Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

Temperature and oxygen dependent metabolite utilization by Salmonella enterica Serovars Derby and Mbandaka

Salmonella enterica is a zoonotic pathogen of clinical and veterinary significance, with over 2500 serovars. In previous work we compared two serovars displaying host associations inferred from isolation statistics. Here, to validate genome sequence data and to expand on the role of environmental metabolite constitution in host range determination we use a phenotypic microarray approach to assess the ability of these serovars to metabolise ~500 substrates at 25°C with oxygen (aerobic conditions) to represent the ex vivo environment and at 37°C with and without oxygen (aerobic/anaerobic conditions) to represent the in vivo environment. A total of 26 substrates elicited a significant difference in the rate of metabolism of which only one, D-galactonic acid-g-lactone, could be explained by the presence (S. Mbandaka) or the absence (S. Derby) of metabolic genes. We find that S. Mbandaka respires more efficiently at ambient temperatures and under aerobic conditions on 18 substrates including: glucosominic acid, saccharic acid, trehalose, fumaric acid, maltotriose, N-acetyl-D-glucosamine, N-acetyl-beta-D-mannosamine, fucose, L-serine and dihydroxy-acetone; whereas S. Derby is more metabolically competent anaerobically at 37°C for dipeptides, glutamine-glutamine, alanine-lysine, asparagine-glutamine and nitrogen sources glycine and nitrite. We conclude that the specific phenotype cannot be reliably predicted from the presence of metabolic genes directly relating to the metabolic pathways under study

Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses

Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs) are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs) from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation). In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life

Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model.

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments

Histone deacetylase inhibitor, butyrate, attenuates lipopolysaccharide-induced acute lung injury in mice

<p>Abstract</p> <p>Background</p> <p>Histone deacetylase (HDAC) inhibitors, developed as promising anti-tumor drugs, exhibit their anti-inflammatory properties due to their effects on reduction of inflammatory cytokines.</p> <p>Objective</p> <p>To investigate the protective effect of butyrate, a HDAC inhibitor, on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice.</p> <p>Methods</p> <p>ALI was induced in Balb/c mice by intratracheally instillation of LPS (1 mg/kg). Before 1 hour of LPS administration, the mice received butyrate (10 mg/kg) orally. The animals in each group were sacrificed at different time point after LPS administration. Pulmonary histological changes were evaluated by hematoxylin-eosin stain and lung wet/dry weight ratios were observed. Concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) and concentrations of nitric oxide (NO) and myeloperoxidase (MPO) activity in lung tissue homogenates were measured by enzyme-linked immunosorbent assay (ELISA). Expression of nuclear factor (NF)-κB p65 in cytoplasm and nucleus was determined by Western blot analysis respectively.</p> <p>Results</p> <p>Pretreatment with butyrate led to significant attenuation of LPS induced evident lung histopathological changes, alveolar hemorrhage, and neutrophils infiltration with evidence of reduced MPO activity. The lung wet/dry weight ratios, as an index of lung edema, were reduced by butyrate administration. Butyrate also repressed the production of TNF-α, IL-1β and NO. Furthermore, the expression of NF-κB p65 in nucleus was markedly suppressed by butyrate pretreatment.</p> <p>Conclusions</p> <p>Butyrate had a protective effect on LPS-induced ALI, which may be related to its effect on suppression of inflammatory cytokines production and NF-κB activation.</p

Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3β, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells

Sensing and Adaptation to Low pH Mediated by Inducible Amino Acid Decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection

Sleeping with the Enemy: How Intracellular Pathogens Cope with a Macrophage Lifestyle

Sleeping with the Enemy: How Intracellular Pathogens Cope with a Macrophage Lifestyl