Molecular Characterization of a Streptococcus gallolyticus Genomic Island Encoding a Pilus Involved in Endocarditis

Camille Danne 0 1 2 3 Jose M. Entenza 2 8 Adeline Mallet 2 7 Romain Briandet 2 9 Michel De barbouille 1 2 3 Farida Nato 2 6 Philippe Glaser 2 5 Gre gory Jouvion 2 4 Philippe Moreillon 2 8 Patrick Trieu-Cuot 1 2 3 Shaynoor Dramsi () 1 2 3 0 Universite Paris 7-Denis Diderot 1 Centre National de la Recherche Scientifique (CNRS) URA 2172 2 Received 14 April 2011; accepted 16 July 2011; electronically published 31 October 2011. Bacte ries Pathoge`nes a` Gram-Positif , CNRS URA 2172, 25, rue du Dr Roux, Paris, France 75015 3 Unite de Biologie des Bacte ries Pathoge`nes a` Gram-Positif 4 Unite Histopathologie Humaine et Mode`les Animaux, Institut Pasteur , Paris 5 Laboratoire Evolution et Ge nomique Bacte riennes and CNRS URA 2171 6 Laboratoire de Production de Prote ines Recombinantes et d'Anticorps 7 Imagopole , Ultrastructural Microscopy Platform 8 University of Lausanne, Department of Fundamental Microbiology , Switzerland 9 Institut National de Recherche Agronomique , Micalis UMR 1319, Massy , France Background. Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. Methods. S. gallolyticus UCN34, Pil11 and Pil12, expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. Results. We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. Conclusions. This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus. - Streptococcus gallolyticus subsp gallolyticus (formerly known as Streptococcus bovis biotype I) is an increasing cause of infective endocarditis (IE). Asymptomatic carriage of S. gallolyticus is commonly observed in the gastrointestinal tract of birds, ruminants, and a small proportion of humans (2.5%15%) [1, 2]. Several studies have shown that endocarditis due to S. gallolyticus are frequently associated with colorectal carcinoma [1,36]. Whether the development of tumors is a cause or a consequence of S. gallolyticus infections remains to be investigated. To address this question, a better understanding of the pathophysiology of these diseases is required with a focus on the bacterial virulence factors responsible for the initiation of infections, that is, adhesion to host tissues. The gallate-degrading strains of S. bovis group have been reassigned to a new species named S. gallolyticus sp [79]. Customarily, human isolates of S. bovis were classified into 3 biotypes designated as I, II/1, and II/2 that corresponds to S. gallolyticus subsp gallolyticus, the closely related subspecies pasteurianus, and the more distant subspecies infantarius, respectively. Finally, Streptococcus macedonicus is considered as a nonpathogenic S. gallolyticus subspecies. However, this taxonomic reclassification is still a matter of debate, and for simplification and readability these subspecies will be considered as species throughout this work. Although the proportion of IE due to S. gallolyticus has increased among streptococci, particularly in Southern Europe [1012], its virulence and colonization factors remain largely unknown. Five serotypes have been described based on capsular typing in strains isolated from pigeons [13]. Electron microscopic studies of S. gallolyticus pigeon strains revealed the existence of filamentous structures known as fimbriae or pili [14]. It was hypothesized that these structures could play a role in virulence. Bacterial pathogens associated with IE possess surface adhesins belonging to MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), which mediate attachment to cardiac vegetations and are involved in valve colonization and infection [15]. Previously characterized collagen-binding proteins include Cna of Staphylococcus aureus [16], Acm of Enterococcus faecium [17], Ace of E. faecalis [18, 19], and Acb from S. gallolyticus strain TX20005 [20]. Development of endocarditis is initiated by injury of the endothelium, which disrupts the normal valve structure and exposes underlying tissues, including extracellular matrix (ECM) material. Deposition of host proteins, such as fibrin and platelets, then leads to the formation of a sterile thrombotic vegetation that may become colonized by circulating bacteria [21]. S. gallolyticus isolates responsible for IE were shown to display heterogeneous profiles of adherence to ECM proteins [2224]. We recently participated in the complete sequencing of S. gallolyticus strain UCN34, isolated from a human IE case associated with colorectal cancer [25]. In silico analyses enabled the identification of 19 putative cell wall-anchored proteins. We focused on 3 putative pilus loci, each encoding 1 sortase C and 2 LPXTG motif proteins, referred herein as pil1 (gallo2179-77), pil2 (gallo1570-68), and pil3 (gallo2040-38). It was recently reported that S. gallolyticus strain TX20005 (draft genome) also encodes 3 pilus loci [20]. Interestingly, only 2 of these loci are identical in both S. gallolyticus strains, namely pil1 (acbsbs7-srtC1 in TX20005) and pil3 (sbs15-sbs14-srtC2 in TX20005). Gram-positive pili were first observed in Corynebacterium renale by electron microscopy [26] and have now been characterized genetically and biochemically in many important pathogens, for example Streptococcus agalactiae [2730]. These pili consist of covalently cross-linked subunit proteins and are anchored to the peptidoglycan (for reviews, see [31, 32]). Sortase-mediated pilus assembly was first demonstrated in Corynebacterium diphteriae [33, 34], and the current model for pilus biogenesis is as follows: the major subunit is assembled into the pilus by a cisencoded class C sortase that catalyzes the covalent attachment between the conserved lysyl residue of the pilin motif (WxxxVxVYPK) of one subunit and the conserved threonyl residue of the LPXTG motif of another subunit [35]. In addition, one or more accessory subunits could also be incorporated into the pilus backbone [36]. Here, we studied the pil1 pilus locus of (...truncated)