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Impact of Lactobacillus plantarum sortase on target protein sorting, gastrointestinal persistence, and host immune response modulation.

ABSTRACT: Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA microarray-based transcriptome analysis revealed that the srtA deletion had only minor impact on the full-genome transcriptome of L. plantarum, while the expression of SDP-encoding genes remained completely unaffected. Mass spectrometry analysis of the bacterial cell surface proteome, which was assessed by trypsinization of intact bacterial cells and by LiCl protein extraction, revealed that SrtA is required for the appropriate subcellular location of specific SDPs and for their covalent coupling to the cell envelope, respectively. We further found that SrtA deficiency did not affect the persistence and/or survival of L. plantarum in the gastrointestinal tract of mice. In addition, an in vitro immature dendritic cell (iDC) assay revealed that the removal of surface proteins by LiCl strongly affected the proinflammatory signaling properties of the SrtA-deficient strain but not of the wild type, which suggests a role of SDPs in host immune response modulation.

SUBMITTER: Remus DM

PROVIDER: S-EPMC3554011 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Impact of Lactobacillus plantarum sortase on target protein sorting, gastrointestinal persistence, and host immune response modulation.

Remus Daniela M DM Bongers Roger S RS Meijerink Marjolein M Fusetti Fabrizia F Poolman Bert B de Vos Paul P Wells Jerry M JM Kleerebezem Michiel M Bron Peter A PA

Journal of bacteriology 20121121 3

Sortases are transpeptidases that couple surface proteins to the peptidoglycan of Gram-positive bacteria, and several sortase-dependent proteins (SDPs) have been demonstrated to be crucial for the interactions of pathogenic and nonpathogenic bacteria with their hosts. Here, we studied the role of sortase A (SrtA) in Lactobacillus plantarum WCFS1, a model Lactobacillus for probiotic organisms. An isogenic srtA deletion derivative was constructed which did not show residual SrtA activity. DNA micr ...[more]

PMID: 23175652

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Project description:BACKGROUND: Lactic acid bacteria (LAB) are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. METHODOLOGY/PRINCIPAL FINDINGS: In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI) survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log) and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na(+)/H(+) antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. CONCLUSION: Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-)robustness, which is important for our molecular understanding of GI-tract survival and could facilitate the design of culture conditions aimed to enhance probiotic culture robustness.

Project description:Reliability of microbial (starter) strains in terms of quality, functional properties, growth performance, and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing, such as acid, osmotic, temperature, and oxidative stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (N-methyl-N'-nitro-N-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be obtained by pretreatment of cultures with other, nonmutagenic stress conditions.

Dataset Information

Impact of Lactobacillus plantarum sortase on target protein sorting, gastrointestinal persistence, and host immune response modulation.

Publications

Impact of Lactobacillus plantarum sortase on target protein sorting, gastrointestinal persistence, and host immune response modulation.

Similar Datasets

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