Dataset Information

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.

SUBMITTER: Klotz C

PROVIDER: S-EPMC5682318 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of <i>Lactobacillus acidophilus</i> Using Quantitative Proteomics.

Klotz Courtney C O'Flaherty Sarah S Goh Yong Jun YJ Barrangou Rodolphe R

Frontiers in microbiology 20171108

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 attempte ...[more]

PMID: 29167661

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Project description:Alphaviruses and flaviviruses are important human pathogens that include Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV), which can cause diseases in humans ranging from arthralgia to hemorrhagic fevers and microcephaly. It was previously shown that treatment with surface layer (S-layer) protein, present on the bacterial cell-envelope of Lactobacillus acidophilus, is able to inhibit viral and bacterial infections by blocking the pathogen's interaction with DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a trans-membrane protein that is a C-type calcium-dependent lectin. DC-SIGN is known to act as an attachment factor for several viruses including alphaviruses and flaviviruses. In the present study, we used alphaviruses as a model system to dissect the mechanism of S-layer inhibition. We first evaluated the protective effect of S-layer using 3T3 cells, either wild type or stably expressing DC-SIGN, and infecting with the alphaviruses Semliki Forest virus (SFV) and CHIKV and the flaviviruses ZIKV and DENV. DC-SIGN expression significantly enhanced infection by all four viruses. Treatment of the cells with S-layer prior to infection decreased infectivity of all viruses only in cells expressing DC-SIGN. In vitro ELISA experiments showed a direct interaction between S-layer and DC-SIGN; however, confocal microscopy and flow cytometry demonstrated that S-layer binding to the cells was independent of DC-SIGN expression. S-layer protein prevented SFV binding and internalization in DC-SIGN-expressing cells but had no effect on virus binding to DC-SIGN-negative cells. Inhibition of virus binding occurred in a time-dependent manner, with a significant reduction of infection requiring at least a 30-min pre-incubation of S-layer with DC-SIGN-expressing cells. These results suggest that S-layer has a different mechanism of action compared to mannan, a common DC-SIGN-binding compound that has an immediate effect in blocking viral infection. This difference could reflect slower kinetics of S-layer binding to the DC-SIGN present at the plasma membrane (PM). Alternatively, the S-layer/DC-SIGN interaction may trigger the activation of signaling pathways that are required for the inhibition of viral infection. Together our results add important information relevant to the potential use of L. acidophilus S-layer protein as an antiviral therapy.

Dataset Information

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

Publications

Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of <i>Lactobacillus acidophilus</i> Using Quantitative Proteomics.

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