Mycobacterium Tuberculosis Genome Research Articles

SRNAdeep: a novel tool for bacterial sRNA prediction based on DistilBERT encoding mode and deep learning algorithms

BackgroundBacterial small regulatory RNA (sRNA) plays a crucial role in cell metabolism and could be used as a new potential drug target in the treatment of pathogen-induced disease. However, experimental methods for identifying sRNAs still require a large investment of human and material resources.MethodsIn this study, we propose a novel sRNA prediction model called sRNAdeep based on the DistilBERT feature extraction and TextCNN methods. The sRNA and non-sRNA sequences of bacteria were considered as sentences and then fed into a composite model consisting of deep learning models to evaluate classification performance.ResultsBy filtering sRNAs from BSRD database, we obtained a validation dataset comprised of 2438 positive and 4730 negative samples. The benchmark experiments showed that sRNAdeep displayed better performance in the various indexes compared to previous sRNA prediction tools. By applying our tool to Mycobacterium tuberculosis (MTB) genome, we have identified 21 sRNAs within the intergenic and intron regions. A set of 272 targeted genes regulated by these sRNAs were also captured in MTB. The coding proteins of two genes (lysX and icd1) are implicated in drug response, with significant active sites related to drug resistance mechanisms of MTB.ConclusionIn conclusion, our newly developed sRNAdeep can help researchers identify bacterial sRNAs more precisely and can be freely available from https://github.com/pyajagod/sRNAdeep.git.

Polymerase Chain Reaction for Detection of Mycobacterium tuberculosis in Granulomatous Anterior Uveitis – An Analysis of 51 Eyes

ABSTRACT Purpose To evaluate the role of polymerase chain reaction (PCR) in detecting Mycobacterium tuberculosis (MTb) in granulomatous anterior uveitis and comparing the results with those of Mantoux test, QuantiFERON-TB Gold test, and High-Resolution Computed Tomography of Chest (HRCT-chest). Methods Data of 51 eyes of 38 patients with clinical features of granulomatous anterior uveitis undergoing anterior chamber fluid aspiration for PCR for MTb targeting IS6110 and MPB64 were studied. Results Out of 38 patients, 25 (65.8%) were positive for MTb genome on PCR. 24 patients were tested for Mantoux, out of which 12 patients (50%) were positive and 12 patients (50%) were negative. 22 out of 38 patients had undergone QuantiFERON-TB Gold (QFT) test, out of which 10 (45.5%) were positive and 12 (54.5%) were negative. 36 out of 38 patients had undergone HRCT. Eight out of 38 (21.1%) patients had significant findings in HRCT-chest, as well as PCR positivity for MTb genome while 12 (31.6%) patients did not have any significant findings on HRCT-chest, but were positive for PCR. Conclusion PCR for MTb is a valuable tool in the diagnosis of tubercular anterior uveitis. PCR can be positive even in cases of negative Mantoux test, QFT, and HRCT-chest in granulomatous anterior uveitis.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R & D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

Comprehensive analysis of Mycobacterium tuberculosis genomes reveals genetic variations in bacterial virulence

Tuberculosis, a disease caused by Mycobacterium tuberculosis (Mtb), is a significant health problem worldwide. Here, we developed a method to detect large insertions and deletions (indels), which have been generally understudied. Leveraging this framework, we performed a comprehensive analysis of single nucleotide variants and small and large indels across 1,960 Mtb clinical isolates. Comparing the distribution of variants demonstrated that gene disruptive variants are underrepresented in genes essential for bacterial survival. An evolutionary analysis revealed that Mtb genomes are enriched in partially deleterious mutations. Genome-wide association studies identified small and large deletions in eccB2 significantly associated with patient prognosis. Additionally, we unveil significant associations with antibiotic resistance in 23 non-canonical genes. Among these, large indels are primarily found in genetic regions of Rv1216c, Rv1217c, fadD11, and ctpD. This study provides a comprehensive catalog of genetic variations and highlights their potential impact for the future treatment and risk prediction of tuberculosis.

Genetic diversity within diagnostic sputum samples is mirrored in the culture of Mycobacterium tuberculosis across different settings

Culturing and genomic sequencing of Mycobacterium tuberculosis (MTB) from tuberculosis (TB) cases is the basis for many research and clinical applications. The alternative, culture-free sequencing from diagnostic samples, is promising but poses challenges to obtain and analyse the MTB genome. Paradoxically, culture is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. To unravel this paradox we generate high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics analysis. Careful downstream comparisons reveal sources of sputum-culture incongruences due to false positive/negative variation associated with factors like low input MTB DNA or variable genomic depths. After accounting for these factors, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants’ frequency (0.98). The combined analysis from five different settings and more than 100 available samples shows that our results can be extrapolated to different TB epidemic scenarios, demonstrating that for the cases tested culture accurately mirrors clinical samples.

Ligand-binding characteristics of CYP51 Mycobacterium tuberculosis in relation to marine steroid compounds

CYP51 steroid-14α-demethylases are members of a large superfamily of cytochrome P450 enzymes found in all kingdoms of living organisms, and catalyze the 14α-demethylation reaction of a number of natural steroids, including lanosterol, obtusifoliol, and 24,25-dihydrolanosterol. CYP51 are important components of the eukaryotic steroid biosynthetic chain, and thus represent one of the main targets for antifungal therapy. A 14α-demethylase CYP51 homologous gene has also been found in the genome of Mycobacterium tuberculosis. At the same time, M. tuberculosis lacks the de novo pathway for steroid biosynthesis. Conservation of CYP51 among the Mycobacterium genus and colocalization in the genome with 3Fe-4S ferredoxin Rv0763c, which maintains its catalytic activity in vitro, may indirectly indicate the involvement of MTCYP51 in a biochemical process important for mycobacteria. In order to characterize the specificity of the MTCYP51 active site to various compounds of isoprenoid nature, we obtained a highly purified MTCYP51 and, using spectrophotometric titration and surface plasmon resonance methods, studied the interaction of MTCYP51 with steroids from marine organisms obtained in the Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences. The investigated compounds represent a wide range of evolutionarily ancient isoprenoids. The results showed that MTCYP51 is able to bind structurally diverse steroid derivatives in the active site. The conducted studies suggest the biological role of MTCYP51 for pathogenic mycobacteria, which consists in the binding and possible metabolism of exogenous bioregulatory isoprenoids in vivo.

Pan-Lineage Mycobacterium tuberculosis Reference Genome for Enhanced Molecular Diagnosis.

In Mycobacterium tuberculosis (MTB) control, whole genome sequencing-based molecular drug susceptibility testing (molDST-WGS) has emerged as a pivotal tool. However, the current reliance on a single-strain reference limits molDST-WGS's true potential. To address this, we introduce a new pan-lineage reference genome, "MtbRf". We assembled "unmapped" reads from 3,614 MTB genomes (751 L1; 881 L2; 1,700 L3; and 282 L4) into 35 shared, annotated contigs (54 CDSs). We constructed MtbRf through: 1) searching for contig homologs among genome database that precipitating results uniquely within Mycobacteria genus; 2) comparing genomes with H37Rv ("lift-over") to define 18 insertions; and 3) filling gaps in H37Rv with insertions. MtbRf adds 1.18% sequences to H37rv, salvaging >60% of previously unmapped reads. Transcriptomics confirmed gene-expression of new CDSs. The new variants provided a moderate DST predictive value (AUROC 0.60-0.75). MtbRf thus unveils previously hidden genomic information, and lays the foundation for lineage-specific molDST-WGS.

A study on factors influencing delayed sputum conversion in newly diagnosed pulmonary tuberculosis based on bacteriology and genomics

Conversion of sputum from positive to negative is one of the indicators to evaluate the efficacy of anti-tuberculosis treatment (ATT). We investigate the factors associated with delayed sputum conversion after 2 or 5 months of ATT from the perspectives of bacteriology and genomics. A retrospective study of sputum conversion in sputum positive 1782 pulmonary tuberculosis (PTB) was conducted from 2021 to 2022 in Beijing, China. We also designed a case-matched study including 24 pairs of delayed-sputum-conversion patients (DSCPs) and timely-sputum-conversion patients (TSCPs), and collect clinical isolates from DSCPs before and after ATT and initial isolates of TSCPs who successfully achieved sputum conversion to negative after 2 months of ATT. A total of 75 strains were conducted drug sensitivity testing (DST) of 13 anti-TB drugs and whole-genome sequencing (WGS) to analyze the risk factors of delayed conversion and the dynamics changes of drug resistance and genomics of Mycobacterium tuberculosis (MTB) during ATT. We found TSCPs have better treatment outcomes and whose initial isolates show lower levels of drug resistance. Clinical isolates of DSCPs showed dynamically changing of resistance phenotypes and intra-host heterogeneity. Single nucleotide polymorphism (SNP) profiles showed large differences between groups. The study provided insight into the bacteriological and genomic variation of delayed sputum conversion. It would be helpful for early indication of sputum conversion and guidance on ATT.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Pangenome-wide association study reveals the selective absence of CRISPR genes (Rv2816c-19c) in drug-resistant Mycobacterium tuberculosis.

The presence of intermittently dispersed insertion sequences and transposases in the Mycobacterium tuberculosis (Mtb) genome makes intra-genome recombination events inevitable. Understanding their effect on the gene repertoires (GR), which may contribute to the development of drug-resistant Mtb, is critical. In this study, publicly available WGS data of clinical Mtb isolates (endemic region n = 2,601; non-endemic region n = 1,130) were de novo assembled, filtered, scaffolded into assemblies, and functionally annotated. Out of 2,601 Mtb WGS data sets from endemic regions, 2,184 (drug resistant/sensitive: 1,386/798) qualified as high quality. We identified 3,784 core genes, 123 softcore genes, 224 shell genes, and 762 cloud genes in the pangenome of Mtb clinical isolates from endemic regions. Sets of 33 and 39 genes showed positive and negative associations (P < 0.01) with drug resistance status, respectively. Gene ontology clustering showed compromised immunity to phages and impaired DNA repair in drug-resistant Mtb clinical isolates compared to the sensitive ones. Multidrug efflux pump repressor genes (Rv3830c and Rv3855c) and CRISPR genes (Rv2816c-19c) were absent in the drug-resistant Mtb. A separate WGS data analysis of drug-resistant Mtb clinical isolates from the Netherlands (n = 1130) also showed the absence of CRISPR genes (Rv2816c-17c). This study highlights the role of CRISPR genes in drug resistance development in Mtb clinical isolates and helps in understanding its evolutionary trajectory and as useful targets for diagnostics development.IMPORTANCEThe results from the present Pan-GWAS study comparing gene sets in drug-resistant and drug-sensitive Mtb clinical isolates revealed intricate presence-absence patterns of genes encoding DNA-binding proteins having gene regulatory as well as DNA modification and DNA repair roles. Apart from the genes with known functions, some uncharacterized and hypothetical genes that seem to have a potential role in drug resistance development in Mtb were identified. We have been able to extrapolate many findings of the present study with the existing literature on the molecular aspects of drug-resistant Mtb, further strengthening the relevance of the results presented in this study.

Large-scale statistical analysis of Mycobacterium tuberculosis genome sequences identifies compensatory mutations associated with multi-drug resistance

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has a significant impact on global health worldwide. The development of multi-drug resistant strains that are resistant to the first-line drugs isoniazid and rifampicin threatens public health security. Rifampicin and isoniazid resistance are largely underpinned by mutations in rpoB and katG respectively and are associated with fitness costs. Compensatory mutations are considered to alleviate these fitness costs and have been observed in rpoC/rpoA (rifampicin) and oxyR’-ahpC (isoniazid). We developed a framework (CompMut-TB) to detect compensatory mutations from whole genome sequences from a large dataset comprised of 18,396 M. tuberculosis samples. We performed association analysis (Fisher’s exact tests) to identify pairs of mutations that are associated with drug-resistance, followed by mediation analysis to identify complementary or full mediators of drug-resistance. The analyses revealed several potential mutations in rpoC (N = 47), rpoA (N = 4), and oxyR’-ahpC (N = 7) that were considered either ‘highly likely’ or ‘likely’ to confer compensatory effects on drug-resistance, including mutations that have previously been reported and validated. Overall, we have developed the CompMut-TB framework which can assist with identifying compensatory mutations which is important for more precise genome-based profiling of drug-resistant TB strains and to further understanding of the evolutionary mechanisms that underpin drug-resistance.

Efflux pump gene single-nucleotide variants associated with resistance in Mycobacterium tuberculosis isolates with discrepant drug genotypes

IntroductionSingle-nucleotide variants (SNVs) in Mycobacterium tuberculosis (M. tuberculosis) genomes can predict multidrug resistance (MDR) but not all phenotype-genotype correlations can be explained. We investigated SNVs in efflux pumps (EPs) in the context of M. tuberculosis drug resistance. MethodsWe analysed 2221 M. tuberculosis genomes from 1432 susceptible and 200 MDR, 172 pre-extensively drug resistant (XDR) and 417 XDR isolates. Analysis of 47 EP genes was conducted using MTB-VCF, an in-house bioinformatics pipeline. SNVs were categorized according to their SIFT/Polyphen scores. Resistance genotypes were also called using the TB-Profiler tool. ResultsGenome comparisons between susceptible and drug resistant (DR) isolates identified 418 unique SNVs in EP of which; 53.5% were in MDR, 68.9% in pre-XDR and 61.3% in XDR isolates. Twenty EPs had unique SNVs with a high SIFT/PolyPhen score, comprising 38 unique SNVs. Sixteen SNVs across 12 EP genes were significantly associated with drug resistance and enriched in pre-XDR and XDR strains. These comprised 12 previously reported SNVs (in Rv0191, Rv0507, Rv0676, Rv1217, Rv1218, Rv1273, Rv1458, Rv1819, and Rv2688) and 4 novel SNVs (in Rv1877 and Rv2333). We investigated their presence in genomes of 52 MDR isolates with phenotype-genotype discrepancies to rifampicin (RIF), isoniazid (INH), or fluoroquinolones. SNVs associated with RIF and INH (Rv1217_1218, Rv1819, Rv0450, Rv1458, Rv3827, Rv0507, Rv0676, Rv1273, and Rv2333), and with fluoroquinolone (Rv2688) resistance were present in these discrepant strains. ConclusionsConsidering SNVs in EPs as part of M. tuberculosis genome-based resistance interpretation may add value, especially in evaluation of XDR resistance in strains with phenotype-genotype discrepancies.

Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

The exceptions that prove the rule—a historical view of bedaquiline susceptibility

In the accompanying study, Nimmo and colleagues estimated the dates of emergence of mutations in mmpR5 (Rv0678), the most important resistance gene to the anti-tuberculosis drug bedaquiline, in over 3500 geographically diverse Mycobacterium tuberculosis genomes. This provided important insights to improve the design and analysis of clinical trials, as well as the World Health Organization catalogue of resistance mutations, the global reference for interpreting genotypic antimicrobial susceptibility testing results.

Pangenome and genomic signatures linked to the dominance of the lineage-4 of Mycobacterium tuberculosis isolated from extrapulmonary tuberculosis patients in western Ethiopia.

The lineage 4 (L4) of Mycobacterium tuberculosis (MTB) is not only globally prevalent but also locally dominant, surpassing other lineages, with lineage 2 (L2) following in prevalence. Despite its widespread occurrence, factors influencing the expansion of L4 and its sub-lineages remain poorly understood both at local and global levels. Therefore, this study aimed to conduct a pan-genome and identify genomic signatures linked to the elevated prevalence of L4 sublineages among extrapulmonary TB (EPTB) patients in western Ethiopia. A cross-sectional study was conducted at an institutional level involving confirmed cases of extrapulmonary tuberculosis (EPTB) patients from August 5, 2018, to December 30, 2019. A total of 75 MTB genomes, classified under lineage 4 (L4), were used for conducting pan-genome and genome-wide association study (GWAS) analyses. After a quality check, variants were identified using MTBseq, and genomes were de novo assembled using SPAdes. Gene prediction and annotation were performed using Prokka. The pan-genome was constructed using GET_HOMOLOGUES, and its functional analysis was carried out with the Bacterial Pan-Genome Analysis tool (BPGA). For GWAS analysis, Scoary was employed with Benjamini-Hochberg correction, with a significance threshold set at p-value ≤ 0.05. The analysis revealed a total of 3,270 core genes, predominantly associated with orthologous groups (COG) functions, notably in the categories of '[R] General function prediction only' and '[I] Lipid transport and metabolism'. Conversely, functions related to '[N] Cell motility' and '[Q] Secondary metabolites biosynthesis, transport, and catabolism' were primarily linked to unique and accessory genes. The pan-genome of MTB L4 was found to be open. Furthermore, the GWAS study identified genomic signatures linked to the prevalence of sublineages L4.6.3 and L4.2.2.2. Apart from host and environmental factors, the sublineage of L4 employs distinct virulence factors for successful dissemination in western Ethiopia. Given that the functions of these newly identified genes are not well understood, it is advisable to experimentally validate their roles, particularly in the successful transmission of specific L4 sublineages over others.

Comparisons of genome assembly tools for characterization of Mycobacterium tuberculosis genomes using hybrid sequencing technologies.

Next-generation sequencing of Mycobacterium tuberculosis, the infectious agent causing tuberculosis, is improving the understanding of genomic diversity of circulating lineages and strain-types, and informing knowledge of drug resistance mutations. An increasingly popular approach to characterizing M. tuberculosis genomes (size: 4.4 Mbp) and variants (e.g., single nucleotide polymorphisms (SNPs)) involves the de novo assembly of sequence data. We compared the performance of genome assembly tools (Unicycler, RagOut, and RagTag) on sequence data from nine drug resistant M. tuberculosis isolates (multi-drug (MDR) n = 1; pre-extensively-drug (pre-XDR) n = 8) generated using Illumina HiSeq, Oxford Nanopore Technology (ONT) PromethION, and PacBio platforms. Our investigation found that Unicycler-based assemblies had significantly higher genome completeness (~98.7%; p values = 0.01) compared to other assembler tools (RagOut = 98.6%, and RagTag = 98.6%). The genome assembly sizes (bp) across isolates and sequencers based on RagOut was significantly longer (p values < 0.001) (4,418,574 ± 8,824 bp) than Unicycler and RagTag assemblies (Unicycler = 4,377,642 ± 55,257 bp, and RagTag = 4,380,711 ± 51,164 bp). RagOut-based assemblies had the fewest contigs (~32) and the longest genome size (4,418,574 bp; vs. H37Rv reference size 4,411,532 bp) and therefore were chosen for downstream analysis. Pan-genome analysis of Illumina and PacBio hybrid assemblies revealed the greatest number of detected genes (4,639 genes; H37Rv reference contains 3,976 genes), while Illumina and ONT hybrid assemblies produced the highest number of SNPs. The number of genes from hybrid assemblies with ONT and PacBio long-reads (mean: 4,620 genes) was greater than short-read assembly alone (4,478 genes). All nine RagOut hybrid genome assemblies detected known mutations in genes associated with MDR-TB and pre-XDR-TB. Unicycler software performed the best in terms of achieving contiguous genomes, whereas RagOut improved the quality of Unicycler's genome assemblies by providing a longer genome size. Overall, our approach has demonstrated that short-read and long-read hybrid assembly can provide a more complete genome assembly than short-read assembly alone by detecting pan-genomes and more genes, including IS6110, and SNPs.

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Utility of Mycobacterium tuberculosis Genome Sequencing Snapshots to Assess Transmission Dynamics Over Time.

We explored the utility of brief Mycobacterium tuberculosis whole-genome sequencing (WGS) "snapshots" at a sentinel site within Lima, Peru, for evaluating local transmission dynamics over time. Within a 17-km2 area, 15 of 70 (21%) isolates with WGS collected during 2011-2012 and 22 of 81 (27%) collected during 2020-2021 were clustered (P = .414), and additional isolates clustered with those from outside the area. Isolates from the later period were disproportionately related to large historic clusters in Lima from the earlier period. WGS snapshots at a sentinel site may not be useful for monitoring transmission, but monitoring the persistence of large transmission clusters might be.