Identifying Bacterial and Host Factors Involved in the Interaction of Mycobacterium bovis with the Bovine Innate Immune Cells

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.Fil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Gravisaco, María José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Bigi, María Mercedes. Universidad de Buenos Aires; ArgentinaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Marquez, Cecilia. No especifíca;Fil: McNeil, Mike. State University of Colorado - Fort Collins; Estados UnidosFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados UnidosFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin

Transcriptional response of bovine monocyte-derived macrophages after the infection with different argentinean mycobacterium bovis Isolates

Infection of bovines with Mycobacterium bovis causes important financial hardship in many countries presenting also a risk for humans. M. bovis is known to be adapted to survive and thrive within the intramacrophage environment. In spite of its relevance, at present the information about macrophage expression patterns is scarce, particularly regarding the bovine host. In this study, transcriptomic analysis was used to detect genes differentially expressed in macrophages derived from peripheral blood mononuclear cells at early stages of infection with two Argentinean strains of M. bovis, a virulent and an attenuated strains. The results showed that the number of differentially expressed genes in the cells infected with the virulent strain (5) was significantly lower than those in the cells infected with the attenuated strain (172). Several genes were more strongly expressed in infected macrophages. Among them, we detected encoding transcription factors, anthrax toxin receptor, cell division and apoptosis regulator, ankyrin proteins, cytoskeleton proteins, protein of cell differentiation, and regulators of endocytic traffic of membrane. Quantitative real-time PCR of a selected group of differentially expressed genes confirmed the microarrays results. Altogether, the present results contribute to understanding the mechanisms involved in the early interaction of M. bovis with the bovine macrophage.Fil: Caimi, Karina Cynthia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Soria, Marcelo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Mycobacterium bovis deleted in mce2 and phoP loci protects C57BL/6 mice against tuberculosis

Here we confirm the efficacy of MbΔmce2-phoP to protect mice against bTB challenge and we demonstrated that this new attenuated strain is safer than our previous bTB vaccine MbΔmce2. Altogether these results support the further assessment of this candidate in cattle model of bTB.Experiments with mice were performed in compliance with the regulations of the Institutional Animal Care and Use Committee (CICUAE) of INTA (61/2014)..Inst. de BiotecnologíaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Bianco, Marí­a Veronica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Good Protection but Excessive Pulmonary Inflammation in Balb/C Mice Vaccinated with Mycobacterium Bovis Mce-2A Mutant after Challenge with Homologous Strains

Tuberculosis (TB) remains a major threat to public and veterinary health. Zoonotic TB (caused by Mycobacterium bovis) is present in wild animals and cattle in most developing countries, and M. bovis is also able to infect humans on a worldwide basis. Thus, the high incidence of bovine TB is a major economic problem and an additional risk to human health, being the development of new vaccines to prevent both human and bovine TB urgent and a major challenge. The aims of the present study were to characterize the pathogenicity and immunogenicity of M. bovis mce2A mutant in BALB/c mice, and then evaluate its potential as vaccine. Mutant M. bovis mce2A produced limited tissue damage (pneumonia) and lower bacilli burdens than its parental strain when administered in high dose by intratracheal inoculation, and showed limited dissemination when used as subcutaneous vaccine. Challenge experiments using low, middle and highly virulent M. tuberculosis or M. bovis strains showed similar protection conferred by mce-2 mutant than BCG. Interestingly, vaccinated animals showed low bacilli loads but high inflammatory response when were challenged with M. bovis strains, while vaccinated mice challenged with M. tuberculosis exhibited low bacilli burdens and scarce inflammation. Thus, in spite of the high genome homology between M. tuberculosis and M. bovis, it seems that there is higher antigenic recognition and in consequence extensive inflammatory response when the strain used as vaccine is homologous to the challenge strain, in this case M. bovis.Fil: Alfonseca Silva, Edgar. Universidad Nacional Autónoma de México; MéxicoFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Hernández Pando, Rogelio. Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”; Méxic

Mycobacterium bovis requires P27 (LprG) to arrest phagosome maturation and replicate within bovine macrophages

Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host.Fil: Vázquez, Cristina Lourdes. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Bianco, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentin

Tuberculosis vaccine candidates based on mycobacterial cell envelope components

Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.Fil: Sarmiento, M.E.. Universiti Sains Malaysia; MalasiaFil: Alvarez, N.. Public Health Research Institute; Estados UnidosFil: Chin, K.L.. Universiti Sains Malaysia; MalasiaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Tirado, Y.. No especifíca;Fil: García, M.A.. No especifíca;Fil: Anis, F.Z.. Universiti Sains Malaysia; MalasiaFil: Norazmi, M.N.. Universiti Sains Malaysia; MalasiaFil: Acosta, A.. Universiti Sains Malaysia; Malasi

Rv2617c and P36 are virulence factors of pathogenic mycobacteria involved in resistance to oxidative stress

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.Instituto de BiotecnologíaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rocha, Rosana Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Villafañe, Luciana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, María Mercedes. Fil: Bigi, María Mercedes. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute, Host-Pathogen Interactions in Tuberculosis Laboratory; Reino UnidoFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

Identification of two proteins that interact with the Erp virulence factor from Mycobacterium tuberculosis by using the bacterial two-hybrid system

<p>Abstract</p> <p>Background</p> <p>The exported repetitive protein (<it>erp</it>) gene encodes a secreted 36-kDa protein with a central domain containing several proline-glycine-leucine-threonine-serine (PGLTS) repeats. It has been demonstrated that <it>erp </it>is a virulence-associated factor since the disruption of this gene impairs the growth of <it>Mycobacterium bovis </it>and <it>Mycobacterium tuberculosis </it>in mice.</p> <p>Results</p> <p>In order to elucidate the function of Erp we searched for Erp-binding proteins from <it>M. tuberculosis </it>by using a bacterial two-hybrid system. Our results indicate that Erp interacts specifically with two putative membrane proteins, Rv1417 and Rv2617c. Further analysis revealed that the latter two interact with each other, indicating that Rv1417, Rv2617c and Erp are connected through multiple interactions. While Rv1417 is disseminated in several <it>Actinomycetales </it>genera, orthologues of Rv2617c are exclusively present in members of the <it>M. tuberculosis </it>complex (MTC). The central and amino-terminal regions of Erp were determined to be involved in the interaction with Rv1417 and Rv2627c. Erp forms from <it>Mycobacterium smegmatis </it>and <it>Mycobacterium leprae </it>were not able to interact with Rv2617c in two-hybrid assays. Immunolocalization experiments showed that Rv1417 and Rv2617c are found on the cell membrane and Erp on the bacterial cell wall. Finally, comparative genomics and expression studies revealed a possible role of Rv1417 in riboflavin metabolism.</p> <p>Conclusion</p> <p>We identified interactive partners of Erp, an <it>M. tuberculosis </it>protein involved in virulence, which will be the focus of future investigation to decipher the function of the Erp family protein.</p

Study of the role of Mce3R on the transcription of mce genes of Mycobacterium tuberculosis

<p>Abstract</p> <p>Background</p> <p><it>mce3 </it>is one of the four virulence-related <it>mce </it>operons of <it>Mycobacterium tuberculosis</it>. In a previous work we showed that the overexpression of Mce3R in <it>Mycobacterium smegmatis </it>and <it>M. tuberculosis </it>abolishes the expression of <it>lacZ </it>fused to the <it>mce3 </it>promoter, indicating that Mce3R represses <it>mce3 </it>transcription.</p> <p>Results</p> <p>We obtained a knockout mutant strain of <it>M. tuberculosis </it>H37Rv by inserting a hygromycin cassette into the <it>mce3R </it>gene. The mutation results in a significant increase in the expression of <it>mce3 </it>genes either <it>in vitro </it>or in a murine cell macrophages line as it was determined using promoter-<it>lacZ </it>fusions in <it>M. tuberculosis</it>. The abundance of <it>mce1</it>, <it>mce2 </it>and <it>mce4 </it>mRNAs was not affected by this mutation as it was demonstrated by quantitative RT-PCR. The <it>mce3R </it>promoter activity in the presence of Mce3R was significantly reduced compared with that in the absence of the regulator, during the <it>in vitro </it>culture of <it>M. tuberculosis</it>.</p> <p>Conclusion</p> <p>Mce3R repress the transcription of <it>mce3 </it>operon and self regulates its own expression but does not affect the transcription of <it>mce1</it>, <it>mce2 </it>and <it>mce4 </it>operons of <it>M. tuberculosis</it>.</p

Assessment of tuberculosis biomarkers in paratuberculosis-infected cattle

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.Instituto de BiotecnologíaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Colombatti Olivieri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moyano, Roberto Damian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Moyano, Roberto Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Romano, Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malovrh, Tadej. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Ocepek, Matjaž. University of Ljubljana. Veterinary Faculty. Institute for Microbiology and Parasitology; EsloveniaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular (IABIMO); ArgentinaFil: Bigi, Fabiana. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin