Insights into Klebsiella pneumoniae type VI secretion system transcriptional regulation

Submitted by Sandra Infurna ([email protected]) on 2019-09-05T13:49:14Z No. of bitstreams: 1 LeticiaSLevy_VictorBarbosa_IOC_2019.pdf: 4019376 bytes, checksum: c24447aa8118e406749803256e556945 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-09-05T13:54:46Z (GMT) No. of bitstreams: 1 LeticiaSLevy_VictorBarbosa_IOC_2019.pdf: 4019376 bytes, checksum: c24447aa8118e406749803256e556945 (MD5)Made available in DSpace on 2019-09-05T13:54:46Z (GMT). No. of bitstreams: 1 LeticiaSLevy_VictorBarbosa_IOC_2019.pdf: 4019376 bytes, checksum: c24447aa8118e406749803256e556945 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ. Brasil.Klebsiella pneumoniae (KP) is an opportunistic pathogen that mainly causes respiratory and urinary tract infections. The frequent occurrence of simultaneously virulent and multiple drug-resistant isolates led WHO to include this species in the list of top priorities for research and development of therapeutic alternatives. The comprehensive knowledge of the molecular mechanisms underlying KP virulence may lead to the proposal of more efficient and specific drugs. One of its virulence factors is the Type VI Secretion System (T6SS), which contributes to bacterial competition, cell invasion and in vivo colonisation. Despite the few studies showing the involvement of T6SS in KP pathogenesis, little is known concerning the regulation of its expression. The understanding of regulatory mechanisms may give more clues about the function of the system and the possibilities of future interference in this process. This work aimed to standardise the annotation of T6SS genes in KP strains and identify mechanisms of their transcriptional regulation through computational predictions

Intracellular Mycobacterium leprae Utilizes Host Glucose as a Carbon Source in Schwann Cells

New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia

Antibody Repertoires Identify β-Tubulin as a Host Protective Parasite Antigen in Mice Infected With Trypanosoma cruzi

Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease

The essential role of cholesterol metabolism in the intracellular survival of Mycobacterium 2 leprae is not coupled to central carbon metabolism and energy production

Submitted by sandra infurna ([email protected]) on 2016-05-17T16:02:04Z No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-05-17T16:50:23Z (GMT) No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5)Made available in DSpace on 2016-05-17T16:50:23Z (GMT). No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil / Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz.de Microbiologia Celular. Rio de Janeiro, RJ, Brasil Instituto Oswaldo Cruz. LaboratórioFundação Oswaldo Cruz.de Microbiologia Celular. Rio de Janeiro, RJ, Brasil Instituto Oswaldo Cruz. LaboratórioColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Laboratório de Bioquímica de Lipídeos e Lipoproteínas. Rio de Janeiro, RJ, Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies

Image_1.tif

<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p

Image_2.tif

<p>Few studies investigate the major protein antigens targeted by the antibody diversity of infected mice with Trypanosoma cruzi. To detect global IgG antibody specificities, sera from infected mice were immunoblotted against whole T. cruzi extracts. By proteomic analysis, we were able to identify the most immunogenic T. cruzi proteins. We identified three major antigens as pyruvate phosphate dikinase, Hsp-85, and β-tubulin. The major protein band recognized by host IgG was T. cruzi β-tubulin. The T. cruzi β-tubulin gene was cloned, expressed in E. coli, and recombinant T. cruzi β-tubulin was obtained. Infection increased IgG reactivity against recombinant T. cruzi β-tubulin. A single immunization of mice with recombinant T. cruzi β-tubulin increased specific IgG reactivity and induced protection against T. cruzi infection. These results indicate that repertoire analysis is a valid approach to identify antigens for vaccines against Chagas disease.</p

Complete genome sequence of the sugarcane nitrogen-fixing endophyte gluconacetobacter diazotrophicus PAL5

17 p. : il.Background: Gluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins. Results: Gluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat. Conclusion: The genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications