Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Transcriptomic Characterization of Tuberculous Sputum Reveals a Host Warburg Effect and Microbial Cholesterol Catabolism.

The crucial transmission phase of tuberculosis (TB) relies on infectious sputum and yet cannot easily be modeled. We applied one-step RNA sequencing (RNA-Seq) to sputum from infectious TB patients to investigate the host and microbial environments underlying transmission of Mycobacterium tuberculosis. In such TB sputa, compared to non-TB controls, transcriptional upregulation of inflammatory responses, including an interferon-driven proinflammatory response and a metabolic shift toward glycolysis, was observed in the host. Among all bacterial sequences in the sputum, approximately 1.5% originated from M. tuberculosis, and its transcript abundance was lower in HIV-1-coinfected patients. Commensal bacterial abundance was reduced in the presence of M. tuberculosis infection. Direct alignment to the genomes of the predominant microbiota species also reveals differential adaptation, whereby firmicutes (e.g., streptococci) displayed a nonreplicating phenotype with reduced transcription of ribosomal proteins and reduced activities of ATP synthases, while Neisseria and Prevotella spp. were less affected. The transcriptome of sputum M. tuberculosis more closely resembled aerobic replication and shared similarity in carbon metabolism to in vitro and in vivo models with significant upregulation of genes associated with cholesterol metabolism and downstream propionate detoxification pathways. In addition, and counter to previous reports on intracellular M. tuberculosis infection in vitro, M. tuberculosis in sputum was zinc, but not iron, deprived, and the phoP loci were also significantly downregulated, suggesting that the pathogen is likely extracellular in location. IMPORTANCE Although a few studies have described the microbiome composition of TB sputa based on 16S ribosomal DNA, these studies did not compare to non-TB samples and the nature of the method does not allow any functional inference. This is the first study to apply such technology using clinical specimens and obtained functional transcriptional data on all three aspects simultaneously. We anticipate that an improved understanding on the biological interactions in the respiratory tract may also allow novel interventions, such as those involving microbiome manipulation or inhibitor targeting disease-specific metabolic pathways.

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

Delayed effects of transcriptional responses in Mycobacterium tuberculosis exposed to nitric oxide suggest other mechanisms involved in survival.

Mycobacterium tuberculosis has succeeded as a human pathogen for tens of thousands of years thanks to its ability to resist and adapt to the adverse conditions it encounters upon infection. Bacterial adaptation to stress is commonly viewed in the context of transcriptional regulation, with the implicit expectation that an initial transcriptomic response is tightly coupled to an ensuing proteomic response. However, after challenging M. tuberculosis with nitric oxide we found that the rapid transcriptional responses, detectable within minutes of nitric oxide exposure, typically took several hours to manifest on the protein level. Furthermore, early proteomic responses were dominated by the degradation of a set of proteins, specifically those containing damaged iron-sulphur clusters. Overall, our findings are consistent with transcriptional responses participating mostly in late-stage recovery rather than in generating an immediate resistance to nitric oxide stress, suggesting that survival of M. tuberculosis under acute stress is contingent on mechanisms other than transcriptional regulation. These findings provide a revised molecular understanding of an important human pathogen.

A Rab20-Dependent Membrane Trafficking Pathway Controls M. tuberculosis Replication by Regulating Phagosome Spaciousness and Integrity.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination.

Characterization of progressive HIV-associated tuberculosis using 2-deoxy-2-[18F]fluoro-D-glucose positron emission and computed tomography.

Tuberculosis is classically divided into states of latent infection and active disease. Using combined positron emission and computed tomography in 35 asymptomatic, antiretroviral-therapy-naive, HIV-1-infected adults with latent tuberculosis, we identified ten individuals with pulmonary abnormalities suggestive of subclinical, active disease who were substantially more likely to progress to clinical disease. Our findings challenge the conventional two-state paradigm and may aid future identification of biomarkers that are predictive of progression.

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site.

Admixture mapping of tuberculosis and pigmentation-related traits in an African-European hybrid cattle population.

Admixture mapping affords a powerful approach to genetic mapping of complex traits and may be particularly suited to investigation in cattle where many breeds and populations are hybrids of the two divergent ancestral genomes, derived from Bos taurus and Bos indicus. Here we design a minimal genome wide SNP panel for tracking ancestry in recent hybrids of Holstein-Friesian and local Arsi zebu in a field sample from a region of high bovine tuberculosis (BTB) endemicity in the central Ethiopian highlands. We first demonstrate the utility of this approach by mapping the red coat color phenotype, uncovering a highly significant peak over the MC1R gene and a second peak with no previously known candidate gene. Secondly, we exploit the described differential susceptibility to BTB between the ancestral strains to identify a region in which Bos taurus ancestry associates, at suggestive significance, with skin test positivity. Interestingly, this association peak contains the toll-like receptor gene cluster on chromosome 6. With this work we have shown the potential of admixture mapping in hybrid domestic animals with divergent ancestral genomes, a recurring condition in domesticated species.

Bovine viral diarrhea virus outbreak in a beef cow herd in South Dakota.

CASE DESCRIPTION: 136 pregnant beef cows were purchased in the fall of 2003. The following spring, 128 cows calved as expected; 8 cows were believed to have aborted with the fetuses unavailable for evaluation. Of the 128 calves born, 8 died within 2 weeks after birth and 9 were born with congenital abnormalities. CLINICAL FINDINGS: Cows and their calves were evaluated for bovine viral diarrhea virus (BVDV) infection. Forty-four of 120 calves, but 0 cows, tested positive for BVDV antigen by immunohistochemical staining of ear notch specimens. TREATMENT AND OUTCOME: Five BVDV test-positive calves died shortly after weaning, and the remaining 39 BVDV test-positive calves were moved to an isolated feedlot and retested for BVDV at 5 to 6 months of age; 36 had positive results, which indicated that they were persistently infected (PI) with BVDV, whereas 3 had negative results, which indicated that they were transiently infected with BVDV at the time of the first test. All PI calves were infected with the same BVDV type 2a strain. As yearlings, 17 of the 36 PI calves died peracutely with lesions consistent with mucosal disease, 6 died without gross lesions, and 2 were euthanized because of chronic ill thrift. The remaining 11 PI calves appeared healthy and were sold for slaughter. Screening of the following year's calf crop for BVDV by use of immunohistochemical staining of ear-notch specimens yielded negative results for all calves. CLINICAL RELEVANCE: Introduction of BVDV into a naïve cow herd resulted in a loss of 44% of the calf crop subsequent to reproductive loss, poor thrift, and mucosal disease.

Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis.

Comparison of genome sequences from clinical isolates of Mycobacterium tuberculosis with phylogenetically-related pathogens Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium leprae reveals diversity amongst genes associated with vitamin B12-related metabolism. Diversity is generated by gene deletion events, differential acquisition of genes by horizontal transfer, and single nucleotide polymorphisms (SNPs) with predicted impact on protein function and transcriptional regulation. Differences in the B12 synthesis pathway, methionine biosynthesis, fatty acid catabolism, and DNA repair and replication are consistent with adaptations to different environmental niches and pathogenic lifestyles. While there is no evidence of further gene acquisition during expansion of the M. tuberculosis complex, the emergence of other forms of genetic diversity provides insights into continuing host-pathogen co-evolution and has the potential to identify novel targets for disease intervention.

Effect of isoniazid on antigen-specific interferon-γ secretion in latent tuberculosis.

Treatment of persons with latent tuberculosis (TB) infection at greatest risk of reactivation is an important component of TB control and elimination strategies. Biomarkers evaluating the effectiveness of treatment of latent TB infection have not yet been identified. This information would enhance control efforts and assist the evaluation of new treatment regimes. We designed a two-group, two-arm, randomised clinical study of tuberculin skin test-positive participants: 26 with documented contact with TB patients and 34 with non-documented contact. Participants in each group were randomly assigned to the immediate- or deferred-isoniazid treatment arms. Assays of in vitro interferon (IFN)-γ secretion in response to recombinant Rv1737 and overlapping synthetic peptide pools from various groups of immunodominant proteins were performed. During isoniazid therapy, a significant increase from baseline in the proportion of IFN-γ responders to the 10-kDa culture filtrate protein, Rv2031, Rv0849, Rv1986, Rv2659c, Rv2693c and the recombinant Rv1737 protein was observed (p⩽0.05). The peptide pool of Rv0849 and Rv1737 recombinant proteins induced the highest percentage of IFN-γ responders after isoniazid therapy. The in vitro IFN-γ responses to these proteins might represent useful markers to evaluate changes associated with treatment of latent TB infection.

Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRP(Mt)) at endogenous expression levels using a specific α-CRP(Mt) antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRP(Mt) binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRP(Mt)-binding site represented only a minor portion of this transcriptional reprogramming with ∼ 19% of those representing transcriptional regulators potentially controlled by CRP(Mt). The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRP(Mt) can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRP(Mt)-binding sites.

Noncoding RNA in Mycobacteria.

Efforts to understand the molecular basis of mycobacterial gene regulation are dominated by a protein-centric view. However, there is a growing appreciation that noncoding RNA, i.e., RNA that is not translated, plays a role in a wide variety of molecular mechanisms. Noncoding RNA comprises rRNA, tRNA, 4.5S RNA, RnpB, and transfer-messenger RNA, as well as a vast population of regulatory RNA, often dubbed "the dark matter of gene regulation." The regulatory RNA species comprise 5' and 3' untranslated regions and a rapidly expanding category of transcripts with the ability to base-pair with mRNAs or to interact with proteins. Regulatory RNA plays a central role in the bacterium's response to changes in the environment, and in this article we review emerging information on the presence and abundance of different types of noncoding RNA in mycobacteria.

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.

Defining the range of pathogens susceptible to Ifitm3 restriction using a knockout mouse model.

The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.

Genome-wide mapping of transcriptional start sites defines an extensive leaderless transcriptome in Mycobacterium tuberculosis.

Deciphering physiological changes that mediate transition of Mycobacterium tuberculosis between replicating and nonreplicating states is essential to understanding how the pathogen can persist in an individual host for decades. We have combined RNA sequencing (RNA-seq) of 5' triphosphate-enriched libraries with regular RNA-seq to characterize the architecture and expression of M. tuberculosis promoters. We identified over 4,000 transcriptional start sites (TSSs). Strikingly, for 26% of the genes with a primary TSS, the site of transcriptional initiation overlapped with the annotated start codon, generating leaderless transcripts lacking a 5' UTR and, hence, the Shine-Dalgarno sequence commonly used to initiate ribosomal engagement in eubacteria. Genes encoding proteins with active growth functions were markedly depleted from the leaderless transcriptome, and there was a significant increase in the overall representation of leaderless mRNAs in a starvation model of growth arrest. The high percentage of leaderless genes may have particular importance in the physiology of nonreplicating M. tuberculosis.

Mapping of genotype-phenotype diversity among clinical isolates of mycobacterium tuberculosis by sequence-based transcriptional profiling.

Genome sequencing has identified an extensive repertoire of single nucleotide polymorphisms among clinical isolates of Mycobacterium tuberculosis, but the extent to which these differences influence phenotypic properties of the bacteria remains to be elucidated. To determine whether these polymorphisms give rise to phenotypic diversity, we have integrated genome data sets with RNA sequencing to assess their impact on the comparative transcriptome profiles of strains belonging to M. tuberculosis Lineages 1 and 2. We observed clear correlations between genotype and transcriptional phenotype. These arose by three mechanisms. First, lineage-specific changes in amino acid sequence of transcriptional regulators were associated with alterations in their ability to control gene expression. Second, changes in nucleotide sequence were associated with alteration of promoter activity and generation of novel transcriptional start sites in intergenic regions and within coding sequences. We show that in some cases this mechanism is expected to generate functionally active truncated proteins involved in innate immune recognition. Finally, genes showing lineage-specific patterns of differential expression not linked directly to primary mutations were characterized by a striking overrepresentation of toxin-antitoxin pairs. Taken together, these findings advance our understanding of mycobacterial evolution, contribute to a systems level understanding of this important human pathogen, and more broadly demonstrate the application of state-of-the-art techniques to provide novel insight into mechanisms by which intergenic and silent mutations contribute to diversity.

A restatement of the natural science evidence base relevant to the control of bovine tuberculosis in Great Britain.

Bovine tuberculosis (bTB) is a very important disease of cattle in Great Britain, where it has been increasing in incidence and geographical distribution. In addition to cattle, it infects other species of domestic and wild animals, in particular the European badger (Meles meles). Policy to control bTB is vigorously debated and contentious because of its implications for the livestock industry and because some policy options involve culling badgers, the most important wildlife reservoir. This paper describes a project to provide a succinct summary of the natural science evidence base relevant to the control of bTB, couched in terms that are as policy-neutral as possible. Each evidence statement is placed into one of four categories describing the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA.

In Mycobacterium tuberculosis, the genes Rv1954A-Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin-antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis.