This adaptation to host cells is reflected in the genome of L. pneumophila, which encodes for an abundance of eukaryotic-like proteins (Cazalet et al., 2004). Lcl is predicted to encode a 49.6 kDa protein with GXY collagen-like repeats. Enzymatic assays were performed to confirm the collagen-like structure. Lcl and rat tail collagen type I reacted in the same way on collagenase and trypsin incubation (data not shown). Furthermore, the GXY repeats were encoded by the VNTR region and a change in the number of repeat units had an influence on the number of GXY repeats

and consequently on the collagen-like protein structure. Some of the Legionella eukaryotic-like proteins have already proven their role in virulence and show that these eukaryotic-like proteins Palbociclib mw are putative candidates to play a role in the L. pneumophila pathogenesis (Cazalet et al., 2004). Therefore, the study of eukaryotic-like proteins, such as Lcl, is important to define

the survival strategies of this intracellular parasite. Virulence factors are also often outer membrane proteins or secreted proteins and previous studies have already identified several outer membrane proteins of L. pneumophila that are involved in the adhesion Rapamycin order and invasion of host cells (Mintz et al., 1992; Chang et al., 2005; D’Auria et al., 2008). Different cellular fractions (the cytoplasm, inner membrane, outer membrane and supernatant) were tested for the presence of Lcl. Separation of the cellular fractions by SDS-PAGE, followed by immunodetection with Lcl-specific antibodies, revealed an immunoreactive band in the outer membrane protein fraction and the extracellular fraction (Fig. 1a). Proteins used as a control were present in the expected fractions (Fig. 1b–d). The results of the cellular fractionation demonstrated that Lcl is an outer membrane protein that can also be found in the extracellular fraction. This could be due to the fragmentation of Lcl, situated at the cell surface, into the

extracellular space, or Lcl could have an additional function as a secreted protein. Other work has also yielded conflicting results regarding the localization of Lcl (DebRoy et al., 2006; Galka et al., 2008; Khemiri et al., 2008), Isoconazole which is probably due to the different techniques used. As Lcl contains the characteristic C-terminal consensus AAVRAVRAF, with a hydrophilic amino acid only in position 3, the outer membrane localization is most likely. The VNTR region of lcl of all 108 strains was amplified by PCR (see Materials and methods). The resulting PCR fragments of different sizes led to the identification of 12 polymorphisms ranging from 7 to 19 repeats of 45 nt. The repeat distribution of lcl in the 108 strains is bimodal, with a preference for 8 or 13 and 14 repeats (Fig. 2a).