Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts.

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette-Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain's vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region

Natural mutations in the sensor kinase of the PhoPR two-component regulatory system modulate virulence of ancestor-like tuberculosis bacilli

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host

Réseau de régulation transcriptionnelle sous-jacent à la différenciation du tissu conjonctif au cours du développement du membre

The musculoskeletal system is composed of muscles, skeletal elements and connective tissues such as tendon and muscle connective tissue. Muscle connective tissue contributes to the elasticity and rigidity of muscles, while tendons transmit forces generated by muscles to the bone to allow body motion. In contrast to muscle and skeleton, connective tissue patterning and formation remain poorly investigated. In order to identify molecular mechanisms underlying connective tissue formation during limb development, five zinc-finger transcription factors were investigated: OSR1, OSR2, EGR1, KLF2 and KLF4. These transcription factors are expressed in distinct subcompartments of the musculoskeletal system and influence the differentiation of limb mesenchymal cells upon overexpression. To further investigate their roles at the molecular level, several genome-wide strategies (RNA-seq, ChIP-seq) were employed. These strategies enabled: (i) to identify that the transcription factors share common regulatory functions and positively regulate biological processes related to signal transduction, cell communication and biological adhesion; (ii) to reveal that the differentially expressed genes were enriched for both active and repressive chromatin signatures at their promoters, suggesting that they are dynamically regulated; (iii) to distinguish between indirect and direct target genes. Altogether, these results provide a framework for future investigations to better understand the interconnectivity between components of the musculoskeletal system.Le système musculo-squelettique se compose des muscles, du squelette et du tissu conjonctif qui comprend, entre autres, les tendons et le tissu conjonctif musculaire. Le tissu conjonctif musculaire contribue à l'élasticité et à la rigidité des muscles, alors que les tendons transmettent les forces musculaires à l'os nécessaires aux mouvements du corps. Contrairement au muscle et au squelette, la mise en place et la formation du tissu conjonctif restent à ce jour peu étudiées. Afin d'identifier les mécanismes moléculaires sous-jacents à la formation du tissu conjonctif au cours du développement du membre, cinq facteurs de transcription à doigt de zinc ont été examinés : OSR1, OSR2, EGR1, KLF2 et KLF4. Ces facteurs de transcription sont exprimés dans différents sous-compartiments du système musculo-squelettique et leur surexpression influence la différentiation des cellules mésenchymateuses du membre. Afin d'élucider leurs rôles au niveau de la régulation génique, plusieurs stratégies à haut-débit (RNA-seq, ChIP-seq) ont été mises en place. Ces stratégies ont permis : (i) d'identifier que les facteurs de transcription partagent des fonctions régulatrices communes liées à la transduction du signal, à la communication cellulaire et à l'adhésion cellulaire ; (ii) de révéler que les gènes différentiellement exprimés étaient enrichis pour des signatures d'activation et de répression chromatiniennes, suggérant qu'ils sont dynamiquement régulés ; (iii) de distinguer les gènes cibles directs des cibles indirectes. Ces résultats fournissent ainsi une base pour des travaux futurs visant à mieux comprendre l'inter-connectivité entre les différents composants de l'appareil locomoteur

Evolution of virulence in the Mycobacterium tuberculosis complex

International audienceMycobacterium tuberculosis, the causative agent of human tuberculosis is one of the most widely spread human pathogens. It has succeeded to infect a quarter of the global human population by developing most sophisticated ways to circumvent innate and adaptive immune defences. This highly specialized, major human pathogen has evolved from a pool of ancestral environmental mycobacteria, whose extant representatives are known under the name of Mycobacterium canettii. Recent whole genome analyses in combination with different phenotypic screens have provided key insights into the evolution of M. tuberculosis and closely related members regrouped in the M. tuberculosis complex (MTBC). They have also elucidated novel virulence determinants that are essential for these obligate pathogens. In this review, we present the most recent evolutionary models of the MTBC and various factors that have contributed to the outstanding evolutionary success of the tuberculosis agent

COV2HTML: A Visualization and Analysis Tool of Bacterial Next Generation Sequencing (NGS) Data for Postgenomics Life Scientists

International audienceCOV2HTML is an interactive web interface, which is addressed to biologists, and allows performing both coverage visualization and analysis of NGS alignments performed on prokaryotic organisms (bacteria and phages). It combines two processes: a tool that converts the huge NGS mapping or coverage files into light specific coverage files containing information on genetic elements; and a visualization interface allowing a real-time analysis of data with optional integration of statistical results. To demonstrate the scope of COV2HTML, the program was tested with data from two published studies. The first data were from RNA-seq analysis of Campylobacter jejuni, based on comparison of two conditions with two replicates. We were able to recover 26 out of 27 genes highlighted in the publication using COV2HTML. The second data comprised of stranded TSS and RNA-seq data sets on the Archaea Sulfolobus solfataricus. COV2HTML was able to highlight most of the TSSs from the article and allows biologists to visualize both TSS and RNA-seq on the same screen. The strength of the COV2HTML interface is making possible NGS data analysis without software installation, login, or a long training period. A web version is accessible at https://mmonot.eu/COV2HTML/. This website is free and open to users without any login requirement

ESX-1-Independent Horizontal Gene Transfer by Mycobacterium tuberculosis Complex Strains

International audienceData on the bacterial sex-mediated impact on mycobacterial evolution are limited. Hence, our results presented here are of importance as they clearly demonstrate the capacity of a wide range of human- and animal-adapted Mycobacterium tuberculosis complex (MTBC) strains to transfer chromosomal DNA to selected strains of Mycobacterium canettii

A dual transcript-discovery approach to improve the delimitation of gene features from RNA-seq data in the chicken model

The sequence of the chicken genome, like several other draft genome sequences, is presently not fully covered. Gaps, contigs assigned with low confidence and uncharacterized chromosomes result in gene fragmentation and imprecise gene annotation. Transcript abundance estimation from RNA sequencing (RNA-seq) data relies on read quality, library complexity and expression normalization. In addition, the quality of the genome sequence used to map sequencing reads, and the gene annotation that defines gene features, must also be taken into account. A partially covered genome sequence causes the loss of sequencing reads from the mapping step, while an inaccurate definition of gene features induces imprecise read counts from the assignment step. Both steps can significantly bias interpretation of RNA-seq data. Here, we describe a dual transcript-discovery approach combining a genome-guided gene prediction and a de novo transcriptome assembly. This dual approach enabled us to increase the assignment rate of RNA-seq data by nearly 20% as compared to when using only the chicken reference annotation, contributing therefore to a more accurate estimation of transcript abundance. More generally, this strategy could be applied to any organism with partial genome sequence and/or lacking a manually-curated reference annotation in order to improve the accuracy of gene expression studies