TgaA, a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75

Self Cite

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.

Section: Resultsmentioning

confidence: 99%

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

Bottacini

Motherway

Casey

et al. 2015

Self Cite

“…The draft genome sequence of L. paracasei DG was obtained through Ion Torrent PGM (Life Technologies, Germany) as described previously (44). The raw sequence data were assembled using MIRA, version 3.9 (http://www.chevreux.org/projects_mira.html), applying the default parameters recommended for Ion Torrent data processing.…”

Section: Methodsmentioning

confidence: 99%

A Novel Rhamnose-Rich Hetero-exopolysaccharide Isolated from Lactobacillus paracasei DG Activates THP-1 Human Monocytic Cells

Balzaretti

Taverniti

Guglielmetti

et al. 2017

Self Cite

127

Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of L-rhamnose, D-galactose, and N-acetyl-D-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host. IMPORTANCEThe consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system. KEYWORDS

“…In fact, most known bacterial molecules governing bacterial adhesion to epithelia or interaction with immune cells are cell wall constituents (lipopolysaccharides, teichoic and lipoteichoic acids, murein, and proteinaceous adhesins) or cell wall appendages (flagella, pili, and fimbriae) (6,8,19,22). Interestingly, TgaA protein was found to be abundantly expressed and located on the outer surface of MIMBb75 bacterial cells (21). Furthermore, notably, a search of the Conserved Domain Database (30) for the TgaA protein produced a significant match (E value of 8.30e-23) between the CHAP module and the conserved domain COG3942, which has been annotated as a surface antigen.…”

Section: Resultsmentioning

confidence: 99%

“…(Milan, Italy). Vectors pET-Tga, pET-⌬CHAP (where CHAP is cysteine-and histidine-dependent amidohydrolase/peptidase) and pET-⌬LT (where LT is lytic murein transglycosylase) (prepared as described in the accompanying paper [21]) were used for the isopropyl-␤-D-thiogalactopyranoside (IPTG)-dependent overexpression in Escherichia coli of the recombinant proteins ⌬SP-TgaA-His (containing both domains of TgaA protein; also named here recombinant TgaA, or rTgaA, and SP PelB -TgaA in the accompanying paper [21]), protein ⌬SP-TgaA-⌬CHAP-His (containing only the LT domain; rLT), and ⌬SP-TgaA-⌬LT-His (containing only the CHAP domain; rCHAP), respectively. In brief, mutant E. coli strains were grown in 2ϫ YT (yeast extract, tryptone) broth at 37°C for 2 h before 1 mM IPTG was added, and incubation was prolonged for 4 h. Afterwards, proteins were extracted under denaturing conditions with PerfectPro Ni-nitrilotriacetic acid (NTA) agarose (5 Prime; Eppendorf Italia, s.r.l., Milan, Italy) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, it is critically important to study the immunological role of microbial molecular cell components to decipher the operating principles of the immune system. In this regard, our use of a reductionist molecular approach allowed us to define the immunological role of an intramolecular region belonging to an outer surface enzyme, TgaA, identified in an accompanying paper (21) by comparative genomic analysis in strain B. bifidum MIMBb75.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

Guglielmetti

Zanoni

Balzaretti

et al. 2014

Self Cite

c Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine-and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system.