Assessment of infant outgrowth of cow's milk allergy in relation to the faecal microbiome and metaproteome.

Previous studies provide evidence for an association between modifications of the gut microbiota in early life and the development of food allergies. We studied the faecal microbiota composition (16S rRNA gene amplicon sequencing) and faecal microbiome functionality (metaproteomics) in a cohort of 40 infants diagnosed with cow's milk allergy (CMA) when entering the study. Some of the infants showed outgrowth of CMA after 12 months, while others did not. Faecal microbiota composition of infants was analysed directly after CMA diagnosis (baseline) as well as 6 and 12 months after entering the study. The aim was to gain insight on gut microbiome parameters in relation to outgrowth of CMA. The results of this study show that microbiome differences related to outgrowth of CMA can be mainly identified at the taxonomic level of the 16S rRNA gene, and to a lesser extent at the protein-based microbial taxonomy and functional protein level. At the 16S rRNA gene level outgrowth of CMA is characterized by lower relative abundance of Lachnospiraceae at baseline and lower Bacteroidaceae at visit 12 months.

Improved annotation of conjugated bile acid hydrolase superfamily members in Gram-positive bacteria.

Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases (EC 3.5.1.24) is of importance, especially for lactobacilli, which are considered to play a major role in bile salt hydrolysis in vivo. In the present study, all enzymes listed in public databases that belong to the Bsh family and the closely related penicillin V acylase (Pva; EC 3.5.1.11) family were compared with the sequences annotated as Bsh in Lactobacillus plantarum WCFS1, as an example. In Gram-positive bacteria, a clear distinction was made between the two families using sequence alignment, phylogenetic clustering, and protein homology modelling. Biochemical and structural data on experimentally verified Bsh and Pva enzymes were used for validation of function prediction. Hidden Markov models were constructed from the sequence alignments to enable a more accurate prediction of Bsh-encoding genes, and their distinction from those encoding members of the Pva family. Many Pva-related sequences appeared to be annotated incorrectly as Bsh in public databases. This refinement in the annotation of Bsh family members influences the prediction of the function of bsh-like genes in species of the genus Lactobacillus, and it is discussed in detail.

Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1.

Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.

Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum.

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.