Bacterial surface-layers (S-layers) are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalog the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose to employ a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag) labeling combined with a triple-stage mass spectrometry (MS3) method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacteria.

The low water activity (a(w) 0.3) of peanut butter prohibits the growth of Salmonella in a product; however, illnesses are reported from peanut butter contaminated with very small doses, suggesting the food matrix itself influences the infectious dose of Salmonella, potentially by improving Salmonella's survival in the gastrointestinal tract. The purpose of our study was to quantify the survival of a peanut butter outbreak-associated strain of Salmonella enterica serotype Tennessee when inoculated into peanut butters with different fat contents and a(w) (high fat, high a(w); high fat, low a(w); low fat, high a(w); low fat, low a(w)) and then challenged with a simulated gastrointestinal system. Exposures to increased fat content and decreased a(w) both were associated with a protective effect on the survival of Salmonella Tennessee in the simulated gastric fluid compared with control cells. After a simulated intestinal phase, the populations of Salmonella Tennessee in the control and low-fat formulations were not significantly different; however, a 2-log CFU/g increase occurred in high-fat formulations. This study demonstrates that cross-protection from low-a(w) stress and the presence of high fat results in improved survival in the low pH of the stomach. The potential for interaction of food matrix and stress adaptations could influence the virulence of Salmonella and should be considered for risk analysis.

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Bacterial surface-layers (S-layers) are crystalline arrays of repeating proteinaceous subunits that coat the exterior of many cell envelopes. S-layers have demonstrated diverse functions in growth and survival, maintenance of cell integrity, and mediation of host interactions. Additionally, S-layers can act as scaffolds for the outward display of auxiliary proteins and glycoproteins. These non-covalently bound S-layer associated proteins (SLAPs) have characterized roles in cell division, adherence to intestinal cells, and modulation of the host immune response. Recently, IgdA (LBA0695), a Lactobacillus acidophilus SLAP that possesses a Group 3 immunoglobulin (Ig)-like domain and GW (Gly-Tryp) dipeptide surface anchor, was recognized for its high conservation among S-layer-forming lactobacilli, constitutive expression, and surface localization. These findings prompted its selection for examination within the present study. Although IgdA and corresponding orthologs were shown to be unique to host-adapted lactobacilli, the Ig domain itself was specific to vertebrate-adapted species suggesting a role in vertebrate adaptation. Using a counterselective gene replacement system, igdA was deleted from the L. acidophilus NCFM chromosome. The resultant mutant, NCK2532, exhibited a visibly disrupted cell surface which likely contributed to its higher salt sensitivity, severely reduced adhesive capacity, and altered immunogenicity profile. Transcriptomic analyses revealed the induction of several stress response genes and secondary surface proteins. Due to the broad impact of IgdA on the cellular physiology and probiotic attributes of L. acidophilus, identification of similar proteins in alternative bacterial species may help pinpoint next-generation host-adapted probiotic candidates.

Background: Surface layers (S-layers) are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost layer of many bacterial cell envelopes. Within the Lactobacillus genus, S-layer presence is frequently associated with probiotic-relevant properties such as improved adherence to host epithelial cells and modulation of the immune response. However, recent studies have demonstrated that certain S-layer functions may be supplemented by a novel subset of proteins embedded within its lattice, termed S-layer associated proteins (SLAPs). In the following study, four Lactobacillus acidophilus NCFM SLAPs (LBA0046, LBA0864, LBA1426, and LBA1539) were selected for in silico and phenotypic assessment. Results: Despite lacking any sequence similarity or catalytic domains that may indicate function, the genes encoding the four proteins of interest were shown to be unique to S-layer-forming, host-adapted lactobacilli species. Likewise, their corresponding deletion mutants exhibited broad, host-relevant phenotypes including decreased inflammatory profiles and reduced adherence to Caco-2 intestinal cells, extracellular matrices, and mucin in vitro. Conclusions: Overall, the data presented in this study collectively links several previously uncharacterized extracellular proteins to roles in the underlying host adaptive mechanisms of L. acidophilus.