Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress

In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.     

Study of simultaneous experimental colonization of Meriones unguiculatus (Mongolian gerbils) by cagPAI+ and cagPAI− strains of Helicobacter pylori

Helicobacter pylori has a chromosomal pathogenicity island (cagPAI), and the presence or absence of this Island places the microorganism into two types of strains: cagPAI+ which is associated to serious infectious processes, and cagPAI? related to mild to moderate infectious events. Simultaneous colonization by cagPAI+ and cagPAI? strains is frequent and these bacteria can interact among themselves.The aim of this project was to analyze the interaction between cagPAI+ and cagPAI? strains of H. pylori in experimental infection, using the Mongolian gerbil as an experimental animal model.We employed J99 (cagPAI+) and 251F (cagPAI?) strains, and obtained 3 derivate strains in successive isolation from experimentally infected gerbils. By RAPD–PCR we found that cagPAI+ and cagPAI? underwent genetic rearrangement during the gerbil-adaptation process. We identified individual isolates from gerbils, and by in situ hybridization we established that both type of strains were able to colonize the same regions of the host’s stomach, and induce a mild to moderate inflammatory process.We studied the competence between cagPAI+ and cagPAI? strains by simultaneous and sequential infections. The study shows that in both colonization experiments, the cagPAI? strains were more efficient than cagPAI+ strains in colonizing the infected host by displacing cagPAI+.     

Role of IL-13 in a model of Strongyloides venezuelensis infection in rats

IL-13 is a cytokine known to play a role in several pulmonary diseases, including asthma and fibrosis. The role of IL-13 in the context of pulmonary changes induced by helminth infection is unclear. Rats experimentally infected with Strongyloides venezuelensis and treated with anti-IL-13 neutralizing antibody were used to evaluate the role of IL-13 on functional and inflammatory changes of host lungs, and on parasite control. S. venezuelensis-induced airway hyperreactivity was IL-13-independent, but IL-13 played an essential role in driving airway mucus production and eosinophil infiltration. IL-13 was important for the control of egg production but not establishment in the intestine.     

Asymmetric microtubule pushing forces in nuclear centering

Dynamic properties of microtubules contribute to the establishment of spatial order within cells. In the fission yeast Schizosaccharomyces pombe, interphase cytoplasmic microtubules are organized into antiparallel bundles that attach to the nuclear envelope and are needed to position the nucleus at the geometric center of the cell. Here, we show that after the nucleus is displaced by cell centrifugation, these microtubule bundles efficiently push the nucleus back to the center. Asymmetry in microtubule number, length, and dynamics contributes to the generation of force responsible for this unidirectional movement. Notably, microtubules facing the distal cell tip are destabilized when the microtubules in the same bundle are pushing from the proximal cell tip. The CLIP-170-like protein tip1p and the microtubule-bundling protein ase1p are required for this asymmetric regulation of microtubule dynamics, indicating contributions of factors both at microtubule plus ends and within the microtubule bundle. Mutants in these factors are defective in nuclear movement. Thus, cells possess an efficient microtubule-based engine that produces and senses forces for centering the nucleus. These studies may provide insights into mechanisms of asymmetric microtubule behaviors and force sensing in other processes such as chromosome segregation and cell polarization.     

A quantitative approach to the experimental transmission success of Echinostoma friedi (Trematoda: Echinostomatidae) in rats

Using a range of parameters, the ability of rats (Rattus norvegicus) to successfully transmit Echinostoma friedi to the next host was examined under experimental conditions. The concept of Experimental Transmission Success (TM), defined as the number of hosts that become successfully infected after exposure to a number of infective stages produced by a previous host per unit of inoculation at which this latter host was exposed, was introduced. Using data for the egg output and miracidium hatching and infectivity, the TM permits us to estimate the ability of a particular definitive host species to successfully transmit a parasite species. This concept may be also useful to compare the transmission fitness of a parasite in different definitive host species. Moreover, variations of the Experimental Transmission Success over the course of the infection were calculated by the use of the Weekly Experimental Transmission Success (TMW). Overall, considering the complete duration of the experiment, the TM of E. friedi using rats as definitive hosts was 0.68 infected snails/metacercaria. However, positive values of the TMW were only obtained from 2 to 4 wk post-infection, with a maximum during the third wk post-infection. When comparing the TM values of E. friedi in rats with those calculated in hamsters on the basis of previously published data, E. friedi appears to be more appropriate to move through this portion of its life cycle when using hamsters (Mesocricetus auratus) as the final host than rats.     

Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).     

Role of GP82 in the Selective Binding to Gastric Mucin during Oral Infection with Trypanosoma cruzi

Oral infection by Trypanosoma cruzi has been the primary cause of recent outbreaks of acute Chagas'' diseases. This route of infection may involve selective binding of the metacyclic trypomastigote surface molecule gp82 to gastric mucin as a first step towards invasion of the gastric mucosal epithelium and subsequent systemic infection. Here we addressed that question by performing in vitro and in vivo experiments. A recombinant protein containing the complete gp82 sequence (J18), a construct lacking the gp82 central domain (J18*), and 20-mer synthetic peptides based on the gp82 central domain, were used for gastric mucin binding and HeLa cell invasion assays, or for in vivo experiments. Metacyclic trypomastigotes and J18 bound to gastric mucin whereas J18* failed to bind. Parasite or J18 binding to submaxillary mucin was negligible. HeLa cell invasion by metacyclic forms was not affected by gastric mucin but was inhibited in the presence of submaxillary mucin. Of peptides tested for inhibition of J18 binding to gastric mucin, the inhibitory peptide p7 markedly reduced parasite invasion of HeLa cells in the presence of gastric mucin. Peptide p7*, with the same composition as p7 but with a scrambled sequence, had no effect. Mice fed with peptide p7 before oral infection with metacyclic forms developed lower parasitemias than mice fed with peptide p7*. Our results indicate that selective binding of gp82 to gastric mucin may direct T. cruzi metacyclic trypomastigotes to stomach mucosal epithelium in oral infection.     

Increased α-Defensins 1-3 Production by Dendritic Cells in HIV-Infected Individuals Is Associated with Slower Disease Progression

Background

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

Methodology/Principal Findings

Monocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated “elite” and “viremic” controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of α-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted α-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher α-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm³, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.

Conclusions/Significance

High production of α-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of α-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.     

cis-Urocanic acid attenuates acute dextran sodium sulphate-induced intestinal inflammation

On exposure to sunlight, urocanic acid (UCA) in the skin is converted from trans to the cis form and distributed systemically where it confers systemic immunosuppression. The aim of this study was to determine if administration of cis-UCA would be effective in attenuating colitis and the possible role of IL-10. Colitis was induced in 129/SvEv mice by administering 5% dextran sodium sulfate (DSS) for 7 days in drinking water. During this period mice received daily subcutaneously injections of cis-UCA or vehicle. To examine a role for IL-10, 129/SvEv IL-10(-/-) mice were injected for 24 days with cis-UCA or vehicle. Clinical disease was assessed by measurement of body weight, stool consistency, and presence of blood. At sacrifice, colonic tissue was collected for histology and measurement of myeloperoxidase and cytokines. Splenocytes were analyzed for CD4+CD25+FoxP3+ T-regulatory cells via flow cytometry. Murine bone-marrow derived antigen-presenting cells were treated with lipopolysaccharide (LPS) ± UCA and cytokine secretion measured. Our results demonstrated that cis-UCA at a dose of 50 μg was effective in ameliorating DSS-induced colitis as evidenced by reduced weight loss and attenuated changes in colon weight/length. This protection was associated with reduced colonic expression of CXCL1, an increased expression of IL-17A and a significant preservation of splenic CD4+CD25+FoxP3+ T-regulatory cells. cis-UCA decreased LPS induced CXCL1, but not TNFα secretion, from antigen-presenting cells in vitro. UCA reduced colonic levels of IFNγ in IL-10(-/-) mice but did not attenuate colitis. In conclusion, this study demonstrates that cis-urocanic acid is effective in reducing the severity of colitis in a chemically-induced mouse model, indicating that pathways induced by ultraviolet radiation to the skin can influence distal sites of inflammation. This provides further evidence for a possible role for sunlight exposure in modulating inflammatory disorders.