Evaluating impacts of syntenic block detection strategies on rearrangement phylogeny using Mycobacterium tuberculosis isolates.

MOTIVATION: The phylogenetic signal of structural variation informs a more comprehensive understanding of evolution. As (near-)complete genome assembly becomes more commonplace, the next methodological challenge for inferring genome rearrangement trees is the identification of syntenic blocks of orthologous sequences. In this article, we studied 94 reference quality genomes of primarily Mycobacterium tuberculosis (Mtb) isolates as a benchmark to evaluate these methods. The clonal nature of Mtb evolution, the manageable genome sizes, along with substantial levels of structural variation make this an ideal benchmarking dataset. RESULTS: We tested several methods for detecting homology and obtaining syntenic blocks and two methods for inferring phylogenies from them, then compared the resulting trees to the standard method's tree, inferred from nucleotide substitutions. We found that, not only the choice of methods, but also their parameters can impact results, and that the tree inference method had less impact than the block determination method. Interestingly, a rearrangement tree based on blocks from the Cactus whole-genome aligner was fully compatible with the highly supported branches of the substitution-based tree, enabling the combination of the two into a high-resolution supertree. Overall, our results indicate that accurate trees can be inferred using genome rearrangements, but the choice of the methods for inferring homology requires care. AVAILABILITY AND IMPLEMENTATION: Analysis scripts and code written for this study are available at https://gitlab.com/LPCDRP/rearrangement-homology.pub and https://gitlab.com/LPCDRP/syntement. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel and reported compensatory mutations in rpoABC genes found in drug resistant tuberculosis outbreaks.

Background: Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by Mycobacterium tuberculosis. RIF resistance is caused by mutations in rpoB, at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in rpoABC genes have been shown to alleviate the fitness cost of rpoB:S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations. Objectives: We performed an association study on a globally representative set of 4,309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations. Results and conclusions: Of the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in rpoABC for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to rpoB:S450L. However, an outbreak of 22 closely related isolates each carried three rpoB mutations, the rare RIFR markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of rpoABC mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional rpoABC mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant M. tuberculosis strains with restored fitness, which pose a greater risk of causing resistant outbreaks.

Structure-Aware Mycobacterium tuberculosis Functional Annotation Uncloaks Resistance, Metabolic, and Virulence Genes.

Accurate and timely functional genome annotation is essential for translating basic pathogen research into clinically impactful advances. Here, through literature curation and structure-function inference, we systematically update the functional genome annotation of Mycobacterium tuberculosis virulent type strain H37Rv. First, we systematically curated annotations for 589 genes from 662 publications, including 282 gene products absent from leading databases. Second, we modeled 1,711 underannotated proteins and developed a semiautomated pipeline that captured shared function between 400 protein models and structural matches of known function on Protein Data Bank, including drug efflux proteins, metabolic enzymes, and virulence factors. In aggregate, these structure- and literature-derived annotations update 940/1,725 underannotated H37Rv genes and generate hundreds of functional hypotheses. Retrospectively applying the annotation to a recent whole-genome transposon mutant screen provided missing function for 48% (13/27) of underannotated genes altering antibiotic efficacy and 33% (23/69) required for persistence during mouse tuberculosis (TB) infection. Prospective application of the protein models enabled us to functionally interpret novel laboratory generated pyrazinamide (PZA)-resistant mutants of unknown function, which implicated the emerging coenzyme A depletion model of PZA action in the mutants' PZA resistance. Our findings demonstrate the functional insight gained by integrating structural modeling and systematic literature curation, even for widely studied microorganisms. Functional annotations and protein structure models are available at https://tuberculosis.sdsu.edu/H37Rv in human- and machine-readable formats. IMPORTANCE Mycobacterium tuberculosis, the primary causative agent of tuberculosis, kills more humans than any other infectious bacterium. Yet 40% of its genome is functionally uncharacterized, leaving much about the genetic basis of its resistance to antibiotics, capacity to withstand host immunity, and basic metabolism yet undiscovered. Irregular literature curation for functional annotation contributes to this gap. We systematically curated functions from literature and structural similarity for over half of poorly characterized genes, expanding the functionally annotated Mycobacterium tuberculosis proteome. Applying this updated annotation to recent in vivo functional screens added functional information to dozens of clinically pertinent proteins described as having unknown function. Integrating the annotations with a prospective functional screen identified new mutants resistant to a first-line TB drug, supporting an emerging hypothesis for its mode of action. These improvements in functional interpretation of clinically informative studies underscore the translational value of this functional knowledge. Structure-derived annotations identify hundreds of high-confidence candidates for mechanisms of antibiotic resistance, virulence factors, and basic metabolism and other functions key in clinical and basic tuberculosis research. More broadly, they provide a systematic framework for improving prokaryotic reference annotations.

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

BACKGROUND: The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. RESULTS: Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome. CONCLUSIONS: Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.