Serge Mostowy

Genomic Deletions Suggest a Phylogeny for the Mycobacterium tuberculosis Complex

To better understand the evolution of the Mycobacterium tuberculosis complex, subspecies were tested for large sequence polymorphisms. Samples with greater numbers of deletions, without exception, were missing all the same regions that were deleted from samples with lesser numbers of deletions. Principal genetic groups based on single-nucleotide polymorphisms were restricted to one of the deletion-based groups, and isolates that shared genotypes based on molecular epidemiological markers were assigned almost exclusively to the same deletion type. The data provide compelling evidence that human tuberculosis did not originate from the present-day bovine form. Genomic deletions present themselves as an attractive modality to study the evolution of the M. tuberculosis complex.

NOD2-Deficient Mice Have Impaired Resistance to Mycobacterium tuberculosis Infection through Defective Innate and Adaptive Immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2−/− mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2−/− mice had decreased production of type 1 cytokines and reduced recruitment of CD8+ and CD4+ T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2−/− mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2−/− mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.

PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence.

Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.

The in vitro evolution of BCG vaccines

The bacillus Calmette-Géurin (BCG) family of vaccines currently implemented to prevent tuberculosis (TB) consist of clonal bacterial strains independently shaped by nearly a half-century of evolution. Derived from virulent Mycobacterium bovis, daughter strains of BCG were additionally passaged under the same laboratory conditions that resulted in its original attenuation. Genomic loss of the RD1 region has been demonstrated to coincide with attenuation from virulence, while deletions occurring after the loss of RD1 are speculated to be responsible for BCGs over-attenuation. To provide a more complete description of their total genomic variation, the genomic content of BCG strains are investigated by Affymetrix GeneChip. Because clinical isolates of M. tuberculosis have previously been characterized via GeneChip interrogation, analysis permits the comparison of in vivo versus in vitro evolution of M. tuberculosis complex subspecies. The contrast between the two modes of evolution are discussed in its relevance towards TB pathogenicity.

THE EVOLUTION OF TRADE‐OFFS: TESTING PREDICTIONS ON RESPONSE TO SELECTION AND ENVIRONMENTAL VARIATION

Abstract The concept of phenotypic trade‐offs is a central element in evolutionary theory. In general, phenotypic models assume a fixed trade‐off function, whereas quantitative genetic theory predicts that the trade‐off function will change as a result of selection. For a linear trade‐off function selection will readily change the intercept but will have to be relatively stronger to change the slope. We test these predictions by examining the trade‐off between fecundity and flight capability, as measured by dorso‐longitudinal muscle mass, in four different populations of the sand cricket, Gryllus firmus. Three populations were recently derived from the wild, and the fourth had been in the laboratory for 19 years. We hypothesized that the laboratory population had most likely undergone more and different selection from the three wild populations and therefore should differ from these in respect to both slope and intercept. Because of geographic variation in selection, we predicted a general difference in intercept among the four populations. We further tested the hypothesis that this intercept will be correlated with proportion macropterous and that this relationship will itself vary with environmental conditions experienced during both the nymphal and adult period. Observed variation in the phenotypic trade‐off was consistent with the predictions of the quantitative genetic model. These results point to the importance of modeling trade‐offs as dynamic rather than static relationships. We discuss how phenotypic models can incorporate such variation. The phenotypic trade‐off between fecundity and dorso‐longitudinal muscle mass is determined in part by variation in body size, illustrating the necessity of considering trade‐offs to be multifactorial rather than simply bivariate relationships.

Reduced expression of antigenic proteins MPB70 and MPB83 in Mycobacterium bovis BCG strains due to a start codon mutation in sigK

Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high‐ and low‐producing strains. By quantitative reverse transcription polymerase chain reaction (RT‐PCR), we determined that transcription of the antigen‐encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50‐fold in low‐producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low‐producing strains compared with high‐producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low‐producing BCG strains. Complementation of a low‐producing strain, BCG Pasteur, with wild‐type sigK fully restored MPB70 and MPB83 production. Microarray‐based analysis and confirmatory RT‐PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low‐producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.

Genomic Analysis Distinguishes Mycobacterium africanum

ABSTRACT Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of “modern” M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of “M. africanum-like” isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.

Genomic interrogation of the dassie bacillus reveals it as a unique RD1 mutant within the Mycobacterium tuberculosis complex.

Despite their remarkable genetic homology, members of the Mycobacterium tuberculosis complex express very different phenotypes, most notably in their spectra of clinical presentation. For example, M. tuberculosis is regarded as pathogenic to humans, whereas members having deleted RD1, such as Mycobacterium microti and Mycobacterium bovis BCG, are not. The dassie bacillus, an infrequent variant of the M. tuberculosis complex characterized as being most similar to M. microti, is the causative agent of tuberculosis (TB) in the dassie (Procavia capensis). Intriguingly, the dassie bacillus is not pathogenic to rabbits or guinea pigs and has never been documented to infect humans. Although it was identified more than a half-century ago, the reasons behind its attenuation are unknown. Because large sequence polymorphisms have presented themselves as the most obvious genomic distinction among members of the M. tuberculosis complex, the DNA content of the dassie bacillus was interrogated by Affymetrix GeneChip to identify regions that are absent from it but present in M. tuberculosis H37Rv. Comparison has led to the identification of nine regions of difference (RD), five of which are shared with M. microti (RDs 3, 7, 8, 9, and 10). Although the dassie bacillus does not share the other documented deletions in M. microti (RD1(mic), RD5(mic), MID1, MID2, and MID3), it has endured unique deletions in the regions of RD1, RD5, N-RD25, and Rv3081-Rv3082c (virS). RD1(das), affecting only Rv3874-Rv3877, is the smallest natural deletion of the RD1 region uncovered and points to genes within this region that are likely implicated in virulence. Newfound deletions from the dassie bacillus are discussed in relation to their evolutionary and biological significance.

Extensive Genomic Polymorphism within Mycobacterium avium

We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms (LSPs) comprising over 700 kb that distinguish M. avium subsp. avium from M. avium subsp. paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains the in vitro mycobactin dependence of M. avium subsp. paratuberculosis.

Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti‐SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis

It has recently been advanced that Mycobacterium tuberculosis sigma factor K (SigK) positively regulates expression of the antigenic proteins MPB70 and MPB83. As expression of these proteins differs between M. tuberculosis (low) and Mycobacterium bovis (high), this study set out to determine whether M. bovis lacks a functional SigK repressor (anti‐SigK). By comparing genes near sigK in M. tuberculosis H37Rv and M. bovis AF2122/97, we observed that Rv0444c, annotated as unknown function, had variable sequence in M. bovis. Analysis of in vitro mpt70/mpt83 expression and Rv0444c sequencing across M. tuberculosis complex (MTC) members revealed that high‐level expression was associated with a mutated Rv0444c. Complementation of M. bovis bacillus Calmette‐Guerin Russia, a high producer of MPB70/MPB83, with wild‐type Rv0444c resulted in a significant decrease in mpb70/mpb83 expression. Conversely, a M. tuberculosis H37Rv mutant which expressed sigK but not Rv0444c manifested the M. bovis phenotype of high‐level MPB70/MPB83 expression. Further support that Rv0444c encodes the anti‐SigK was obtained by yeast two‐hybrid studies, where the N‐terminal region of Rv0444c‐encoded protein interacted with SigK. Together these findings indicate that Rv0444c encodes the regulator of SigK (RskA) and mutations in this gene explain high‐level MPT70/MPT83 expression by certain MTC members.