, and elicits antibody that cross-reacts with S. aureus. We identify a comprehensive examination of this hypothesis without pre- the staphylococcal target of cross-reactive antibody as 1-pyrro- selection of candidate antigens has not yet been performed. Here we investigate whether the host immune response to line-5-carboxylate dehydrogenase (P5CDH), and the homologous fl immunogen in S. pneumoniae as SP_1119, both of which are S. pneumoniae carriage can in uence S. aureus colonization in vivo. conserved dehydrogenases. These antigens are necessary and We demonstrate that antibodies elicited during pneumococcal sufficient to inhibit the acquisition of S. aureus colonization in a colonization in a mouse model cross-react with S. aureus, leading to MICROBIOLOGY a reduction in S. aureus nasal colonization. We identify the staph- mouse model. Our findings demonstrate that immune-mediated ylococcal target of cross-reactive antibody and the homologous cross-reactivity between S. pneumoniae and S. aureus protects immunogen in S. pneumoniae and confirm that these antigens are against S. aureus nasal acquisition and thus reveal a paradigm for fi S. aureus necessary and suf cient to limit the acquisition of S. aureus nasal identifying protective antigens against . colonization in vivo. pneumococcus | methicillin-resistant S. aureus | vaccine Results Pneumococcal Colonization in Mice Reduces Subsequent S. aureus he Gram-positive bacterial pathogen Staphylococcus aureus is Carriage in an Antibody-Dependent Manner. To recapitulate the Tresponsible for significant morbidity, mortality, and excess observed interference between S. pneumoniae and S. aureus col- healthcare costs worldwide. The management of S. aureus dis- onization, we developed a mouse model of S. aureus nasal colo- fi ease has become increasingly dif cult because of the rising nization using strain 502A, a clinical isolate known for superior prevalence of methicillin-resistant S. aureus (MRSA), which can nasal colonization in humans (25). Unlike previously described account for 60% of S. aureus infections in hospital and com- models of S. aureus nasal carriage, which are highly variable, nasal munity settings (1, 2). Given the limited treatment options for colonization by 502A is established reproducibly in naive C57BL/6 MRSA infection, novel preventative approaches are needed to mice with higher and less variable densities than seen with other protect against S. aureus infection and transmission. strains (Fig. S1 A and B). 502A colonization was achieved with A predominant risk factor for S. aureus infection and trans- a dose of 105 cfu, but levels were highest and most reproducible at mission is asymptomatic colonization of the anterior nares (3). day 1 postinoculation with a dose of 108 cfu (Fig. S1 C and D). For Eighty percent of S. aureus invasive infections in humans are all further experiments, we chose to use these latter conditions, caused by the host’s colonizing strain (4). However, the specific which reproducibly model the first step in colonization—nasal host and bacterial determinants of S. aureus nasal carriage are not acquisition—but do not model the long-term human carrier state. well understood (5). In children, significantly reduced S. aureus Therefore, our studies with this model focus on the initial estab- colonization rates have been associated with carriage of another S. aureus member of the upper respiratory tract flora, Streptococcus pneu- lishment of nasal colonization rather than on persistent moniae (6–14). These large and geographically diverse cohorts carriage. Under these conditions, the levels of S. aureus detected in have demonstrated reproducibly that colonization with S. pneu- our model are comparable to those recovered from experimentally moniae reduces the risk of S. aureus carriage by approximately half. colonized humans (26). This interference phenomenon has been reported for both vaccine We next combined our 502A acquisition model with an estab- and nonvaccine serotypes of S. pneumoniae (13). Moreover, lished murine model of pneumococcal nasopharyngeal colonization pneumococcal vaccination, which reduces S. pneumoniae carriage, that has colonization dynamics and immune responses similar to has been associated with an increased incidence of S. aureus- those observed in humans, including a robust antibody response to induced otitis media in children (15). The etiology of this interference phenomenon between S. pneu- moniae and S. aureus colonization is unknown. Although in vitro Author contributions: R.S.L. and J.N.W. designed research; R.S.L., S.L.L., Q.L., and T.B. studies have demonstrated that hydrogen peroxide secreted by performed research; R.S.L., D.P., and T.B. contributed new reagents/analytic tools; R.S.L. S. pneumoniae is bactericidal to S. aureus in coculture (16–18), and J.N.W. analyzed data; and R.S.L. and J.N.W. wrote the paper. neither hydrogen peroxide secretion by S. pneumoniae nor hydro- The authors declare no conflict of interest. gen peroxide sensitivity of S. aureus is predictive of cocolonization *This Direct Submission article had a prearranged editor. patterns in vivo (19–21). Moreover, any direct competitive effect 1To whom correspondence should be addressed. E-mail: [email protected]. in vivo is unlikely, because S. aureus is found primarily in the an- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. terior nares (5), whereas S. pneumoniae colonizes the nasopharynx 1073/pnas.1208075109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208075109 PNAS Early Edition | 1of6 Downloaded by guest on October 3, 2021 p = 0.029 closely homologous protein was identified in the S. pneumoniae TIGR4 genome by tBLASTn. The homologous loci in the p = 0.038 N.S. − 105 TIGR4 genome are sp_1161 (E value = 1e 56)andsp_1119 − (E value = 6e 68), respectively, and both encode putative but 104 uncharacterized dehydrogenases which we refer to hereafter as “SP_1161” and “SP_1119.” 103 Candidate Antigen Is SP_1119 in S. pneumoniae and Its S. aureus 102 Homolog, P5CDH. Each candidate antigen was cloned, recombi- nantly expressed, purified, and used to generate specific antisera. 1 IgG to P5CDH and DLDH bound to the surface of live S. aureus, 10 indicating that these proteins are antibody accessible (Fig. 3A). In contrast, incubation of S. pneumoniae TIGR4 with antisera 100 specific to the pneumococcal proteins did not result in surface Prior PBS TIGR4 P1121 PBS TIGR4 IgG binding (Fig. 3B). However, elimination of the antiopsonic colonization capsular polysaccharide in TIGR4cps facilitated surface binding by anti-SP_1119 and anti-SP_1161 IgG, suggesting that these Mouse strain WT antigens are surface associated but masked by capsule (Fig. 3C). We investigated whether antibodies raised against each can- Fig. 1. Pneumococcal colonization in mice reduces subsequent S. aureus didate protein could cross-react with the heterologous species. carriage in an antibody-dependent manner. Colonization density of S. aureus When S. aureus was incubated with antisera to the pneumococcal 502A in C57BL/6 wild-type (closed circles) or antibody-deficient μMT mice (open circles) 5 wk after prior colonization with S. pneumoniae TIGR4, proteins, we observed cross-reactive binding with anti-SP_1119 S. pneumoniae P1121, or sham (PBS) colonization. 502A carriage levels were but not with anti-SP_1161 IgG (Fig. 3A). Similarly, antisera to the assessed in lavages of the upper respiratory tract at day 1 postchallenge. Hor- staphylococcal homolog of SP_1119, P5CDH, bound to the sur- izontal solid lines indicate median values; dotted line indicates limit of de- face of unencapsulated S. pneumoniae, but antisera to DLDH did tection, N.S., not significant. not (Fig. 3C). Together, these data suggest that antisera to the homologous pair P5CDH