Thepil2operon has a low conservation amongS. based on antibody titers against any of these four antigens resulted in assay sensitivities between 16% and 43% for the detection of early-stage CRC. Together these findings underscore the potential of a multi-antigen approach to complement diagnosis ofS. gallolyticussubsp.gallolyticusassociated CRC. == Introduction == The human gastrointestinal tract is the habitat for a large and dynamic bacterial community, which is essential for digestion of food and the control of intestinal epithelial homeostasis (1,2). Remarkably, although hundreds of bacterial species reside in the human intestinal tract, NAV3 only the opportunistic pathogenStreptococcus gallolyticussubsp.gallolyticus(S. bovisbiotype I), seems to benefit from the presence of premalignant colonic lesions to invade the human body (27). In this respect,S. gallolyticussubsp.gallolyticuscan be regarded as a whistle-blower for colorectal cancer (CRC) as multiple studies showed that a (precursor of) CRC is detected in 33% to 100% of the individuals that undergo full bowel examination following diagnosis of this contamination. Notably, our recent meta-analysis showed that this percentage is far above the prevalence of this disease in the asymptomatic age-matched population (8).S. gallolyticussubsp.gallolyticusis a known causative agent for infective endocarditis (IE), however, due to its mild virulence characteristics this bacterium can only establish a clinical infection in patients with preexisting heart valve abnormalities. Molecular studies suggested thatS. gallolyticussubsp.gallolyticusis relatively invisible for the innate immune system due to its inert surface structure (9,10; our unpublished observations), which implies thatS. gallolyticussubsp.gallolyticusmay cause subclinical infections in a substantial part of CRC patients (11). The latter idea was supported by our previous finding that the humoral immune response against the ribosomal protein L7/L12 fromS. gallolyticussubsp.gallolyticuswas significantly increased in early-stage CRC patients (1214), which is indicative for an increased exposure to this bacterium. However, a drawback of this approach was the fact that this high conservation of this antigen in the bacterial kingdom was associated with a considerable amount of cross-reactivity in the immunoassay (14). This study aimed at the development of new ELISAs exploiting antigens that are specific forS. gallolyticussubsp.gallolyticusstrains. These candidate antigens concerned 4 cell wall peptidoglycan-anchored proteins that form pilin-like structures on theS. gallolyticussubsp.gallolyticuscell surface (15). Two of these proteins, annotated Gallo2178 (major pilin) and Gallo2179 (collagen-binding adhesin) are encoded by thepil1locus that also encodes a sortase (Gallo2177) which is specifically responsible for the polymerization of these 2 LPTG into a pilus structure. Thepil1locus is present in the majority of clinicalS. gallolyticussubsp.gallolyticusIE isolates and involved in binding to collagen type I, biofilm formation, and virulence in a rat model of experimental endocarditis GSK1904529A (to be published elsewhere). Interestingly, collagen I-binding capacity has also been proposed as a distinguished virulence feature ofS. gallolyticussubsp.gallolyticusstrains GSK1904529A to facilitate its adherence to premalignant colonic sites (9). GSK1904529A Collagen binding is likely to be mediated by Gallo2179, which contains a collagen-binding domain name. The other 2 candidate antigens, Gallo1569 and Gallo2039, are major pilins related to Gallo2178, but encoded by thepil2andpil3operons, respectively (16). Thepil2operon has a low conservation amongS. gallolyticussubsp.gallolyticusstrains, whereas homologouspil3operons can also be found inStreptococcus infantariussubsp.infantariusstrains. Our current data showed that ELISAs with these 4 antigens were indeed specific forS. gallolyticussubsp.gallolyticusinfections. Furthermore, our data showed a highly selective humoral immune response to these antigens in CRC patients. However, a multimarker approach could identify a substantial number of these patients. This obtaining argues in favor of developing extended multiplex assays based on specific antigens from CRC-associated bacteria as screening tool GSK1904529A for CRC. == Materials and Methods == == Patient material == Blood samples were derived from the same collections as used before in our studies (14). However, here, we primarily focused on the early stages of CRC (i.e., colorectal adenomas and local stage of colorectal cancer). Serum samples from 37 CRCs, 12 polyp patients (6 adenomas, 2 villous adenomas, and 4 undefined polyps), and 15 patients with a clinical bacterial infection [Escherichia coli. (3),Klebsiella pneumoniae(3),Salmonella typhimurium(3),Streptococcus pneumonia(3), orS. gallolyticussubsp.gallolyticus(3; CRC diagnosed in 1 patient] who had been admitted to the Radboud University Nijmegen Medical Centre (Nijmegen, the Netherlands) were used. Patients suffering from bacterial infections were recognized as such by a positive blood culture and routine microbial typing. As control, serum samples from 27 healthy blood donors (>50 years), who did not undergo colonic evaluation, were used. In addition, plasma samples from 33 CRC, 11 polyp patients, and 47 healthy controls who participated in a population-based casecontrol study in Metropolitan Detroit, were included as a second independent study population. CRC samples concerned localized disease (stage I or II), with the exception of 7 Detroit cases with unknown stage. All cases in both the Nijmegen and Detroit population underwent colonic evaluation. The use of the samples was approved by the Medical.