Treatment of Yersinia similis with the cationic lipid DOTAP enhances adhesion to and invasion into intestinal epithelial cells - A proof-of-principle study.

The monocationic quaternary surfactant DOTAP has been used for the delivery of nucleic acids and peptides into mammalian cells. This study tested the applicability of DOTAP for the enhancement of adhesion and invasion frequencies of Yersinia (Y.) similis to enable the analysis of the effects of low-pathogenic bacteria on intestinal epithelial cells. Incubation of Y. similis with DOTAP ahead of infection of C2BBe1 intestinal epithelial cells increased invasion and adhesion frequency four- and five-fold, respectively, in plating assays. Proteomic approaches confirmed the increased bacterial load on infected cells: analysis of protein extracts by two-dimensional difference gel electrophoresis (2D-DIGE) revealed higher amounts of bacterial proteins present in the cells infected with DOTAP-treated bacteria. MALDI-TOF mass spectrometry of selected spots from gel-separated protein extracts confirmed the presence of both bacterial and human cell proteins in the samples. Label-free quantitative proteomics analysis identified 1170 human cell proteins and 699 bacterial proteins. Three times more bacterial proteins (279 vs. 93) were detected in C2BBe1 cells infected with DOTAP-treated bacteria compared to infections with untreated bacteria. Infections with DOTAP-treated Y. similis led to a significant upregulation of the stress-inducible ubiquitin-conjugating enzyme UBE2M in C2BBe1 cells. This points towards a stronger impact of the stress and infection responsive transcription factor AP-1 by enhanced bacterial load. DOTAP-treatment of uninfected C2BBe1 cells led to a significant downregulation of the transmembrane trafficking protein TMED10. The application of DOTAP could be helpful for investigating the impact of otherwise low adherent or invasive bacteria on cultivated mammalian cells without utilisation of genetic modifications.

Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP).

The needle length of the Yersinia spp. injectisome is determined by Yop secretion protein P (YscP), an early substrate of the injectisome itself. There is a linear correlation between the length of YscP and the length of the needle, suggesting that YscP acts as a molecular ruler. However, it is not known whether one single molecule of YscP suffices to control the length of one needle or whether several molecules of YscP are exported in alternation with the needle subunit YscF until the needle length matches the ruler length, which would stop needle growth. To address this question, three different strains expressing simultaneously a short and a long version of YscP were engineered. The experimentally obtained needle length distribution was compared with the distributions predicted by stochastic modeling of the various possible scenarios. The experimental data are compatible with the single ruler model and not with the scenarios involving more than one ruler per needle.

A transition from strong right-handed to canonical left-handed supercoiling in a conserved coiled-coil segment of trimeric autotransporter adhesins.

Trimeric autotransporter adhesins (TAAs) represent an important class of pathogenicity factors in proteobacteria. Their defining feature is a conserved membrane anchor, which forms a 12-stranded beta-barrel through the outer membrane. The proteins are translocated through the pore of this barrel and, once export is complete, the pore is occluded by a three-stranded coiled coil with canonical heptad (7/2) sequence periodicity. In many TAAs this coiled coil is extended by a segment of varying length, which has pentadecad (15/4) periodicity. We used X-ray crystallography and biochemical methods to analyze the transition between these two periodicities in the coiled-coil stalk of the Yersinia adhesin YadA. Our results show how the strong right-handed supercoil of the 15/4-periodic part locally undergoes further over-winding to 19/5, before switching at a fairly constant rate over 14 residues to the canonical left-handed supercoil of the 7/2-periodic part. The transition region contains two YxD motifs, which are characteristic for right-handed coiled-coil segments of TAAs. This novel coiled-coil motif forms a defined network of inter- and intrahelical hydrogen bonds, thus serving as a structural determinant. Supercoil fluctuations have hitherto been described in coiled coils whose main sequence periodicity is disrupted locally by discontinuities. Here we present the first detailed analysis of two fundamentally different coiled-coil periodicities being accommodated in the same structure.

Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon.

Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca(2+)-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca(2+) levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca(2+), and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca(2+) levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.

Identification of a domain in Yersinia virulence factor YadA that is crucial for extracellular matrix-specific cell adhesion and uptake.

For many pathogens, cell adhesion factors are critical virulence determinants. Enteropathogenic Yersinia species express the afimbrial adhesin YadA, the prototype of a class of homotrimeric outer membrane adhesins, which mediates adherence to host cells by binding to extracellular matrix components. In this study, we demonstrate that different pathogenic functions are attributable to highly homologous YadA proteins. YadA of Yersinia pseudotuberculosis (YadA(pstb)) and Yersinia enterocolitica (YadA(ent)) exhibit fundamental differences in their specificity of extracellular matrix substrate binding, they cause dissimilar bacterial aggregation behaviors, and YadA(pstb), but not YadA(ent), promotes efficient uptake into human cells. Evidence is presented here that a unique N-terminal amino acid sequence of YadA(pstb), which is absent in YadA(ent), acts as an "uptake domain" by mediating tight binding to fibronectin bound on alpha(5)beta(1) integrin receptors, which are crucial for initiating the entry process. Deleting this motif in YadA(pstb) generated all features of the YadA(ent) protein, i.e., the molecule lost its adhesiveness to fibronectin and its invasiveness, but gained adhesion potential to collagen and laminin. Loss of the "uptake region" also attenuated host tissue colonization by Y. pseudotuberculosis during oral infections of mice, demonstrating that this motif plays a crucial role in defining pathogen-host cell interaction and pathogenesis. We conclude that even small variations in adhesion factors can provoke major differences in the virulence properties of related pathogens.

Detecçäo de uma substância semelhante a bacteriocina em Yersina spp / Detection of a bacteriocin-like substance in Yersina Spp

Vinte e duas amostras de Yersina spp foram testadas quanto à capacidade de produzir substância semelhante à bacteriocina pelo método do "spot-test" usando dupla camada de ágar.O resultado mostrou a existência de uma atividade isoinibitória somente em Y.intermedia à temperatura de 25ºC e näo a 37ºC, e contra Y.entericolitica sorotipo 0:8.

Long-term starvation survival of Yersinia ruckeri at different salinities studied by microscopical and flow cytometric methods.

Cultures of three strains of the fish pathogenic bacterium Yersinia ruckeri survived starvation in unsupplemented water for at least 4 months. At salinities of 0 to 20/1000 there were no detectable changes in CFU during the first 3 days of starvation and only a small decrease during the following 4 months, whereas at 35/1000 salinity, the survival potential of the cultures was markedly reduced. These results suggest that Y. ruckeri may survive for long periods in freshwater and brackish environments after an outbreak of enteric redmouth disease. Survival was also examined by use of the direct viable count method, and we show that this method can be combined with flow cytometry for automatic counting of viable bacteria. By flow cytometry, it was shown that genome replication initiated before the onset of starvation was completed, during the initial phase of starvation, and that starved cells could contain up to six genomes per cell.

[Effect of acidin-pepsin on Yersinia pseudotuberculosis]. / Deistvie atsidin-pepsina na Yersinia pseudotuberculosis.

The effect of acidin-pepsin solution at a concentration of 1 : 100 on newly isolated Yersinia cultures has been tested in three series of experiments. Acidin-pepsin has been found to exert a bactericidal effect on Yersinia and to induce the appearance of involutionary forms and large rod-shaped Yersinia cells with a better capacity for survival. The surviving Yersinia cells do not pass their resistance to acidin-pepsin on to their progeny and show low invasiveness and pathogenicity in white mice.

[Fermentative mechanisms of the psychrophilicity of Yersinia tuberculosis]. / Fermentativnye mekhanizmy psikhrofil'nosti psvedotuberkuleznogo mikroba.

The specific activity of urease, nitrogenase, hialuronidase and neuraminidase in Y. pseudotuberculosis grown in different culture media and at different temperature has been studied. These enzymes have been found capable of functioning at both relatively low (2-8 degrees C) and high (37 degrees C) temperatures. The thermoadaptive properties of Y. pseudotuberculosis within a wide range of temperatures are ensured by the constant presence of isoenzymes, functioning only at low temperatures or only at high temperatures, in the microbial cells. Low temperature in combination with a definite culture medium triggers the activity of certain enzymatic systems, which explains, to some extent, the biochemical mechanisms of the psychrophilic properties of Y. pseudotuberculosis.