Host-adapted lactobacilli: evolution, molecular mechanisms and functional applications

• Author / Creator

  Duar, Rebbeca M

• Bacteria of the genus Lactobacillus can be found associated with plants, insects and vertebrate hosts, and their lifestyle can range from free-living to strictly host specific. Of the lactobacilli associated with vertebrates, the lifestyle of L. reuteri is particularly well understood. The species has been studied by population genetics, comparative genomic and functional analyses in animal models. The phylogenetic structure of L. reuteri suggests that lineages evolved alongside with rodents, poultry, swine and humans. For rodent strains, co-evolution resulted in host-adaptation. The first goal of this dissertation was to determine whether host-adaptation extended to non-rodent lineages and also to resolve open questions regarding the evolutionary relationships within lineage VI, which is shared by human and poultry isolates. An experimental approach was devised to determine the ability of strains to propagate under the ecological conditions of the gastrointestinal tract (GIT) of different hosts. Rodent isolates became enriched in the GIT of mice and poultry isolates in chickens. Moreover, human isolates of the lineage VI were found to be competitive in the GIT of chickens but not in humans. These findings revealed that L. reuteri evolved host-specialization in rodents and chicken, while open questions remain about the exact evolutionary consequences in humans and pigs. Biofilm formation is a common strategy by which lactobacilli maintain stable associations with their hosts. Only rodent isolates of L. reuteri can produce biofilms in the forestomach of mice. The second goal of this dissertation was to determine the role of a rodent-specific two component system (TCS70529-30) in biofilm formation of the rat isolate L. reuteri 100-23. Experiments in monoassociated mice revealed that mutation of the response regulator, but not the histidine kinase impaired biofilm formation. In vitro experiments confirmed in vivo and findings and further revealed significant alterations in the architecture of the mutant biofilms. Compared to the wildtype, histidine kinase mutants produced thick and robust biofilms, while the response regulator mutants formed thinner and less adherent biofilms. These findings provide empirical evidence of rodent specific signal transduction system playing a role in biofilm formation of L reuteri, likely by regulating genes responsible for development of the biofilm matrix. Contrary to rodent strains, human isolates of L. reuteri lack the genetic machinery to form biofilms, but conserve a 58-gene pdu-cbi-cob-hem cluster (pdu-cluster). Encoded in the pdu-cluster is the PduCDE diol dehydratase involved in utilization of 1,2 propanediol (1,2 PD). In the human gut, 1,2 PD is readily available as a result of fermentation of rhamnose and fucose found in dietary and host-derived glycans, respectively. The third goal of this dissertation was to determine the role of the pdu-cluster in utilization of 1,2 PD by human isolates of L. reuteri. The ability of the human isolate L. reuteri ATCC 6475 to cross-feed from 1,2 PD produced by Escherichia coli MG1655 and Bifidobacterium breve UCC2003 was determined in vitro and compared to a pduCDE mutant. We found that during fermentation of hexoses, 1,2 PD serves as an electron acceptor increasing the metabolic efficiency of L. reuteri, a factor that could be pivotal to the competitiveness of human isolates of the human GIT. The fourth goal of this dissertation was to identify and characterize bacterial isolates from the proximal GI tract of pigs capable of degrading peptides involved in the etiology of celiac disease. Strains were selected from the GIT tract of pigs fed a 20% gluten diet and after an in vitro process aimed to enrich for gluten degrading bacteria. Pigs were selected as these animals harbor large amounts of lactobacilli. Strains of the species L. amylovorus, L. johnsonii, L. ruminis, and L. salivarius were identified as having the highest proteolytic activity against several well characterized gluten immunotoxic peptides. Since these strains are adapted to the conditions in the proximal GI tract, they are likely to be good candidates for probiotics aimed at removing gluten epitopes before they reach the epithelium of the small intestine in celiac patients. Together findings in this dissertation contribute to our understanding of the evolution of L. reuteri with different vertebrate hosts, reveal insights into lineage-specific functions underlying adaptation to the vertebrate GIT, and provide a basis for the selection of lactobacilli adapted to GIT for functional applications.

• Subjects / Keywords

  • Celiac
  • Symbiosis
  • Lactobacillus
  • Probiotics
  • Biofilms
  • Cross-feed

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3DN40915

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.