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

Virulence factors of the mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. Introduction.Fil: Forrellad, Marina Andrea. 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: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Gioffré, Andrea Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Sabio y García, Julia Verónica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Escuela de Ciencias Médicas. Cátedra de Microbiología, Parasitología y Virología; ArgentinaFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Cataldi, Ángel Adrián. 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: 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

Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the causative agent of human tuberculosis, has developed several strategies involving proteins and other compounds known collectively as virulence factors to subvert human host defences and invade the human host. The Mce proteins are among these virulence-related proteins and are encoded by the mce1, mce2, mce3 and mce4 operons in the genome of M. tuberculosis. It has been proposed that these operons encode ABC-like lipid transporters; however, the nature of their substrates has only been revealed in the case of the Mce4 proteins. Here we found that the knockout of the mce1 operon alters the lipid profile of M. tuberculosis H37Rv and the uptake of palmitic acid. Thin layer chromatography and liquid chromatography-mass spectrometry analysis showed that the mce1 mutant accumulates more mycolic acids than the wild type and complemented strains. Interestingly, this accumulation of mycolic acid is exacerbate when bacteria are cultured in the presence of palmitic acid or arachidonic acid, growth conditions that may mimic the intracellular environment. These results suggest that the mce1 operon may serve as a mycolic acid re-importer.Fil: Forrellad, Marina Andrea. 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: McNeil, Michael. State University of Colorado - Fort Collins; Estados UnidosFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Blanco, Federico Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Garcia, Elizabeth Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Klepp, Laura Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Huff, Jason. University of Alabama at Birmingahm; Estados UnidosFil: Niederweis, Michael. University of Alabama at Birmingahm; Estados UnidosFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados UnidosFil: Bigi, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

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

Impact of the deletion of the six mce operons in Mycobacterium smegmatis

The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4 operon is involved in cholesterol transport in M. smegmatis.Fil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Osella, Ana Virginia. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Stella, Emma Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Bianco, María Verónica. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Santangelo, María de la Paz. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Sassetti, Cristopher. Massachusetts Institute of Technology; Estados UnidosFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados UnidosFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. CNIA Castelar. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Mycobacterial MCE proteins as transporters that control lipid homeostasis of the cell wall

Mammalian cell entry (mce) genes are not only present in genomes of pathogenic mycobacteria, including Mycobacterium tuberculosis (the causative agent of tuberculosis), but also in saprophytic and opportunistic mycobacterial species. MCE are conserved cell-wall proteins encoded by mce operons, which maintain an identical structure in all mycobacteria: two yrbE genes (A and B) followed by six mce genes (A, B, C, D, E and F). Although these proteins are known to participate in the virulence of pathogenic mycobacteria, the presence of the operons in nonpathogenic mycobacteria and other bacteria indicates that they play another role apart from host cell invasion. In this respect, more recent studies suggest that they are functionally similar to ABC transporters and form part of lipid transporters in Actinobacteria. To date, most reviews on mce operons in the literature discuss their role in virulence. However, according to data from transcriptional studies, mce genes, particularly the mce1 and mce4 operons, modify their expression according to the carbon source and upon hypoxia, starvation, surface stress and oxidative stress; which suggests a role of MCE proteins in the response of Mycobacteria to external stressors. In addition to these data, this review also summarizes the studies demonstrating the role of MCE proteins as lipid transporters as well as the relevance of their transport function in the interaction of pathogenic Mycobacteria with the hosts. Altogether, the evidence to date would indicate that MCE proteins participate in the response to the stress conditions that mycobacteria encounter during infection, by participating in the cell wall remodelling and possibly contributing to lipid homeostasis.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: Sabio Y Garcia, Julia Veronica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Sabio Y Garcia, Julia Veronica. 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

ERAP1 and PDE8A are downregulated in cattle protected against bovine tuberculosis

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that is responsible for significant economic losses worldwide. In spite of its relevance, the limited knowledge about the host immune responses that provide effective protection against the disease has long hampered the development of an effective vaccine. The identification of host proteins with an expression that correlates with protection against bTB would contribute to the understanding of the cattle defence mechanisms against M. bovis infection. In this study, we found that ERAP1 and PDE8A were downregulated in vaccinated cattle that were protected from experimental M. bovis challenge. Remarkably, both genes encode proteins that have been negatively associated with immune protection against bTB.Instituto de BiotecnologíaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin