Abstract
Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli.
Highlights
The high diversity of extracellular proteins means that their function, even in pathogenic bacteria, is not possible to identify by bioinformatics, in particular because the sequence similarity between proteins that have the same function is often limited to a genus, species, and even strain
This is important in construction of bacterial genomic shot-gun phage display libraries, to avoid resistance to infection with the helper phage when the inserts contain bacterial promoters upstream of the open readinf frames (ORFs) encoding displayed proteins
We report a discovery of two extracellular proteins that appear to form a complex on the surface of the probiotic bacterium L. rhamnosus HN001 This complex was identified by affinity screenings of an L. rhamnosus HN001 phage display library
Summary
Bacterial surface proteins have been studied extensively in pathogenic bacteria over the last several decades, demonstrating their role in host colonization and cell invasion as well as very sophisticated manipulation of the host immune response pathways that often neutralize microbial-pattern-induced signaling pathways (Patti et al 1994; Beckmann et al 2002; Selbach and Backert 2005; Lilic et al 2006; Luck et al 2006; Timmer et al 2006; Eskan et al 2007; Gillen et al 2008; Kulkarni et al 2009; McGhie et al 2009). The high diversity of extracellular proteins means that their function, even in pathogenic bacteria, is not possible to identify by bioinformatics, in particular because the sequence similarity between proteins that have the same function is often limited to a genus, species, and even strain (e.g., proteins implicated in binding to the mucosal surfaces, or manipulation of immune response). These proteins appear to represent recent evolutionary adaptations (Baltrus et al 2011; Carroll et al 2011).
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