Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominantMycobacterium tuberculosismutations in therpoBgene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains ofM. tuberculosis(wild type) to those of their respective rifampin-resistant,rpoBmutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon- and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. BothrpoBmutants showed significant upregulation of the polyketide synthase genesppsA-ppsEanddrrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids inM. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins.ppsA(Rv2931),ppsB(Rv2932), andppsC(Rv2933) were also found to be upregulated more than 10-fold in the BeijingrpoBmutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in bothrpoBmutant isolates. These data suggest thatrpoBmutation inM. tuberculosismay trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistantM. tuberculosisinfections.

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m/z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalase-peroxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance-conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels.

Mycobacterium tuberculosis isolates of the Manila sublineage are genetically homogeneous. In this study, we used whole-genome sequencing (WGS) to type a collection of 36 M. tuberculosis isolates of the Manila family. WGS enabled the subtyping of these 36 isolates into at least 10 distinct clusters. Our results indicate that WGS is a powerful approach to determining the relatedness of Manila family M. tuberculosis isolates.

The transmission and persistence of Mycobacterium tuberculosis within high risk populations is a threat to tuberculosis (TB) control. In the current study, we used whole genome sequencing (WGS) to decipher the transmission dynamics and microevolution of M. tuberculosis ON-A, an endemic strain responsible for an ongoing outbreak of TB in an urban homeless/under-housed population. Sixty-one M. tuberculosis isolates representing 57 TB cases from 1997 to 2013 were subjected to WGS. Sequencing data was integrated with available epidemiological information and analyzed to determine how the M. tuberculosis ON-A strain has evolved during almost two decades of active transmission. WGS offers higher discriminatory power than traditional genotyping techniques, dividing the M. tuberculosis ON-A strain into 6 sub-clusters, each defined by unique single nucleotide polymorphism profiles. One sub-cluster, designated ON-A^NM (Natural Mutant; 26 isolates from 24 cases) was also defined by a large, 15 kb genomic deletion. WGS analysis reveals the existence of multiple transmission chains within the same population/setting. Our results help validate the utility of WGS as a powerful tool for identifying genomic changes and adaptation of M. tuberculosis.