Stress responses and digestive tract robustness of Lactobacillus plantarum

Research output: Thesisinternal PhD, WU

Abstract

 

Lactobacillus plantarumis one of the most versatile lactic acid bacteria that can successfully inhabit a variety of environmental niches. It is a common inhabitant of the human and animal gastrointestinal (GI) tract and it is used as starter culture in various fermentation processes for different food raw-materials, including milk, fruits, vegetables, and meat. Moreover, L. plantarum is marketed as a health-promoting culture, i.e. a probiotic. In these different environments and processes the bacteria encounter stress conditions, such as heat, cold, acid, salt, and oxygen stress. Since starter cultures and probiotics require metabolic activity to contribute to the taste and texture of the fermented products, and/or viability to exert their in situ beneficial effect on the consumer, it is important to understand and improve the gene-regulatory adaptation that sustains their function and viability under these challenging conditions. Nowadays, genomic approaches are available that enable the global, genome-wide analysis of stress responses in lactic acid bacteria. The work presented in this thesis employs such tools and also developed some novel strategies to understand stress responses in L. plantarum.

During wine fermentation, L. plantarum is exposed to ethanol and global transcriptome profiling demonstrated the gene expression adaptation of this microorganism upon short and long term exposure to sublethal levels of this solvent. The results suggested that the ethanol induced activation of the CtsR-related stress regulon contributes to its adaptation to ethanol exposure which also provides cross-protection against heat stress. Transcriptome analyses under different growth conditions of gene deletion derivatives of the L. plantarum WCFS1 strain that lack the genes encoding the stress response regulators ctsR and/or hrcA, enabled the refinement of the gene regulation repertoire that is controlled by these central regulators of stress responses in this species. Notably, the deletion of both stress-regulators, elicited transcriptome changes that affected a large variety of additional gene-functions in a temperature-dependent manner, which prominently included genes related to cell-envelope remodelling.

Culturing  of L. plantarum WCFS1 under different fermentation conditions led to large differences in GI-tract survival and robustness, which was addressed using a simple in vitro survival assay. Enhanced GI-tract survival and robustness could be associated with low salt and low pH conditions during the fermentations.  The transcriptomes obtained for each of the fermentation conditions employed, were correlated with the observed GI-tract survival rates, enabling the identification of candidate genes involved in the robustness phenotype, including a transcription regulator involved in capsular polysaccharide remodelling (Lp_1669), a penicillin-binding protein (Pbp2A) involved in peptidoglycan biosynthesis, and a Na+/H+ antiporter (NapA3). A role of these candidate genes in actual survival in the GI-tract assay could be confirmed by mutation analysis, further confirming their contribution to GI-tract stress robustness in L. plantarum.

This thesis also describes the use of a novel, next-generation sequencing-based method, for the assessment of the in vivo GI-tract persistence of different L. plantarum strains that were administered to healthy human volunteers in specifically designed strain-mixtures. A remarkable consistency of the strain-specific in vivo persistence curves was observed when comparing data obtained from different volunteers. Moreover, a striking congruency was observed between the strain-specific in vivo persistence curves and the predicted GI-tract survival based on the simple in vitro assay. Finally, evolutionary adaptation of L. plantarum WCFS1 to the murine GI-tract was studied by extended exposure of the strain to the mice digestive tract through consecutive rounds of (re)feeding of the longest persisting bacterial colonies. Re-sequencing of the genomes of more persistent derivatives of the original strain, and the evaluation of the genomic modifications identified, implied that genes encoding cell envelope-associated functions and energy metabolism play an important role in the determination of GI-tract persistence in L. plantarum.

The results described in this thesis strive to obtain an improved understanding of the gene-regulatory adaptations of L. plantarum that allow its survival under stress conditions, including those associated with residence in the gastrointestinal tract of animals and humans, with the intention to exploit such understanding to rationally improve the robustness of these bacteria.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Kleerebezem, Michiel, Promotor
  • Bron, P.A., Co-promotor
Award date3 Jul 2013
Place of Publication[S.l.
Print ISBNs9789461736291
Publication statusPublished - 2013

Keywords

  • lactobacillus plantarum
  • stress response
  • digestive tract
  • gene regulation
  • adaptation

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