Biology subjects

Ha, D. T. M.

Publications and source records attributed to Ha, D. T. M..

6 recordsLinked to original sources

bioRxiv2026

Whole blood transcriptional responses associated with bacterial burden in pulmonary tuberculosis

BackgroundPulmonary TB (PTB) patients present with a wide range of pre-treatment Mycobacterium tuberculosis (Mtb) burdens, which predict poor treatment outcomes. We sought to identify immune pathways and biomarkers associated with pre-treatment Mtb burden. MethodsWe conducted whole-blood RNA sequencing in 295 Vietnamese adults with PTB, quantifying bacterial load using GeneXpert Ct values. Weighted gene co-expression network analysis (WGCNA) identified gene modules, pathways, and hub genes associated with Mtb burden. Deconvolution analysis assessed contributions of immune cell types. Key findings were validated in independent PTB (n=171) and TB meningitis (TBM, n=281) cohorts, and publicly available animal datasets. We used non-linear regression for variable selection to identify gene predictors of Mtb burden and hurdle regression to model Mtb loads below the detection limit. FindingsHigher Mtb burden correlated with prolonged symptom duration, elevated neutrophil and monocyte counts, and severe lung pathology. WGCNA identified a 1,093-gene module associated with Mtb burden, characterized by coordinated innate-adaptive pathway interactions. Within this module, IFN-{gamma} signaling participates in modulating the increase of innate signaling (Toll-like, Nod-like receptors, TNF) and the decrease of adaptive signaling (T- and B-cell receptor) pathways in high-burden patients. These responses were primarily driven by neutrophils and classical monocytes. CNIH4 emerged as the strongest hub-gene and a top predictor of bacterial burden, with consistent validation across independent PTB and TBM cohorts. InterpretationOur study reveals systemic innate-adaptive immune dynamics underlying bacterial burden in PTB and identifies CNIH4 as a potential biomarker for treatment monitoring as well as a therapeutic target. FundingNational Institute of Health; Wellcome Trust, UK. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed from Jan 1, 2000, to Dec 10, 2025, without language restrictions, for human studies examining the relationship between Mycobacterium tuberculosis (Mtb) burden and host blood transcriptional responses in pulmonary tuberculosis. Search terms were used in combination as follows: ("Tuberculosis, Pulmonary" OR "pulmonary tuberculosis" OR PTB) AND ("Mycobacterium tuberculosis" OR mycobacter*) AND ("bacterial load" OR "bacterial burden" OR "sputum smear" OR "smear grade" OR xpert OR genexpert OR "cycle threshold" OR ct OR "time to positivity" OR TTP OR CFU OR "molecular bacterial load" OR MBLA)) AND (blood OR "whole blood" OR "peripheral blood") AND (RNA-seq OR "RNA sequencing" OR transcriptom* OR "gene expression" OR microarray). We included studies of active pulmonary tuberculosis that measured quantitative or semi-quantitative bacterial burden and profiled host blood transcriptome-wide responses at baseline, reporting either differential expression by burden strata or associations between bacterial burden and host gene expression. We excluded studies limited to latent tuberculosis, animal or in vitro models, diagnostic or prognostic signature studies without bacterial burden measurement, studies focused on treatment response, and studies using targeted assays without transcriptome-wide profiling. This search identified 12 articles describing blood transcriptional signatures for tuberculosis diagnosis, prognosis, and treatment response. However, only one study directly examined the relationship between pre-treatment bacterial burden and whole-blood transcriptome-wide profiles. That study demonstrated differences in systemic gene expression between patients with higher and lower sputum mycobacterial load and proposed a 20-gene blood signature associated with bacterial burden. However, the analysis was limited by small sample size, lack of pathway-level and cellular interpretation or assessment of correlation between signature with bacterial load. Added value of this studyOur study advances existing evidence by leveraging the wide spectrum of pre-treatment bacterial burden observed in routine clinical populations, quantified using GeneXpert Ct values, and integrating this with whole-blood RNA sequencing in large, well-characterized clinical cohorts. Through network-based transcriptomic analysis, immune cell deconvolution, and non-linear modelling, we identify a bacterial burden-associated gene network characterized by enhanced innate inflammatory signaling and relative suppression of adaptive immune pathways, predominantly driven by neutrophils and classical monocytes and modulated by IFN-{gamma} signaling. Within this network, CNIH4 emerges as a central hub gene and a robust predictor of bacterial burden, with consistent validation across independent pulmonary tuberculosis and tuberculous meningitis cohorts. Implications of all the available evidenceTaken together, the available evidence indicates that host blood transcriptional responses correlate with bacterial burden in pulmonary tuberculosis, but previous studies have provided limited insight into the underlying immune processes. Our findings strengthen the biological link between pre-treatment mycobacterial burden and systemic immune dysregulation, showing that higher bacterial burden is associated with transcriptional state marked by coordinated upregulation of innate immune responses and downregulation of adaptive immune pathways. These results support the use of host transcriptomic profiling as a biologically informative complement to sputum-based measures of bacterial burden and highlight burden-associated immune pathways, particularly CNIH4, as a potential target for treatment monitoring and host-directed therapeutic development.

immunology↗

bioRxiv2025

Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INHR) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc2155 (Msm) as a model, we show that brief exposure to sublethal INH (2x IC50) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (> 500x IC50) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INHR strains (p-value = 1.09x10-7). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis.

microbiology↗

bioRxiv2025

Evolution of Mycobacterium tuberculosis transcription regulation is associated with increased transmission and drug resistance

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for thousands of years causing variation in virulence, transmissibility, and disease phenotypes. To identify bacterial contributors to phenotypic diversity, we developed new RNA-seq and phylogenomic tools to capture hundreds of Mtb isolate transcriptomes, link transcriptional and genetic variation, and find associations between variants and epidemiologic traits. Across 274 Mtb clinical isolates, we uncovered unexpected diversity in expression of virulence genes which we linked to known and previously unrecognized regulators. Surprisingly, we found that many isolates harbor variants associated with decreased expression of EsxA (Esat6) and EsxB (Cfp10), which are virulence effectors, dominant T cell antigens, and immunodiagnostic targets. Across >55,000 isolates, these variants associate with increased transmissibility, especially in drug resistant Mtb strains. Our data suggest expression of key Mtb virulence genes is evolving across isolates in part to optimize fitness under drug pressure, with sobering implications for immunodiagnostics and next-generation vaccines.

microbiology↗

bioRxiv2023

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. We determined the in-vitro rifampicin survival fraction by minimum duration of killing assay in isoniazid susceptible (IS, n=119) and resistant (IR, n=84) M. tuberculosis isolates. Then we correlated the rifampicin tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal IR isolates collected from patients were analyzed for changes in rifampicin tolerance and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation. This indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed IR association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P-trend=0.0003). The high tolerance in IR isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Furthermore, the high rifampicin tolerant IR isolates have survival potential similar to multi-drug resistant isolates. These findings suggest that IR tuberculosis needs to be evaluated for high rifampicin tolerance to improve treatment regimen and prevent the risk of MDR-TB emergence.

microbiology↗

bioRxiv2023

Whole blood transcriptional profiles and the pathogenesis of tuberculous meningitis

BackgroundMortality and morbidity from tuberculous meningitis (TBM) are frequent and strongly associated with the inflammatory response to Mycobacterium tuberculosis infection. However, the mechanisms driving the associations are uncertain. We sought to identify the gene modules, hubs and pathways associated with the pathogenesis and mortality from TBM, and to identify which best-predicted death. MethodsWe used whole blood RNA sequencing to obtain transcriptional profiles from 281 Vietnamese adults with TBM (207 HIV-negative; 74 HIV-positive), 295 with pulmonary TB (PTB), and 30 healthy controls. The TBM cohort was divided randomly into a discovery cohort (n=142) and a validation cohort (n=139). Weighted gene co-expression network analysis identified clusters of genes (or modules) and hub genes associated with death or disease severity. An overrepresentation analysis identified pathways associated with TBM mortality, with a consensus analysis identifying consensual patterns between HIV-positive and HIV-negative individuals. A multivariate elastic-net Cox regression model selected the candidate predictors of TBM mortality, then model prediction performance using logistic regression and internal bootstrap validation to choose best predictors. ResultsOverall, TBM mortality was associated with increased neutrophil activation and decreased T and B cell activation pathways. Death from TBM was associated with increased angiogenesis in HIV-positive adults, and with activated TNF signaling and down-regulated extracellular matrix organization in HIV-negative adults. PTB and TBM have similar transcriptional profiles compared to healthy controls, although inflammatory genes were more activated in HIV-positive than HIV-negative TBM. The expression of four hub genes - MCEMP1, NELL2, ZNF354C and CD4 - were strongly predictive of death from TBM (AUC 0.80 and 0.86 for HIV-negative and HIV-positive, respectively). ConclusionsWhole blood transcriptional profiling revealed that TBM is associated with a characteristic systemic inflammatory response, similar to that invoked by pulmonary tuberculosis, but with key gene modules, hubs and pathways strongly associated with death. Our analysis suggests a novel 4-gene biomarker for predicting death from TBM, but also opens a new window into TBM pathogenesis that may reveal novel therapeutic targets for this lethal disease.

molecular biology↗

bioRxiv2023

High-throughput phenogenotyping of Mycobacteria tuberculosis clinical strains reveals bacterial determinants of treatment outcomes

BackgroundCombatting the tuberculosis (TB) epidemic caused by Mycobacterium tuberculosis (Mtb) necessitates a better understanding of the factors contributing to patient clinical outcomes and transmission. While host and environmental factors have been evaluated, the impact of Mtb genetic background and phenotypic diversity is underexplored. Previous work has made associations between Mtb genetic lineages and some clinical and epidemiological features, but the bacterial traits underlying these connections are largely unknown. MethodsWe developed a high-throughput functional genomics platform for defining genotype-phenotype relationships across a panel of Mtb clinical isolates. These phenotypic fitness profiles function as intermediate traits which can be linked to Mtb genetic variants and associated with clinical and epidemiological outcomes. We applied this approach to a collection of 158 Mtb strains from a study of Mtb transmission in Ho Chi Minh City, Vietnam. Mtb strains were genetically tagged in multiplicate, which allowed us to pool the strains and assess in vitro competitive fitness using deep sequencing across a set of 14 host-relevant antibiotic and metabolic conditions. Phylogenetic and monogenic associations with these intermediate traits were identified and then associated with clinical outcomes. FindingsMtb clinical strains have a broad range of growth and drug response dynamics that can be clustered by their phylogenetic relationships. We identified novel monogenic associations with Mtb fitness in various metabolic and antibiotic conditions. Among these, we find that mutations in Rv1339, a phosphodiesterase, which were identified through their association with slow growth in glycerol, are further associated with treatment failure. We also identify a previously uncharacterized subclade of Lineage 1 strains (L1.1.1.1) that is phenotypically distinguished by slow growth under most antibiotic and metabolic stress conditions in vitro. This clade is associated with cavitary disease, treatment failure, and demonstrates increased transmission potential. InterpretationHigh-throughput phenogenotyping of Mtb clinical strains enabled bacterial intermediate trait identification that can provide a mechanistic link between Mtb genetic variation and patient clinical outcomes. Mtb strains associated with cavitary disease, treatment failure, and transmission potential display intermediate phenotypes distinguished by slow growth under various antibiotic and metabolic conditions. These data suggest that Mtb growth regulation is an adaptive advantage for host bacterial success in human populations, in at least some circumstances. These data further suggest markers for the underlying bacterial processes that govern these clinical outcomes. FundingNational Institutes of Allergy and Infectious Diseases: P01 AI132130 (SS, SMF); P01 AI143575 (XW, SMF); U19 AI142793 (QL, SMF); 5T32AI132120-03 (SS); 5T32AI132120-04 (SS); 5T32AI049928-17 (SS) Wellcome Trust Fellowship in Public Health and Tropical Medicine: 097124/Z/11/Z (NTTT) National Health and Medical Research Council (NHMRC)/A*STAR joint call: APP1056689 (SJD) The funding sources had no involvement in study methodology, data collection, analysis, and interpretation nor in the writing or submission of the manuscript. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe used different combinations of the words mycobacterium tuberculosis, tuberculosis, clinical strains, intermediate phenotypes, genetic barcoding, phenogenomics, cavitary disease, treatment failure, and transmission to search the PubMed database for all studies published up until January 20th, 2022. We only considered English language publications, which biases our search. Previous work linking Mtb determinants to clinical or epidemiological data has made associations between bacterial lineage, or less frequently, genetic polymorphisms to in vitro or in vivo models of pathogenesis, transmission, and clinical outcomes such as cavitary disease, treatment failure, delayed culture conversion, and severity. Many of these studies focus on the global pandemic Lineage 2 and Lineage 4 Mtb strains due in part to a deletion in a polyketide synthase implicated in host-pathogen interactions. There are a number of Mtb GWAS studies that have led to novel genetic determinants of in vitro drug resistance and tolerance. Previous Mtb GWAS analyses with clinical outcomes did not experimentally test any predicted phenotypes of the clinical strains. Published laboratory-based studies of Mtb clinical strains involve relatively small numbers of strains, do not identify the genetic basis of relevant phenotypes, or link findings to the corresponding clinical outcomes. There are two recent studies of other pathogens that describe phenogenomic analyses. One study of 331 M. abscessus clinical strains performed one-by-one phenotyping to identify bacterial features associated with clearance of infection and another details a competition experiment utilizing three barcoded Plasmodium falciparum clinical isolates to assay antimalarial fitness and resistance. Added value of this studyWe developed a functional genomics platform to perform high-throughput phenotyping of Mtb clinical strains. We then used these phenotypes as intermediate traits to identify novel bacterial genetic features associated with clinical outcomes. We leveraged this platform with a sample of 158 Mtb clinical strains from a cross sectional study of Mtb transmission in Ho Chi Minh City, Vietnam. To enable high-throughput phenotyping of large numbers of Mtb clinical isolates, we applied a DNA barcoding approach that has not been previously utilized for the high-throughput analysis of Mtb clinical strains. This approach allowed us to perform pooled competitive fitness assays, tracking strain fitness using deep sequencing. We measured the replicative fitness of the clinical strains in multiplicate under 14 metabolic and antibiotic stress condition. To our knowledge, this is the largest phenotypic screen of Mtb clinical isolates to date. We performed bacterial GWAS to delineate the Mtb genetic variants associated with each fitness phenotype, identifying monogenic associations with several conditions. We then defined Mtb phenotypic and genetic features associated with clinical outcomes. We find that a subclade of Mtb strains, defined by variants largely involved in fatty acid metabolic pathways, share a universal slow growth phenotype that is associated with cavitary disease, treatment failure and increased transmission potential in Vietnam. We also find that mutations in Rv1339, a poorly characterized phosphodiesterase, also associate with slow growth in vitro and with treatment failure in patients. Implications of all the available evidencePhenogenomic profiling demonstrates that Mtb strains exhibit distinct growth characteristics under metabolic and antibiotic stress conditions. These fitness profiles can serve as intermediate traits for GWAS and association with clinical outcomes. Intermediate phenotyping allows us to examine potential processes by which bacterial strain differences contribute to clinical outcomes. Our study identifies clinical strains with slow growth phenotypes under in vitro models of antibiotic and host-like metabolic conditions that are associated with adverse clinical outcomes. It is possible that the bacterial intermediate phenotypes we identified are directly related to the mechanisms of these outcomes, or they may serve as markers for the causal yet unidentified bacterial determinants. Via the intermediate phenotyping, we also discovered a surprising diversity in Mtb responses to the new anti-mycobacterial drugs that target central metabolic processes, which will be important in considering roll-out of these new agents. Our study and others that have identified Mtb determinants of TB clinical and epidemiological phenotypes should inform efforts to improve diagnostics and drug regimen design.

microbiology↗