Defective Innate Cell Response and Lymph Node Infiltration Specify Yersinia pestis Infection

Since its recent emergence from the enteropathogen Yersinia pseudotuberculosis, Y. pestis, the plague agent, has acquired an intradermal (id) route of entry and an extreme virulence. To identify pathophysiological events associated with the Y. pestis high degree of pathogenicity, we compared disease progression and evolution in mice after id inoculation of the two Yersinia species. Mortality studies showed that the id portal was not in itself sufficient to provide Y. pseudotuberculosis with the high virulence power of its descendant. Surprisingly, Y. pseudotuberculosis multiplied even more efficiently than Y. pestis in the dermis, and generated comparable histological lesions. Likewise, Y. pseudotuberculosis translocated to the draining lymph node (DLN) and similar numbers of the two bacterial species were found at 24 h post infection (pi) in this organ. However, on day 2 pi, bacterial loads were higher in Y. pestis-infected than in Y. pseudotuberculosis-infected DLNs. Clustering and multiple correspondence analyses showed that the DLN pathologies induced by the two species were statistically significantly different and identified the most discriminating elementary lesions. Y. pseudotuberculosis infection was accompanied by abscess-type polymorphonuclear cell infiltrates containing the infection, while Y. pestis-infected DLNs exhibited an altered tissue density and a vascular congestion, and were typified by an invasion of the tissue by free floating bacteria. Therefore, Y. pestis exceptional virulence is not due to its recently acquired portal of entry into the host, but is associated with a distinct ability to massively infiltrate the DLN, without inducing in this organ an organized polymorphonuclear cell reaction. These results shed light on pathophysiological processes that draw the line between a virulent and a hypervirulent pathogen

The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue

International audienceIntestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and-3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis

H. pylori-induced promoter hypermethylation downregulates USF1 and USF2 transcription factor gene expression

International audienceHelicobacter pylori infection is associated with the development of gastric adenocarcinoma. Upstream stimulatory factors USF1 and USF2 regulate the transcription of genes related to immune response, cell cycle and cell proliferation. A decrease in their expression is observed in human gastric epithelial cells infected with H. pylori, associated to a lower binding to their DNA E-box recognition site as shown by electrophoretic mobility shift assay. DNA methylation leads to gene silencing. The treatment of cells with 5′-azacytidine, an inhibitor of DNA methylation, restored the USF1 and USF2 gene expression in the presence of infection. Using promoter PCR methylation assay, a DNA hypermethylation was shown in the promoter region of USF1 and USF2 genes, in infected cells. The inhibition of USF1 and USF2 expression by H. pylori and the DNA hypermethylation in their gene promoter region was confirmed in gastric tissues isolated from 12 to 18 months infected mice. Our study demonstrated the involvement of USF1 and USF2 as molecular targets of H. pylori and the key role of DNA methylation in their regulation. These mechanisms occurred in the context of metaplastic lesions, suggesting that alteration of USF1 and USF2 levels could participate in the promotion of neoplastic process during H. pylori infection

Aspergillus fumigatus Does Not Require Fatty Acid Metabolism via Isocitrate Lyase for Development of Invasive Aspergillosis

Aspergillus fumigatus is the most prevalent airborne filamentous fungus causing invasive aspergillosis in immunocompromised individuals. Only a limited number of determinants directly associated with virulence are known, and the metabolic requirements of the fungus to grow inside a host have not yet been investigated. Previous studies on pathogenic microorganisms, i.e., the bacterium Mycobacterium tuberculosis and the yeast Candida albicans, have revealed an essential role for isocitrate lyase in pathogenicity. In this study, we generated an isocitrate lyase deletion strain to test whether this strain shows attenuation in virulence. Results have revealed that isocitrate lyase from A. fumigatus is not required for the development of invasive aspergillosis. In a murine model of invasive aspergillosis, the wild-type strain, an isocitrate lyase deletion strain, and a complemented mutant strain were similarly effective in killing mice. Moreover, thin sections demonstrated invasive growth of all strains. Additionally, thin sections of lung tissue from patients with invasive aspergillosis stained with anti-isocitrate lyase antibodies remained negative. From these results, we cannot exclude the use of lipids or fatty acids as a carbon source for A. fumigatus during invasive growth. Nevertheless, test results do imply that the glyoxylate cycle from A. fumigatus is not required for the anaplerotic synthesis of oxaloacetate under infectious conditions. Therefore, an antifungal drug inhibiting fungal isocitrate lyases, postulated to act against Candida infections, is assumed to be ineffective against A. fumigatus

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

International audienceActivation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host

Illustrations of some of the criteria used for scoring.

<p>Panels A–E, G, H and J–L display HE stained sections, panels F and I show sections immunostained with <i>Y. pestis</i> specific antibodies. In the text below, the criteria are referred to according to the numbering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001688#pone-0001688-t001" target="_blank">Table 1</a>. A: Wedge shaped abscess (criterion 4) indicated by arrows. Within the abscess bacterial colonies are visible as pink patches. B: Peripheral layer of PMNs containing bacterial foci (criterion 9), with a blunt demarcation (arrows) from the lymph node tissue (criterion 11). Inset: higher magnification to show the characteristic horseshoe shaped nuclei of the PMNs, and a bacterial focus. C: Layer of PMNs bordering a peripheral band of bacteria, cell debris and PMNs (criterion 10). The inset shows the typical PMN morphology of the cells within the layer. Behind this layer, bacterial aggregates are seen as pink areas (arrowheads) containing purple dots that are, as seen as higher magnification (not shown here) PMNs and cell debris. D: Patch of densely packed bacteria, bordered by PMNs (criterion14). Packed bacteria form a pink 8-shaped area at the centre of the picture (star). E: Atypical bacterial patch (criterion 15). At the center of the picture an aggregate of bacterial rods, not as densely packed as the preceding one, is loosely surrounded by inflammatory cells. F: A large bacterial zone (criterion 17), stained brownish on the preparation. G: Isolated host cells within a bacterial zone (criterion 19). Isolated host cells and cell remnants are seen amid a sea of bacteria which gives a “ground glass” appearance to this part of the LN section. H, <i>left</i>: bacterial infiltration around host cells (criterion 20). A bacterial strand (arrow), that seems to originate from a nearby colony (star), passes between host cells. H, <i>right</i>: Free floating bacterium, indicated by an arrow (criterion 21). I: Zone of reduced tissular density (arrows) outside bacterial areas, which are brownish on this preparation (criterion 26). This image also shows flame-like inward bacterial projections (criterion 18). J: Area of reduced host cell density with a reticular pattern (criterion 27) and containing numerous pycnotic cells (criterion 32). K: Moth eaten appearance (criterion 33) of a lymph node with areas of contrasting tissular densities. L: Vascular congestion (criterion 40), showing bright red on this preparation. <i>Magnifications</i>: Panels A–C, F, I–L : bar = 200 µm. Insets of panels B and C: bar = 50 µm. Panels D, E, G and H: bar = 10 µm.</p

Bacterial loads at injection site and in draining lymph node.

<p>Data are means of log₁₀ of cfu numbers recovered from 77 mice infected over six independent experiments. Bars = Standard Errors. Stars indicate that the data are significantly different between the two <i>Yersinia</i> species (p≤0.0001). The dashed line shows the detection limit (10 cfu). The cross symbol indicates that most <i>Y. pestis</i>-infected mice died between days 2 and 3.</p

Criteria used to score sections of lymph nodes infected with <i>Y. pestis</i> or <i>Y. pseudotuberculosis</i>, and their discriminating indexes.

<p>Discriminating indexes are the test values in the right column. Only significant test values (i.e. >1.96 or <−1.96) are given. Positive and negative test-values are distinctive of, respectively, <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i> infections.</p

Deficiency in OGG1 Protects against Inflammation and Mutagenic Effects Associated with H. pylori Infection in Mouse

International audienceBackground: Helicobacter pylori infection is associated with gastric cancer. Study with the Big Blue mouse model has reported a mutagenic effect associated with the H. pylori infection, as a result in part of oxidative DNA damage. The present work investigates the consequences of a deficiency in the OGG1 DNA glycosylase, responsible for the excision of 8-oxo guanine, on the inflammatory and genotoxic host response to the infection.Materials and Methods: Big Blue Ogg1–/– C57BL/6 mice were orally inoculated with H. pylori strain SS1 or vehicle only, and sacrificed after 1, 3, or 6 months. The serologic response, histologic lesions, mutant frequency, and spectra of mutations were assessed in the stomach and compared to what observed in the wild-type (Wt) context.Results: Inflammatory lesions induced in the gastric mucosa of H. pylori-infected mice, corresponding to a moderate gastritis, were less severe in Ogg1–/– than in Wt Big Blue mice. Analysis of antimicrobial humoral immunity exhibited a lower IgG2a serum level (Th1 response) after 6 months of infection in Ogg1–/– than in the Wt mice. In these conditions, the H. pylori-SS1 infection in the Ogg1–/– mice did not induce a mutagenic effect at the gastric epithelial cells level, either after 3 or 6 months.Conclusions: The inactivation of the OGG1 DNA glycosylase in mouse leads to less severe inflammatory lesions and abolished the mutagenic effect at the gastric epithelial cells level, induced by the H. pylori infection. These data suggest for the OGG1deficiency a protective role against inflammation and genotoxicity associated to the H. pylori infection