Biochemistry & MCB.

The final aim at this area of BIFI is to understand and to control biological systems depending on proteins that have interest for chemical, biotechnological, pharmacological and biomedical applications. Knowledge of protein behaviour at molecular and cellular levels allows interpretation of the macroscopic mechanisms of the cellular functions in which they are involved, but many of the parameters controlling these processes still remain unknown. Proteins adopt an organized three-dimensional structure closely related with their function that can be regulated by the interaction with other biomolecules and/or small organic molecules. Defective structural arrangements can prevent protein interaction with other molecules, provoking many different illnesses in human beings.

Many other illnesses are also produced by infectious viruses and microorganisms, and a way of stopping them could be to block a step of their vital cycle involving a protein or biomolecule. Moreover, small molecules from the environment can also be either toxic inhibitors or activatiors of particular activities in different organisms, and in many cases they might be used as effectors of the expression some genes, and the production or action of particular proteins. The research lines in Biochemistry and Molecular and Cellular Biology at BIFI study different biological systems involved in key metabolic routes that serve as model for other systems combining classic methodologies from this area with biophysical and computational methods. Applications of the obtained knowledge are additionally being used to control and to modulate the behaviour of particular systems with benefits for the society.

Genetic and evolution of Mycobacterium tuberculosis

Head of the Research Line:

Jesús Gonzalo-Asensio

Researchers:

Carlos Martín Montañés (Full Professor)
Esther Broset Blasco (Postdoc)
Irene Pérez Sanchez (DGA Grant)
Ana Picó Marco (Lab Technician)
Juan Calvet Seral (DGA Grant)
Elena Campos Pardos (FPU)

SUMMARY OF THE RESEARCH LINE

According to the last WHO report, Tuberculosis (TB) is responsible for 1.8 million deaths and causes 10.4 million new TB cases worldwide per year. Cumulative data indicate that TB has killed 1 billion people in the past 200 years, which turns it as the biggest killer compared to other infectious diseases as plague, influenza, smallpox, malaria, cholera or AIDS.

TB in humans is mainly caused by the Mycobacterium tuberculosis bacillus. However, other Mycobacterium species are also able to infect humans. These include M. canettii and M. africanum, responsible for human cases geographically restricted to East- and West-African countries respectively. In addition, a number of Mycobacterium species are also able to cause TB in mammals, which represents an economic problem as well as a zoonotic risk for humans. Overall, these species constitute the M. tuberculosis Complex (MTBC). (Figure 1)

Figure 1: Schematic phylogenetic relationships of the MTBC. Phylogenetic distribution of M. canettii, the L1-L7 lineages of M. tuberculosis and the animal-adapted species. The figure also shows the geographic distribution of each lineage and the preferred host. Adaptado de Broset et al. mBio 2015.

To gain a complete evolutionary perspective, it is highly recomended to extend genomic studies to the whole MTBC. Our cumulative knowledge in mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired during the natural evolution of mycobacterial species probably to ensure adaptation to different hosts (Gonzalo-Asensio et al, PNAS 2014). PhoPR is a well-known regulator of pathogenic phenotypes in the Mycobacterium tuberculosis complex (MTBC) including secretion of the virulent factor ESAT-6 (Broset et al. mBio 2015), biosynthesis of acyltrehalose-based lipids (Gonzalo-Asensio et al. JBC 2008) or modulation of antigen export (Solans et al. PLoS pathogens 2014) (Figure 2).

Figure 2. Molecular characterization of the PhoPR Two-Component System. A. Positioning of the PhoP transcription factor relative to the RNA polymerase and its target genes measured as the most probable values from ChIP-seq data. The PhoP consensus motif (TCACAG-N5-TCACAG) is also indicated. B. ChIP-seq reads from representative promoters of PhoP-regulated genes. Note the significant increase in ChIP-seq peaks in the wild-type strain relative to its phoP mutant indicative of a specific interaction of PhoP with these regions. C. PhoP binding motifs elucidated from ChIP-seq data shown in panel B. Distance to the start codon of target genes is also indicated. D. Genomic location of relevant phoP polymorphisms in the MTBC. Insertions of IS6110 in the promoter region and their positions relative to the phoP start codon are shown. Amino acid positions of the Asp71 residue involved in the phosphotransfer reaction and the Ser219Leu substitution in H37Ra are also indicated. E. Ribbon model of the DNA-binding domain of PhoP superimposed on the structure of a PhoB-DNA complex. Solid spheres show the wild-type serine 219 in H37Rv (left) or the leucine residue that appears in H37Ra (right) (Adapted from Gonzalo-Asensio et al. J. Bacteriol 2008). The mutant leucine residue is expected to interfere with DNA binding and/or recognition. F. Secondary structure of the PhoR sensor kinase indicating its membrane topology. Each domain has been coloured individually indicating the presence of α-helices (“racetrack” ovals) and β-strands (arrows). Note the presence of PhoR polymorphisms in the sensor loop located in the periplasmic space. Position of the Histidine involved in the phosphotransfer reaction is also indicated. Adapted from Broset et al. mBio 2015.

Evolutionarily conserved polymorphisms in PhoPR from M. africanum and animal-adapted species result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and likely to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream phoPR in a particular M. bovis strain able to transmit between humans or polymorphisms in M. africanum and M. bovis affecting the regulatory region of the espACD operon, which allow PhoPR independent ESAT-6 secretion (Gonzalo-Asensio et al. PNAS 2014) (Figure 3).

Figure 3. Comparative illustration of PhoPR-regulated phenotypes in M. tuberculosis, M. bovis, M. africanum L6 and a M. tuberculosis phoP mutant. M. tuberculosis carrying a functional PhoR is able to sense its cognate stimulus and subsequently phosphorylate PhoP. Phosphorylated PhoP regulates three well-known phenotypes including synthesis of SL and DAT/PAT (via pks2 and pks3 regulation), secretion of ESAT-6 (through espA regulation) and post-transcriptional regulation of tatC (mediated by the mcr7 non-coding RNA). M. bovis and M. africanum L6 carrying a defective PhoR G71I allele are expected to have defects in PhoP phosphorylation, as a consequence these strains lacks SL, DAT and PAT. However, ESAT-6 secretion in these strains is restored by compensatory mutations in the espACD promoter region that include RD8 deletion and species-specific polymorphisms (asterisks). M. tuberculosis phoP mutants lack the aforementioned PhoP-regulated phenotypes and consequently does not synthesize SL, DAT and PAT or secrete ESAT-6. These mutants also have a deregulated TAT system and consequently secrete higher amounts of TAT substrates that include the antigens Ag85A and Ag85C. Consequently, adequately attenuated M. tuberculosis phoP-based vaccine strains, such as MTBVAC, are expected to induce long-lasting immunogenicity in clinical trials. Adapted de Broset et al. mBio 2015

Our research line is focused on the rising significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation (Figure 3). In this context, the live-attenuated MTBVAC based on a phoP/fadD26 deletion mutant of M. tuberculosis is the first vaccine of this kind to successfully enter clinical development, representing a historic milestone in the field of vaccinology.

Relevant publications

1.- New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages. Jesús Gonzalo-Asensio*, Irene Pérez, Nacho Aguiló, Santiago Uranga, Ana Picó, Carlos Lampreave, Alberto Cebollada, Isabel Otal, Sofía Samper, Carlos Martín. 2018.. PLoS Genetics, IF= 6,1, D1 Genética y Herencia

2.- Reactogenicity to major tuberculosis antigens absent in BCG is essential to improve protection against Mycobacterium tuberculosis. Nacho Aguiló, Jesús Gonzalo-Asensio, Samuel Alvarez-Arguedas, Dessislava Marinova, Ana Belen Gomez, Santiago Uranga, Ralf Spallek, Mahavir Singh, Régine Audran, François Spertini, Carlos Martin. 2017. “” Nature Communications, IF=12,124, D1 Ciencias Multidisciplinares

3.- Evolutionary landscape of the Mycobacterium tuberculosis complex from the viewpoint of PhoPR: implications in virulence regulation and application to vaccine development. Esther Broset, Carlos Martín and Jesús Gonzalo-Asensio*. 2015. mBio, IF=6.786, D1 microbiology.

4.- Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator. Jesús Gonzalo-Asensio, Wladimir Malaga, Alexandre Pawlik, Catherine Astarie-Dequeker, Charlotte Passemar, Flavie Moreau, Françoise Laval, Mamadou Daffé, Carlos Martin, Roland Brosch, Christophe Guilhot. 2014 PROC. NATL. ACAD. SCI. USA, IF=9.809, D1 multidisciplinary sciences.

5.- A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component. Luis Solans, Nacho Aguiló, Sofía Samper, Alexandre Pawlik, Wafa Frigui, Carlos Martín, Roland Brosch, Jesús Gonzalo-Asensio*. 2014. Infection And Immunity, IF=4,156, Q1 Infectious Diseases.

6.- The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis. Luis Solans*, Jesús Gonzalo-Asensio*, Claudia Sala*, Andrej Benjak, Swapna Uplekar, Jacques Rougemont, Christophe Guilhot, Wladimir Malaga, Carlos Martín, Stewart T. Cole. 2014. PLoS PATHOGENS, IF=8,136 D1 Microbiology, Virology and Parasitology.

7.- Tuberculosis Vaccine. Carlos Martín, Brigitte Gicquel, Esther Pérez, Jesús Gonzalo Asensio, Ainhoa Arbués. INTERNATIONAL PATENT. PCT/ES 2007/070051. Spain (ES7730491) / Europe (1997881) / USA (US12/294,199) / Canada (CA2,647,287) / Japan (JP2009-500878) / China (CN200780010366.5) / Russia (RU2008142140 (0)) / India (IN8123/DELN/2008) / Brasil (BRPI-0709106-0).

Main research projects

1.- Advancing Novel and Promising TB Vaccine Candidates From Discovery to Preclinical and Early Clinical Development (Reference TBVAC2020) H2020, IP: Carlos Martín Montañés. Participant.

2.- Multiplex Iterative Genome Engineering (MIGE) in Mycobacterium. Applications to development of genetic tools for testing new antibiotics Myco-MIGE (Reference BFU2015-72190-EXP). Ministerio de Economía y Competitividad, IP: Jesús Gonzalo-Asensio.

3.- Análisis de las diferencias de IS6110 entre los miembros del complejo Mycobacterium tuberculosis y el papel de su localización en el origen de replicación. (Reference PI15/00317) FIS-Instituto de Salud Carlos III, IP: Sofía Samper Blasco. Participant.

4.- Polimorfismos genómicos y transcriptómicos en M. tuberculosis complex y su significado en la clínica. (Reference PI12/01970) FIS-Instituto de Salud Carlos III. IP: Sofía Samper. Participant