Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics

Abstract

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.

Probiotics alter the immune response of gingival epithelial cells challenged by Porphyromonas gingivalis

“…Adhesion of probiotics to host cells is mediated by extracellular polysaccharides and surface layer proteins . The expression of these surface components is regulated by environmental conditions, thus affecting their hydrophobic properties and adhesion efficiency . Furthermore, certain adhesins of lactobacilli are proteases‐sensitive, whereas others are resistant, suggesting that the adhesive properties of probiotics may be altered by P. gingivalis proteolytic activity.…”

“…However, our in vitro data suggested that P. gingivalis colonized sites may be more susceptible to colonization by these organisms. Therefore, further experiments are required to reveal how the interactions between probiotic strains, pathogens, and host cells can affect their phenotypes at the proteomic level and the immunological response in complex biofilms …”

Deletion of S-Layer Associated Ig-Like Domain Protein Disrupts the Lactobacillus acidophilus Cell Surface

“…In addition to surface proteins, salt stress also triggered the upregulation of a previously characterized bile-inducible operon encoding a two-component regulatory system (Pfeiler et al, 2007). This system includes two-component response regulator, LBA1431, which possess an OmpR/PhoBtype DNA-binding domain (Pfeiler et al, 2007) and putative SLAP LBA1426 (Klotz et al, 2017). Interestingly, this cluster of genes was not induced in the salt-stressed parent strain.…”

“…To complement these results, COGs were assigned using the EggNOG Database (Huerta-Cepas et al, 2016; Figure 7B). Additionally, a previously curated list of the most abundant log phase SLAPs (Klotz et al, 2017), and corresponding fold change, is detailed in Table 2. In MRS, the absence of igdA triggered the significant upregulation of 44 genes, while none were downregulated (Figure 7A, left).…”

“…The Ig domain-containing SLAP of L. acidophilus NCFM (LBA0695), which we have designated IgdA, was recently recognized for its high constitutive expression on the cell exterior (Klotz et al, 2017). Its conservation among S-layer-forming lactobacilli, compelling domain architecture, and high surface abundance, prompted its selection for further investigation.…”