Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.Instituto de BiotecnologíaFil: Carneiro, Paulo A. M. Michigan State University. Center for Comparative Epidemiology; Estados UnidosFil: Pasquatti, Taynara N. Dom Bosco Catholic University; BrasilFil: Takatani, Haruo. Agencia de Defesa Agropecuaria do Amazonas; BrasilFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Marfil, Maria Jimena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Barnard, Christian. Agencia de Defesa Agropecuaria do Amazonas; BrasilFil: Fitzgerald, Scott D. Michigan State University. Veterinary Diagnostic Laboratory; Estados UnidosFil: Abramovitch, Robert B. Michigan State University. Department of Microbiology and Molecular Genetics; Estados UnidosFil: Araujo, Flabio Ribeiro de. Centro Nacional de Pesquisa de Gado de Corte; BrasilFil: Kaneene, John B. Michigan State University. Center for Comparative Epidemiology; Estados Unido

Mycobacterium tuberculosis Responds to Chloride and pH as Synergistic Cues to the Immune Status of its Host Cell

PubMed ID: 23592993This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Genomewide identification of \u3ci\u3ePseudomonas syringae\u3c/i\u3e pv.\u3ci\u3etomato\u3c/i\u3e DC3000 promoters controlled by the HrpL alternative sigma factor

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpLresponsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulenceimplicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpLdependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), N-(indole-3-acetyl)-L-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

Diverse AvrPtoB Homologs from Several Pseudomonas syringae Pathovars Elicit Pto-Dependent Resistance and Have Similar Virulence Activities

AvrPtoB is a type III effector protein from Pseudomonas syringae pv. tomato that physically interacts with the tomato Pto kinase and, depending on the host genotype, either elicits or suppresses programmed cell death associated with plant immunity. We reported previously that avrPtoB-related sequences are present in diverse gram-negative phytopathogenic bacteria. Here we describe characterization of avrPtoB homologs from P. syringae pv. tomato T1, PT23, and JL1065, P. syringae pv. syringae B728a, and P. syringae pv. maculicola ES4326. The avrPtoB homolog from P. syringae pv. maculicola, hopPmaL, was identified previously. The four new genes identified in this study are designated avrPtoB(T1), avrPtoB(PT23), avrPtoB(JL1065), and avrPtoB(B728a). The AvrPtoB homologs exhibit 52 to 66% amino acid identity with AvrPtoB. Transcripts of each of the avrPtoB homologs were detected in the Pseudomonas strains from which they were isolated. Proteins encoded by the homologs were detected in all strains except P. syringae pv. tomato T1, suggesting that T1 suppresses accumulation of AvrPtoB(T1). All of the homologs interacted with the Pto kinase in a yeast two-hybrid system and elicited a Pto-dependent defense response when they were delivered into leaf cells by DC3000ΔavrPtoΔavrPtoB, a P. syringae pv. tomato strain with a deletion of both avrPto and avrPtoB. Like AvrPtoB, all of the homologs enhanced the ability of DC3000ΔavrPtoΔavrPtoB to form lesions on leaves of two susceptible tomato lines. With the exception of HopPmaL which lacks the C-terminal domain, all AvrPtoB homologs suppressed programmed cell death elicited by the AvrPto-Pto interaction in an Agrobacterium-mediated transient assay. Thus, despite their divergent sequences, AvrPtoB homologs from diverse P. syringae pathovars have conserved avirulence and virulence activities similar to AvrPtoB activity

Immune Activation Of The Host Cell Induces Drug Tolerance In Mycobacterium Tuberculosis Both In Vitro And In Vivo

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb\u27s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs\u27 primary targets. Mtb\u27s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb\u27s response to drug pressure and suggest novel routes for future drug discovery programs

Multiple-stage Precursor Ion Separation and High Resolution Mass Spectrometry toward Structural Characterization of 2,3-Diacyltrehalose Family from Mycobacterium tuberculosis

Mass spectrometry (MS)-based precursor ion isolation, collision-induced dissociation (CID) fragmentation, and detection using linear ion-trap multiple-stage mass spectrometry (LIT MSn) in combination with high resolution mass spectrometry (HRMS) provides a unique tool for structural characterization of complex mixture without chromatographic separation. This approach permits not only separation of various lipid families and their subfamilies, but also stereoisomers, thereby, revealing the structural details. In this report, we describe the LIT MSn approach to unveil the structures of a 2,3-diacyl trehalose (DAT) family isolated from the cell envelope of Mycobacterium tuberculosis, in which more than 30 molecular species, and each species consisting of up to six isomeric structures were found. LIT MSn performed on both [M + Na]+ and [M + HCO2]− ions of DAT yield complimentary structural information for near complete characterization of the molecules, including the location of the fatty acyl substituents on the trehalose backbone. This latter information is based on the findings of the differential losses of the two fatty acyl chains in the MS2 and MS3 spectra; while the product ion spectra from higher stage LIT MSn permit confirmation of the structural assignment