Bile salt hydrolase in Lactobacillus plantarum: functional analysis and delivery to the intestinal tract of the host

In the liver of mammals, bile salts are synthesised from cholesterol and conjugated to either taurine or glycine. Following release into the intestine, conjugated bile salts can be deconjugated by members of the endogenous microbiota that produce an enzyme called bile salt hydrolase (Bsh). Bsh appears to play an important role in both host intestinal physiology and bacterial survival and persistence in the intestinal tract; especially lactobacilli have been suggested to be of importance for in vivo bile salt hydrolysis in the small intestine.
In this thesis, a functional analysis of Bsh in Gram-positive bacteria, and in particular, the model organism Lactobacillus plantarum WCFS1 was performed. In-depth investigation of the annotation of bsh genes in Gram-positive bacteria using a combination of in silico methods led to the re-annotation of eight conjugated bile acid hydrolase superfamily members in various lactobacilli. Furthermore, these analyses provided a robust methodology for accurate annotation of this enzyme superfamily. L. plantarum WCSF1 was previously predicted to contain four bsh genes (bsh1, bsh2, bsh3, and bsh4), but according to our in silico analyses, three of these genes appeared to be penicillin acylase-related.
To unravel the functionality of each of the separate bsh genes, the generation of multiple isogenic bsh-deletion strains was required. Therefore, a Cre-lox-based toolbox for the construction of multiple deletions and selectable-marker removal in Gram-positive organisms was designed and implemented in L. plantarum WCFS1. Using heterologous over-expression and multiple bsh-deletion derivatives of L. plantarum WCFS1, Bsh1 was shown to be the major bile salt hydrolase in this strain, where it appeared to be involved in glycodeoxycholic acid tolerance. Although these experiments validated the prediction that bsh2, bsh3, and bsh4 do not encode true Bsh enzymes, the in vivo functionality of Bsh2, Bsh3, and Bsh4 was not entirely clarified. Bsh2, Bsh3, and Bsh4 appeared to encode enzymes with acylase activity possibly using penicillin-like chemicals as their preferred substrate.
To investigate the influence of Bsh-producing probiotics on host physiology, two modes of Bsh delivery to the small intestine were investigated in this work; delivery of Bsh activity by viable L. plantarum was compared to delivery using Bsh-whey protein/gum arabic microencapsulates in an in vitro model. The microencapsulates provided excellent protection of the enzyme during transit through gastric conditions, however, under pancreatin pressure during intestinal conditions, the Bsh enzyme was subject to proteolytic degradation. In contrast, L. plantarum was able to withstand both gastric and intestinal conditions, however, enzyme delivery levels are limited when compared to the capacity of microencapsulates. Finally, the influence of delivery of bile salt hydrolase activity by viable bacteria and whey protein/gum arabic microencapsulates on the host was investigated in vivo using a rat model. However, no effect of delivery of Bsh on the intestinal bile salt composition or mucin excretion was detected. These results may indicate that the physiological relevance and magnitude of bile salt hydrolase activity of probiotics in the small intestine is limited.