Publications by authors named "Fabiana Bigi"

62 Publications

The subunit vaccine H65 + CAF01 increased the BCG- protection against Mycobacterium bovis infection in a mouse model of bovine tuberculosis.

Res Vet Sci 2021 May 14;136:595-597. Epub 2021 Apr 14.

Instituto de Agrobiotecnología y Biología Molecular, (IABIMO) INTA-CONICET, Argentina; Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria, Institute of Biotechnology, National Institute of Agricultural Technology, Argentina. Electronic address:

H65, a fusion protein of three pairs of ESX-secreted antigens of Mycobacterium tuberculosis and Mycobacterium bovis, formulated with the liposomal adjuvant CAF01 has been shown to confer protection against M. tuberculosis infection in mice. In this study, we evaluated the impact of combining BCG with H65 + CAF01 immunization in a M. bovis mouse model of infection. We found that a BCG-H65 + CAF01/ H65 + CAF01 prime-boost scheme induced higher protection than BCG and H65 + CAF01 alone. Altogether, H65 antigen formulated in liposomal adjuvant improved the BCG-induced immune protection, thus making this vaccine strategy a promising tool to control bovine tuberculosis.
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http://dx.doi.org/10.1016/j.rvsc.2021.04.014DOI Listing
May 2021

Elimination of ESAT-6 and CFP-10 from a candidate vaccine against bovine tuberculosis impaired its protection efficacy in the BALBc mouse model.

Int J Mycobacteriol 2020 Oct-Dec;9(4):417-421

Institute of Agrobiotechnology and Molecular Biology, INTA-CONICET; CICVyA, Institute of Biotechnology, National Institute of Agricultural Technology, Buenos Aires, Argentina.

Background: Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium bovis that mainly affects cattle. Although vaccination is the most effective strategy to control bTB, it may interfere with the diagnosis of the infection. Therefore, ancillary tests to differentiate vaccinated from infected animals (DIVA) are essential in a cattle vaccination scenario. ESAT-6 and CFP-10 are the most promissory DIVA antigens.

Method: In this study, we deleted esat6 and cfp10 genes from the M. bovis Δ mce2 live-attenuated vaccine candidate and evaluated its protection level against bTB in BALBc mice.

Results: We found that the M. bovis strain mutant in mce2, esat-6 and cfp-10 failed to confer protection against virulent M. bovis challenge in a mouse model of tuberculosis.

Conclusions: This result highlights the relevant role of ESAT-6 and CFP-10 in the induction of protective immune response against M. bovis infection and reveals the need of evaluating different strategies to compensate for the lack of these DIVA antigens in new vaccine formulations.
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http://dx.doi.org/10.4103/ijmy.ijmy_180_20DOI Listing
December 2020

Role of PhoPR in the response to stress of Mycobacterium bovis.

Comp Immunol Microbiol Infect Dis 2021 Feb 21;74:101593. Epub 2020 Nov 21.

Instituto de Agrobiotecnología y Biología Molecular, (IABIMO) INTA-CONICET, Argentina; Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria Institute of Biotechnology, National Institute of Agricultural Technology, Argentina. Electronic address:

PhoP is part of the two-component PhoPR system that regulates the expression of virulence genes of Mycobacteria. The goal of this work was to elucidate the role of PhoP in the mechanism that Mycobacterium bovis, the causative agent of bovine tuberculosis, displays upon stress. An analysis of gene expression and acidic growth curves indicated that M. bovis neutralized the external acidic environment by inducing and secreting ammonia. We found that PhoP is essential for ammonia production/secretion and its role in this process seems to be the induction of asparaginase and urease expression. We also demonstrated that the lack of PhoP negatively affected the synthesis of phthiocerol dimycocerosates. This finding is consistent with the role of the lipid anabolism in maintaining the redox environment upon stress in mycobacteria. Altogether the results of this study indicate that PhoP plays an important role in the response mechanisms to stress of M. bovis.
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http://dx.doi.org/10.1016/j.cimid.2020.101593DOI Listing
February 2021

FasR Regulates Fatty Acid Biosynthesis and Is Essential for Virulence of .

Front Microbiol 2020 27;11:586285. Epub 2020 Oct 27.

Laboratory of Physiology and Genetics of Actinomycetes, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during infection. In this work, we constructed and characterized a FasR-deficient mutant in and demonstrated that FasR positively regulates and expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of infection.
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http://dx.doi.org/10.3389/fmicb.2020.586285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652896PMC
October 2020

Rv2577 of Is a Virulence Factor With Dual Phosphatase and Phosphodiesterase Functions.

Front Microbiol 2020 22;11:570794. Epub 2020 Oct 22.

Instituto de Agrobiotecnología y Biología Molecular (IABIMO), Instituto Nacional de Tecnología Agropecuaria-Consejo Nacional de Investigaciones Científicas y Técnicas (INTA-CONICET), INTA, Buenos Aires, Argentina.

Tuberculosis, a lung disease caused by , is one of the ten leading causes of death worldwide affecting mainly developing countries. can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and . In this study, we characterized the Rv2577 of as a potential alkaline phosphatase/phosphodiesterase enzyme. By an kinetic assay, we demonstrated that purified Rv2577 expressed in displays both enzyme activities, as evidenced by using the artificial substrates NPP and bis-(NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in virulence.
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http://dx.doi.org/10.3389/fmicb.2020.570794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642983PMC
October 2020

Does Mycobacterium bovis persist in cattle in a non-replicative latent state as Mycobacterium tuberculosis in human beings?

Vet Microbiol 2020 Aug 21;247:108758. Epub 2020 Jun 21.

(Instituto de Biotecnología-IABIMO, INTA-CONICET), Institute of Biotechnology-IABIMO, National Institute of Agricultural Technology (INTA) and National Scientific and Technical Research Council (CONICET), Argentina. Electronic address:

Members of the Mycobacterium tuberculosis complex (MTBC) are responsible for tuberculosis in several mammals. In this complex, Mycobacterium tuberculosis and Mycobacterium bovis, which are closely related, show host preference for humans and cattle, respectively. Although human and bovine tuberculosis are clinically similar, M. tuberculosis mostly causes latent infection in humans, whereas M. bovis frequently leads to an acute infection in cattle. This review attempts to connect the pathology in experimental animal models as well as the cellular responses to M. bovis and M. tuberculosis regarding the differences in protein expression and regulatory mechanisms of both pathogens that could explain their apparent divergent latency behaviour. The occurrence of latent bovine tuberculosis (bTB) would represent a serious complication for the eradication of the disease in cattle, with the risk of onward transmission to humans. Thus, understanding the physiological events that may lead to the state of latency in bTB could assist in the development of appropriate prevention and control tools.
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http://dx.doi.org/10.1016/j.vetmic.2020.108758DOI Listing
August 2020

A Phenotypic Characterization of Two Isolates of a Multidrug-Resistant Outbreak Strain of with Opposite Epidemiological Fitness.

Biomed Res Int 2020 8;2020:4741237. Epub 2020 Apr 8.

Institute of Biotechnology, National Institute of Agricultural Technology (INTA)/IABIMO-CONICET (Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria), Argentina.

Tuberculosis (TB) is an infectious disease, caused by , primarily affecting the lungs. The strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.
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http://dx.doi.org/10.1155/2020/4741237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168692PMC
January 2021

Production of Mycobacterium bovis Antigens Included in Recombinant Occlusion Bodies of Baculovirus.

J Mol Microbiol Biotechnol 2019 7;29(1-6):83-90. Epub 2020 Apr 7.

Institute of Biotechnology, National Institute of Agricultural Technology (INTA, Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria) and IABIMO-National Scientific and Technical Research Council (CONICET, Consejo Nacional de Investigaciones Científicas y Tecnológicas), Buenos Aires, Argentina,

Bovine tuberculosis (bTB) is a disease produced by Mycobacterium bovis that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of M. bovis (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used M. bovis antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from Escherichia coli, because this bacterium produces a high level of recombinant proteins. However, E. coli recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c M. bovis antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same M. bovis proteins produced in E. coli.
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http://dx.doi.org/10.1159/000506687DOI Listing
April 2021

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

Virulence 2019 12;10(1):1026-1033

Institute of Biotechnology, National Institute of Agricultural Technology (INTA, Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria) and IABIMO-National Scientific and Technical Research Council (CONICET, Consejo Nacional de Investigaciones Científicas y Tecnológicas), Hurlingham, Buenos Aires, Argentine.

In this study, we characterized the role of Rv2617c in the virulence of . Rv2617c is a protein of unknown function unique to complex (MTC) and , 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 gene in 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 hydrogen peroxide treatment in and , respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.
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http://dx.doi.org/10.1080/21505594.2019.1693714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930017PMC
December 2019

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Vet Microbiol 2019 Dec 4;239:108482. Epub 2019 Nov 4.

Instituto de Biotecnología, IABIMO, CICVyA/INTA, Argentina. Electronic address:

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.
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http://dx.doi.org/10.1016/j.vetmic.2019.108482DOI Listing
December 2019

Editorial: Cellular and Molecular Mechanisms of Virulence.

Front Cell Infect Microbiol 2019 9;9:331. Epub 2019 Oct 9.

Institute of Biotechnology, National Institute of Agricultural Technology, Buenos Aires, Argentina.

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http://dx.doi.org/10.3389/fcimb.2019.00331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794420PMC
June 2020

ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

Front Immunol 2019 26;10:929. Epub 2019 Apr 26.

Laboratorio de Fisiología de los Procesos Inflamatorios, Instituto de Medicina Experimental (IMEX)- Consejo Nacional de investigaciones Científicas y Tecnológicas (CONICET)/Academia Nacional de Medicina de Buenos Aires, Buenos Aires, Argentina.

The epidemic clone of (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.
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http://dx.doi.org/10.3389/fimmu.2019.00929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497972PMC
September 2020

Characterization of the two component regulatory system PhoPR in Mycobacterium bovis.

Vet Microbiol 2018 Aug 21;222:30-38. Epub 2018 Jun 21.

Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria, Institute of Biotechnology, National Institute of Agricultural Technology (INTA), N. Repetto and de los Reseros, Hurlingham, CP1686, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Tecnológicas, National Scientific and Technical Research Council (CONICET), Argentina. Electronic address:

Mycobacterium bovis is the causative agent of bovine tuberculosis and is a member of Mycobacterium tuberculosis complex, which causes tuberculosis in a number of mammals including humans. Previous studies have shown that the genes encoding the two-component system PhoPR, which regulates several genes involved in the virulence of M. tuberculosis, are polymorphic in M. bovis, when compared to M. tuberculosis, which results in a dysfunctional two-component system. In this study we investigated the role of PhoPR in two M. bovis strains with differing degrees of virulence. We found that the deletion of phoP in an M. bovis isolate reduced its capacity of inducing phagosomal arrest in bovine macrophages. By gene expression analysis, we demonstrated that, in both M. bovis strains, PhoP regulates the expression of a putative lipid desaturase Mb1404-Mb1405, a protein involved in redox stress AhpC, the sulfolipid transporter Mmpl8 and the secreted antigen ESAT-6. Furthermore, the lack of PhoP increased the sensitivity to acidic stress and alteration of the biofilm/pellicle formation of M. bovis. Both these phenotypes are connected to bacterial redox homeostasis. Therefore, the results of this study suggest a role of PhoPR in M. bovis to be linked to the mechanisms that mycobacteria display to maintain their redox balance.
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http://dx.doi.org/10.1016/j.vetmic.2018.06.016DOI Listing
August 2018

ERAP1 and PDE8A Are Downregulated in Cattle Protected against Bovine Tuberculosis.

J Mol Microbiol Biotechnol 2017 14;27(4):237-245. Epub 2017 Sep 14.

Instituto de Biotecnología, CICVyA-INTA, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1159/000479183DOI Listing
June 2018

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain.

Tuberculosis (Edinb) 2017 03 4;103:28-36. Epub 2017 Jan 4.

Universidad de Buenos Aires, Facultad de Agronomía, Cátedra de Microbiología Agrícola.INBA-CONICET, Av. San Martín 4453, C1417DSE, Buenos Aires, Argentina. Electronic address:

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8 cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-l-methionine-dependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.
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http://dx.doi.org/10.1016/j.tube.2016.12.007DOI Listing
March 2017

Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages.

Infect Immun 2017 03 23;85(3). Epub 2017 Feb 23.

Instituto de Biotecnología, CICVyA-INTA, Nicolás Repetto y De Los Reseros, Buenos Aires, Argentina

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 and Here, we describe a novel function of P27 in the interaction of with its natural host cell, the bovine macrophage. We found that a deletion in the operon impairs the replication of in bovine macrophages. Importantly, we show for the first time that arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type but not fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of infection. In addition, we also showed that the presence of P27 from 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.
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http://dx.doi.org/10.1128/IAI.00720-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328499PMC
March 2017

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

J Infect Dev Ctries 2016 Aug 31;10(8):892-4. Epub 2016 Aug 31.

Instituto de Biotecnología, CICVyA-INTA, Hurlingham, Buenos Aires, Argentina.

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http://dx.doi.org/10.3855/jidc.7721DOI Listing
August 2016

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis.

Microbiol Immunol 2016 Aug;60(8):552-60

Biotechnology Institute, National Institute of Agricultural Technology (INTA), Hurlingham 1686, Argentina.

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosis.
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http://dx.doi.org/10.1111/1348-0421.12402DOI Listing
August 2016

Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

Tuberculosis (Edinb) 2015 Dec 8;95(6):795-801. Epub 2015 Aug 8.

Laboratorio de Tuberculosis Bovina, Instituto de Biotecnología, CICVyA, INTA-Castelar, Buenos Aires, Argentina. Electronic address:

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.
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http://dx.doi.org/10.1016/j.tube.2015.07.009DOI Listing
December 2015

Evaluation of Mycobacterium bovis double knockout mce2-phoP as candidate vaccine against bovine tuberculosis.

Tuberculosis (Edinb) 2015 Mar 9;95(2):186-9. Epub 2015 Jan 9.

Instituto de Biotecnología, CICVyA-INTA, N. Repetto and De los Reseros, 1686 Hurlingham, Argentina. Electronic address:

In this study, a Mycobacterium bovis knockout strain in phoP-phoR and mce2 operons was tested as an antituberculosis experimental vaccine in animal models. The double mutant strain was significantly more attenuated than the wild type strain in inmunocompetent and inmunodeficient mice. Vaccination with the double mutant protected mice against challenge with a virulent M. bovis strain.
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http://dx.doi.org/10.1016/j.tube.2015.01.001DOI Listing
March 2015

IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis.

Autophagy 2014 ;10(12):2109-21

a Departamento de Química Biológica; Facultad de Ciencias Exactas y Naturales; Universidad de Buenos Aires (UBA) ; Buenos Aires , Argentina.

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.
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http://dx.doi.org/10.4161/15548627.2014.981791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502660PMC
December 2015

Differential expression of immunogenic proteins on virulent Mycobacterium tuberculosis clinical isolates.

Biomed Res Int 2014 7;2014:741309. Epub 2014 Jul 7.

IMEX-CONICET, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 CABA, Argentina.

Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB.
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http://dx.doi.org/10.1155/2014/741309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109345PMC
April 2015

Identification of potential biomarkers of disease progression in bovine tuberculosis.

Vet Immunol Immunopathol 2014 Aug 6;160(3-4):177-83. Epub 2014 May 6.

Microbiología Agrícola, Facultad de Agronomía, Universidad de Buenos Aires, INBA Consejo Nacional de Investigaciones Científicas y Técnicas, Ciudad de Buenos Aires, Argentina. Electronic address:

Bovine tuberculosis (bTB) remains an important animal and zoonotic disease in many countries. The diagnosis of bTB is based on tuberculin skin test and IFN-γ release assays (IGRA). Positive animals are separated from the herd and sacrificed. The cost of this procedure is difficult to afford for developing countries with high prevalence of bTB; therefore, the improvement of diagnostic methods and the identification of animals in different stages of the disease will be helpful to control the infection. To identify biomarkers that can discriminate between tuberculin positive cattle with and without tuberculosis lesions (ML+ and ML-, respectively), we assessed a group of immunological parameters with three different classification methods: lineal discriminant analysis (LDA), quadratic discriminant analysis (QDA) and K nearest neighbors (k-nn). For this purpose, we used data from 30 experimentally infected cattle. All the classifiers (LDA, QDA and k-nn) selected IL-2 and IL-17 as the most discriminatory variables. The best classification method was LDA using IL-17 and IL-2 as predictors. The addition of IL-10 to LDA improves the performance of the classifier to discriminate ML-individuals (93.3% vs. 86.7%). Thus, the expression of IL-17, IL-2 and, in some cases, IL-10 would serve as an additional tool to study disease progression in herds with a history of bTB.
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http://dx.doi.org/10.1016/j.vetimm.2014.04.008DOI Listing
August 2014

Experimental selection of long-term intracellular mycobacteria.

Cell Microbiol 2014 Sep 2;16(9):1425-40. Epub 2014 Jun 2.

Research Group Phagosome Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany.

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.
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http://dx.doi.org/10.1111/cmi.12303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4283733PMC
September 2014

Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

Biomed Res Int 2014 21;2014:951978. Epub 2014 Jan 21.

Instituto de Biotecnología, CICVyA-INTA, N. Repetto and De los Reseros, 1686 Hurlingham, Argentina.

A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.
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http://dx.doi.org/10.1155/2014/951978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918748PMC
October 2014

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

Tuberculosis (Edinb) 2014 Mar 2;94(2):170-7. Epub 2014 Jan 2.

Instituto de Biotecnología, CICVyA - INTA, N. Repetto and De los Reseros, Hurlingham 1686, Argentina. Electronic address:

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 exacerbated when bacteria are cultured in the presence of palmitic acid or arachidonic acid. These results suggest that the mce1 operon may serve as a mycolic acid re-importer.
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http://dx.doi.org/10.1016/j.tube.2013.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951760PMC
March 2014

Draft Genome Sequence of Mycobacterium bovis 04-303, a Highly Virulent Strain from Argentina.

Genome Announc 2013 Nov 27;1(6). Epub 2013 Nov 27.

School of Computing, UFMS, Campo Grande, Mato Grosso do Sul, Brazil.

Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.
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http://dx.doi.org/10.1128/genomeA.00931-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869323PMC
November 2013

Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

BMC Microbiol 2013 Sep 5;13:200. Epub 2013 Sep 5.

Instituto de Biotecnología, CICVyA-INTA, N, Repetto and De los Reseros, Hurlingham 1686, Argentina.

Background: Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor.

Results: We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied.

Conclusions: The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis.
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http://dx.doi.org/10.1186/1471-2180-13-200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847441PMC
September 2013

Mycobacterium bovis Δmce2 double deletion mutant protects cattle against challenge with virulent M. bovis.

Tuberculosis (Edinb) 2013 May 19;93(3):363-72. Epub 2013 Mar 19.

Instituto de Biotecnología, INTA, N. Repetto y De los Reseros, 1686 Hurlingham, Buenos Aires, Argentina.

A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an experimental vaccine in cattle challenged with a virulent M. bovis strain. Three-and-a-half-month old calves (n = 5 to 6 per group) were vaccinated and challenged with a virulent strain of M. bovis by the intratracheal route 9 weeks after vaccination. A non-vaccinated group and a group vaccinated with BCG were included as controls. Blood samples were collected to measure IFN-γ by an interferon-gamma release assay (IGRA), cytometry and cytokine responses of bovine purified protein derivative (PPD) restimulated peripheral blood mononuclear cells (PBMCs). The IGRA test showed IFN-γ values similar to pre-vaccination except for the animals vaccinated with M. bovis Δmce2, where a significant increase was observed at 30 days post-vaccination. The expression of IL-2R on CD4(+) cells in response to PPD from the animals vaccinated with Δmce2 increased at 15 days post-vaccination compared to cells from non-vaccinated group. Vaccination of cattle with M. bovis Δmce2 induced the highest (P < 0.05) expression of IFN-γ and IL-17 mRNA upon PPD stimulation of PBMCs compared to vaccination with BCG or that for the non-vaccinated group. There was a weak positive correlation between the production of these proinflammatory cytokines post-vaccination and reduced pathology scores post-challenge. The animals were euthanized and necropsied 100 days after challenge. The group vaccinated with M. bovis Δmce2 displayed a significantly lower histopathological score for lesions in lungs and pulmonary lymph nodes than for the other groups (P < 0.05). A marked positive reaction to tuberculin intradermal test was observed post-vaccination in animals vaccinated with M. bovis Δmce2 compared to those vaccinated with BCG or the non-vaccinated group. In contrast, after challenge, non-vaccinated animals had greater skin test responses than the vaccinated animals. In summary, M. bovis Δmce2 is a promising vaccine candidate to control M. bovis pathogenesis in cattle.
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http://dx.doi.org/10.1016/j.tube.2013.02.004DOI Listing
May 2013
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