The role of fimbriae and flagella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants

To investigate the role of fimbriae and flagella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coil O78:K80 strain EC34195 defective in the elaboration of type-1 and curli fimbriae and flagella were constructed by allelic exchange, Single and multiple non-fimbriate and non-flagellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant, Mutant strains defective in the elaboration of type-1 fimbriae were significantly less adherent - in the order of 90% reduction - than the wild-type strain in all assays. Mutant strains defective in the elaboration of flagella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a significant reduction of adherence - of the order of 90% - compared with the wild-type strain was observed. Mutant strains defective for the elaboration of curb fimbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a significant reduction of adherence - of the order of 80% - compared with the wild-type strain was observed, Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants, Together, these data support the hypothesis that type-1 fimbriae are significant factors in adherence, aided by flagella for penetration of mucus and curli fimbriae for adherence to the gut.

Influence of Yersinia pseudotuberculosis outer proteins (Yops) on interleukin-12, tumor necrosis factor alpha and nitric oxide production by peritoneal macrophages

An essential key to pathogenicity in Yersinia is the presence of a 70 kb plasmid (pYV) which encodes a type-III secretion system and several virulence outer proteins whose main function is to enable the bacteria to survive in the host. Thus, a specific immune response is needed in which cytokines are engaged. The aim of this study was to assess the influence of Yersinia outer proteins (Yops) released by Yersinia pseudotuberculosis on the production of the proinflammatory cytokines, interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO) by murine peritoneal macrophages. To this end, female Swiss mice were infected intravenously with wild-type Y pseudotuberculosis or with mutant strains unable to secrete specific Yops (YopE, YopH, YopJ, YopM, and YpkA). on the 7th, 14th, 21st, and 28th days after infection, the animals were sacrificed and the cytokines and NO were assayed in the peritoneal macrophages culture supernatants. A fall in NO production was observed during the course of infection with all the strains tested, though during the infection with the strains that did not secrete YopE and YopH, the suppression occurred later. There was, in general, an unchanged or sometimes increased production of TNF-alpha between the 7th and the 21st day after infection, compared to the control group, followed by an abrupt decrease on the last day of infection. The IL-12 production was also suppressed during the infection, with most of the strains tested, except with those that did not secrete YopJ and YopE. The results suggest that Yops may suppress IL-12, TNF-alpha, and NO production and that the most important proteins involved in this suppression are YopE and YopH. (C) 2004 Elsevier B.V. All rights reserved.

THE PHO-2A MUTANT OF NEUROSPORA-CRASSA WHICH IS DEFICIENT IN PI-REPRESSIBLE ALKALINE-PHOSPHATASE (EC 3.1.3.1) IS ALSO DEFECTIVE IN PI-REPRESSIBLE ACID-PHOSPHATASE (EC 3.1.3.2)

1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively.2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54-degrees-C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54-degrees-C, pH 5.4).3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50-degrees-C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50-degrees-C, pH 5.4).4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54-degrees-C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min al 54-degrees-C, pH 5.4).5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.

Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Invasin of Yersinia pseudotuberculosis is not a polyclonal activator of mouse B lymphocytes

It is known that the invasin molecule of Yersinia pseudotuberculosis stimulates human peripheral B cells in vitro. In this work we evaluated the in vivo role of invasin as polyclonal activator of B lymphocytes in the mouse experimental model, by comparing strains of Y. pseudotuberculosis expressing invasin and isogenic inv mutants. Swiss mice were infected intravenously with two strains expressing invasin (YpIII pIB1 and an isogenic virulence plasmid-cured strain, YpIII) and with two invasin mutant strains (Yp100 pIB1 and Yp100, plasmid-cured). Spleen cells were sampled on days 7, 14, 21 and 28 after infection. Immunoglobulin (Ig)-secreting spleen cells were detected by protein A plaque assay and specific antibodies were detected in sera by ELISA. The virulent strain YPIII pIB1 (wild type) did not provoke polyclonal activation of B lymphocytes in vivo. In general, fewer Ig-secreting spleen cells of all isotypes were found in the infected animals than in the control animals. Specific IgG antibodies were detected in the sera of animals infected with all strains. The peak response occurred on the 21 st day post-infection, and the Yp100 strain provoked the highest level of these antibodies. We concluded that invasin is not a polyclonal activator of murine B cells.

Glycogen storage and muscle glucose transporters (GLUT-4) of mice selectively bred for high voluntary wheel running

To examine the evolution of endurance-exercise behaviour, we have selectively bred four replicate lines of laboratory mice (Mus domesticus) for high voluntary wheel running ('high runner' or HR lines), while also maintaining four non-selected control (C) lines. By generation 16, HR mice ran ∼2.7-fold more than C mice, mainly by running faster (especially in females), a differential maintained through subsequent generations, suggesting an evolutionary limit of unknown origin. We hypothesized that HR mice would have higher glycogen levels before nightly running, show greater depletion of those depots during their more intense wheel running, and have increased glycogen synthase activity and GLUT-4 protein in skeletal muscle. We sampled females from generation 35 at three times (photophase 07:00 h-19:00 h) during days 5-6 of wheel access, as in the routine selection protocol: Group 1, day 5, 16:00 h-17:30 h, wheels blocked from 13:00 h; Group 2, day 6, 02:00 h-03:30 h (immediately after peak running); and Group 3, day 6, 07:00 h-08:30 h. An additional Group 4, sampled 16:00 h-17:30 h, never had wheels. HR individuals with the mini-muscle phenotype (50% reduced hindlimb muscle mass) were distinguished for statistical analyses comparing C, HR normal, and HR mini. HR mini ran more than HR normal, and at higher speeds, which might explain why they have been favored by the selective-breeding protocol. Plasma glucose was higher in Group 1 than in Group 4, indicating a training effect (phenotypic plasticity). Without wheels, no differences in gastrocnemius GLUT-4 were observed. After 5 days with wheels, all mice showed elevated GLUT-4, but HR normal and mini were 2.5-fold higher than C. At all times and irrespective of wheel access, HR mini showed approximately three-fold higher [glycogen] in gastrocnemius and altered glycogen synthase activity. HR mini also showed elevated glycogen in soleus when sampled during peak running. All mice showed some glycogen depletion during nightly wheel running, in muscles and/or liver, but the magnitude of this depletion was not large and hence does not seem to be limiting to the evolution of even-higher wheel running.

Influência das proteínas Yops de Yersinia pseudotuberculosis na resposta imune humoral murina

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Papel das Yops de yersinia pseudotuberculosis na modulação da resposta imune celular durante infecção experimental

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor α chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn−/−) and lyn-deficient (Lyn−/−) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor α chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn−/−) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn−/− mice, and it was severely impaired in B cells from Lyn−/− and Fyn/Lyn−/− mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn−/− and Fyn/Lyn−/− mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Immunologic basis of transplant-associated arteriosclerosis.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.