No abstract

Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.

The low-calcium response (lcr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37 degrees C in Ca(2+)-depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence-plasmid-encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature-regulated activator and by Ca(2+)-regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface-located when Yersinia is grown at 37 degrees C. yopN was found to be part of an operon that is induced during the low-calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild-type phenotype of the yopN mutant. Thus the surface-located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 Å resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel ninecoiled left-handed parallel b-roll. Before the b-roll, the polypeptide loops from one monomer to the rest, and after the b-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA b-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.

One of the most virulent and feared bacterial pathogens is Yersinia pestis, the aetiologic agent of bubonic plague. Characterization of the O‐antigen gene clusters of 21 serotypes of Yersinia pseudotuberculosis and the cryptic O‐antigen gene cluster of Y. pestis showed that the plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype O:1b. The nucleotide sequences of both gene clusters (about 20.5 kb each) were determined and compared to identify the differences that caused the silencing of the Y. pestis gene cluster. At the nucleotide sequence level, the loci were 98.9% identical and, of the 17 biosynthetic genes identified from the O:1b gene cluster, five were inactivated in the Y. pestis cluster, four by insertions or deletions of one nucleotide and one by a deletion of 62 nucleotides. Apparently, the expression of the O‐antigen is not beneficial for the virulence or to the lifestyle of Y. pestis and, therefore, as one step in the evolution of Y. pestis, the O‐antigen gene cluster was inactivated.

A chromosomally encoded protein, which mediates invasion into HeLa cells was recently identified in Yersinia pseudotuberculosis. The role of this protein (invasin) in the virulence process was not, however, investigated. We show that mutation of the invasin gene in Y. pseudotuberculosis abolishes the ability of the bacteria to invade HeLa cells. When mice were challenged by intraperitoneal injection both the mutant and the wild-type strain produced infections of similar virulence but mutant showed a slower rate of infection after oral challenge. A double mutant, carrying an additional mutation in the gene coding for the Yop1 protein, was also constructed. The double mutant was significantly more virulent than either the wild-type or the corresponding single mutants. Y. pestis, in contrast to Y. pseudotuberculosis lacks the ability to express either invasin or Yop1, sequence analysis of the yopA gene from both Y. pestis and Y. pseudotuberculosis shows that the yopA gene of Y. pestis contains a point-mutation leading to a reading-frame shift. When the yopA+ gene was introduced into Y. pestis the virulence of this strain was reduced. These results may provide insight into the rise and fall of plague epidemics caused by Y. pestis.

No abstract

The YadA surface protein of enteropathogenic Yersinia species contains two highly hydrophobic regions: one close to the amino terminal, and the other at the carboxy-terminal end of the YadA polypeptide. To study the role of these hydrophobic regions, we constructed 66 bp deletion mutants of the yadA genes of Yersinia enterocolitica serotype O:3 strain 6471/76 (YeO3) and of O:8 strain 8081 (YeO8). The mutant proteins, YadAYeO3-delta 83-104 and YadAYeO8-delta 8O-101, lacked 22 amino acids from the amino-terminal hydrophobic region, formed fibrillae and were expressed on the cell surface. Bacteria expressing the mutated protein lost their auto-agglutination potential as well as their collagen-binding property. Binding to fibronectin and laminin was affected differently in the YeO3 and the YeO8 constructs. The deletion did not influence YadA-mediated complement inhibition. Loss of the collagen-binding property was associated with loss of virulence in mice. We also constructed a number of YadAYeO3 deletion mutants lacking the hydrophobic carboxy-terminal end of the protein. Deletions ranging from 19 to 79 amino acids from the carboxy terminus affected polymerization of the YadA subunits, and also resulted in the loss of the YadA expression on the cell surface. This suggests that the carboxy terminus of YadA is involved in transport of the protein to the bacterial outer surface.