Carlos Martín

Instituto de Salud Carlos III, Madrid, Madrid, Spain

Are you Carlos Martín?

Claim your profile

Publications (66)239.32 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis is the causal agent of the tuberculosis (TB) and causes about 2 million deaths a year. The recent rise of the multi-drug resistant forms of TB together with the loss of efficacy of the current vaccine BCG, have made TB a health priority. Deepen the understanding on the host-pathogen interactions during the illness could be key in the development of new drugs and vaccines. Lungs are the site of entry of M. tuberculosis and the first interactions host-pathogen occur in them. The characteristics of this interactions and the ability of the immune system to recognize bacteria mark the success or failure of the infection (1). For this reason, we are interested in knowing the expression profile of the pathogen and the host through the extraction of the RNA directly of the lungs. In addition, the possibility of simultaneously overseeing the expression of both organism by the Dual RNA-Seq technique allow us to know different points of the infection (2). For our purpose, we infected mice by intranasal inoculation and we hope the chronicity of the disease at least 3 weeks prior to euthanize animals. From this point, we tested different protocols found in the literature (3, 4) and other established in our laboratory (5). After each extraction, we check the purity and integrity of the genetic material obtained. Low bacterial load, high content of RNases in lungs and the required purity for the RNA-Seq are the main challenges of the process. References: 1. Talaat AM, Lyons R, Howard ST, Johnston SA. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(13):4602-7. 2. Westermann AJ, Gorski SA, Vogel J. Nature reviews Microbiology. 2012;10(9):618-30. 3. Bold TD, Banaei N, Wolf AJ, Ernst JD. PLoS pathogens. 2011;7(5):e1002063. 4. Chomczynski P, Sacchi N. Analytical biochemistry. 1987;162(1):156-9. 5. Solans L, Gonzalo-Asensio J, Sala C, Benjak A, Uplekar S, Rougemont J, et al. PLoS pathogens. 2014;10(5):e1004183
    VII Workshop of Training of the Center of Biomedical Research in Respiratory Diseases Network (CIBERES), Valladolid; 10/2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis is the causal agent of the tuberculosis (TB) and causes about 2 million deaths a year. The recent rise of the multi-drug resistant forms of TB together with the loss of efficacy of the current vaccine BCG, have made TB a health priority. Deepen the understanding on the host-pathogen interactions during the illness could be key in the development of new drugs and vaccines. Lungs are the site of entry of M. tuberculosis and the first interactions host-pathogen occur in them. The characteristics of this interactions and the ability of the immune system to recognize bacteria mark the success or failure of the infection (1). For this reason, we are interested in knowing the expression profile of the pathogen and the host through the extraction of the RNA directly of the lungs. In addition, the possibility of simultaneously overseeing the expression of both organism by the Dual RNA-Seq technique allow us to know different points of the infection (2). For our purpose, we infected mice by intranasal inoculation and we hope the chronicity of the disease at least 3 weeks prior to euthanize animals. From this point, we tested different protocols found in the literature (3, 4) and other established in our laboratory (5). After each extraction, we check the purity and integrity of the genetic material obtained. Low bacterial load, high content of RNases in lungs and the required purity for the RNA-Seq are the main challenges of the process.
    VII Workshop of Training of the Center of Biomedical Research in Respiratory Diseases Network (CIBERES), Valladolid, Spain; 10/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Safety of individuals at risk of immune suppression is an important concern for live vaccines. The new-generation tuberculosis vaccine candidate MTBVAC, a genetically engineered doubly attenuated Mycobacterium tuberculosis mutant with deletions in phoP and fadD26 virulence genes has demonstrated comparable safety in different relevant animal models and superior protection in mice as compared to the only currently licensed tuberculosis vaccine Mycobacterium bovis BCG. Here we describe the construction of a highly attenuated MTBVAC-based live vaccine by an additional gene inactivation generated in erp of MTBVAC. The gene product of erp is an exported repeated protein (Erp), a virulence factor described to be involved in intracellular replication of M. tuberculosis. The resultant strain, MTBVAC erp(-), was tested in severe combined immunodeficiency (SCID) mouse model showing to be severely attenuated when compared to BCG and MTBVAC. Experiments conducted in immunocompetent mice revealed that the hyper-attenuated profile observed with MTBVAC erp(-) strain did not compromise its protective efficacy profile in comparison with BCG. These results postulate MTBVAC erp(-) as a potential tuberculosis vaccine candidate for use in high-risk populations of immune suppression (e.g., due to HIV infection), where the use of BCG is not recommended.
    Vaccine 07/2014; · 3.49 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.
    Proceedings of the National Academy of Sciences 07/2014; · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: The use of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as treatment of superficial bladder cancer is one of the most successful immunotherapy for cancer (1). In the development of a new vaccine candidate for replacing the actual BCG (2), we studied the effect of our genetically attenuated strain of Mycobacterium tuberculosis MTBVAC on the viability of bladder cancer cell lines. Methods: The T24, J82 (both from human) and MB49 (from mouse) bladder tumour cell lines (3) were cultured at different multiplicities of infection (MOI) with BCG Pasteur-GFP or MTBVAC-GFP (these strains express the GFP constitutively). Percentage of infection and cell death at different time points were measured by Flow Cytometry. Additionally, we studied the localization and the basic characteristics of the organelles that contain the BCG or MTBVAC by the use of Confocal microscopy. Results: The study of percentage of infection shows a very significant increase in the case of MTBVAC vaccine at 96 hours and 7 days in contrast of the low percentages in BCG Pasteur-GFP. Moreover, the study of cell death by Flow Cytometry is in accordance with the observed results of infection. Finally, we have obtained images in which MTBVAC-GFP shows clearly their localization in the acid compartments of the cell inside while the BCG Pasteur-GFP are in not acidic organelles in the inner of the cell. The number of bacilli of MTBVAC-GFP in the inner of the any cell line of bladder cancer is significantly higher than BCG Pasteur-GFP and the integrity of the cells is worse, mainly at 7 days of infection. Conclusions: We show that our vaccine candidate MTBVAC is a promising potentially alternative to BCG in treatment of bladder cancer. References: 1. Brandau S, Suttmann H. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2007;61(6):299-305. 2. Arbues A, Aguilo JI, Gonzalo-Asensio J, Marinova D, Uranga S, Puentes E, et al. Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials. Vaccine. 2013;31(42):4867-73. 3. Secanella-Fandos S, Luquin M, Julian E. Connaught and Russian strains showed the highest direct antitumor effects of different Bacillus Calmette-Guerin substrains. The Journal of urology. 2013;189(2):711-8.
    10th Meeting of Molecular Microbiology, Group Specializing in the Microbiology Spanish Society (SEM)., Segovia, Spain; 06/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis given that its inactivation severely attenuates virulent mycobacteria. In this work we show that clinical isolates of M. tuberculosis produce and secrete higher amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 (rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publically available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP down-regulates whiB6 expression in H37Rv while it up-regulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis - with the exception of the H37Rv strain - ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.
    Infection and Immunity 06/2014; · 4.16 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.
    ALTEX. 05/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.
    PLoS Pathogens 05/2014; 10(5):e1004183. · 8.14 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The BCG vaccine is one of the most widely used vaccines worldwide. However, it appears ineffective in preventing pulmonary tuberculosis. Here we show that pulmonary BCG vaccination of mice over a broad dose range provides superior protection against Mycobacterium tuberculosis challenge compared with subcutaneous vaccination.
    Clinical and vaccine Immunology: CVI 02/2014; · 2.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing. The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG463, mgtC182, Ag85C103 and RDRio deletion were detected. This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates "SCG-6c" reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.
    BMC Microbiology 02/2014; 14(1):21. · 2.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Substantial efforts have been made over the past decade to develop vaccines against tuberculosis. We review recent developments in tuberculosis vaccines in the global portfolio, including those designed for use in a prophylactic setting, either alone or as boosts to Bacille Calmette-Guérin, and therapeutic vaccines designed to improve chemotherapy. While there is no doubt that progress is still being made, there are limitations to our animal model screening processes, which are further amplified by the lack of understanding of the immunological responses involved and the precise type of long-lived immunity that new vaccines need to induce. The challenge ahead is to optimize the planning for advanced clinical trials in poor endemic settings, which could be greatly facilitated by identifying correlates of protection.
    Expert Review of Vaccines 12/2013; 12(12):1431-48. · 4.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The insertion sequence (IS) 6110, besides being a very useful tool in molecular epidemiology seems to have an impact on the biology of the bacillus. In the present work, we mapped and localized the 12 points of insertion of IS6110 in the genome of a successful strain named Mycobacterium tuberculosis Zaragoza "MTZ". This strain belonging to the principal genetic group 3 caused a large and unsuspected tuberculosis outbreak involving 85 patients in Zaragoza (Spain) from 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of MTZ strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described in a successful Beijing sub-lineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.
    Journal of clinical microbiology 08/2013; · 4.23 Impact Factor
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.
    Vaccine 08/2013; · 3.49 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Apoptosis modulation is a procedure amply utilized by intracellular pathogens to favour the outcome of the infection. Nevertheless, the role of apoptosis during infection with Mycobacterium tuberculosis, the causative agent of human tuberculosis, is subject of an intense debate and still remains unclear. In this work, we describe that apoptosis induction in host cells is clearly restricted to virulent M. tuberculosis strains, and is associated with the capacity of the mycobacteria to secrete the 6-kD early secreted antigenic target ESAT-6 both under in vitro and in vivo conditions. Remarkably, only apoptosis-inducing strains are able to propagate infection into new cells, suggesting that apoptosis is used by M. tuberculosis as a colonization mechanism. Finally, we demonstrate that in vitro modulation of apoptosis affects mycobacterial cell-to-cell spread capacity, establishing an unambiguous relationship between apoptosis and propagation of M. tuberculosis. Our data further indicate that BCG and MTBVAC vaccines are inefficient in inducing apoptosis and colonizing new cells, correlating with the strong attenuation profile of these strains previously observed in vitro and in vivo.
    Cellular Microbiology 07/2013; · 4.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains.
    BMC Genomics 06/2013; 14(1):422. · 4.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The major Mycobacterium tuberculosis virulence factor ESAT-6 exported by the ESX-1 secretion system has been described as a pro-apoptotic factor by several independent groups in recent years, sustaining a role for apoptosis in M. tuberculosis pathogenesis. This role has been supported by independent studies in which apoptosis has been shown as a hallmark feature in human and mouse lungs infected with virulent strains. Nevertheless, the role of apoptosis during mycobacterial infection is subject to an intense debate. Several works maintain that apoptosis is more evident with attenuated strains, whereas virulent mycobacteria tend to inhibit this process, suggesting that apoptosis induction may be a host mechanism to control infection. In this review, we summarize the evidences that support the involvement of ESX-1-induced apoptosis in virulence, intending to provide a rational treatise for the role of programmed cell death during M. tuberculosis infection.
    Frontiers in Cellular and Infection Microbiology 01/2013; 3:88. · 2.62 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.
    PLoS ONE 09/2012; 7(9):e45213. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The insertion element IS6110 is one of the main sources of genomic variability in Mycobacterium tuberculosis, the etiological agent of human tuberculosis. Although IS 6110 has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. We identified a total of 6,976 IS6110 flanking regions on the different isolates. When validated using reference strains, the method had 100% specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed. This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in M. tuberculosis strains.
    BMC Genomics 06/2012; 13:249. · 4.04 Impact Factor

Publication Stats

2k Citations
239.32 Total Impact Points

Institutions

  • 2012–2014
    • Instituto de Salud Carlos III
      • CIBER of Respiratory Diseases (CIBERES)
      Madrid, Madrid, Spain
  • 2001–2014
    • University of Zaragoza
      • • Department of Microbiology, Preventive Medicine and Public Health
      • • Faculty of Medicine
      Caesaraugusta, Aragon, Spain
  • 2011
    • Hospital Universitario Miguel Servet
      Caesaraugusta, Aragon, Spain
  • 2009
    • University of British Columbia - Vancouver
      Vancouver, British Columbia, Canada
    • Centro de Investigacion Biomédica en Red de Enfermedades Respiratorias
      Bunyola, Balearic Islands, Spain
  • 2008
    • National University of Colombia
      • Departamento de Química (Bogotá)
      Μπογκοτά, Bogota D.C., Colombia
    • University of Central Florida
      • Graduate Program in Molecular Biology and Microbiology
      Orlando, Florida, United States
  • 2003–2006
    • Institut Pasteur
      Lutetia Parisorum, Île-de-France, France