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Chao, M. C.

Publications and source records attributed to Chao, M. C..

11 recordsLinked to original sources
bioRxiv2026

Spatial transcriptomics of pediatric tuberculosis lymph nodes reveals a conserved hypoxia_SPP1 niche

Pediatric tuberculosis (TB) often presents as primary disease involving the mediastinal lymph nodes; however, its granuloma microenvironment remains poorly characterized because these population patient tissues are rarely available for study. Here, we use spatial transcriptomics to profile 60 granulomas from four children aged 4 to 36 months. We identified nine recurrent transcriptional microenvironments shared across granulomas, despite their marked histological heterogeneity. One macrophage-rich microenvironment showed strong hypoxia-associated activity and secreted phosphoprotein 1 (SPP1) enrichment. This microenvironment was present in both non-necrotic and necrotic lesions and overlapped with a Mtb-persistence macrophage signature previously identified in murine TB lymph nodes. Pathway analysis revealed the enrichment of both antimicrobial and regulatory programs within this microenvironment, suggesting partial immune control rather than uniform inflammatory or suppressive activity in this region. SPP1 expression was associated with regions enriched for both M1- and M2-like macrophages, suggesting a macrophage program that extends beyond conventional polarization classifications and may be influenced by the local granuloma microenvironment. We further observed a similar spatial association between hypoxia-associated activity and SPP1 expression in an independent adult TB dataset (pooled Spearman {rho}=0.65) and validated this association at protein level by multiplex immunofluorescence in our pediatric TB granuloma sample. Overall, this study identified a hypoxia-associated SPP1-enriched microenvironment that may contribute to incomplete bacterial clearance and could provide a framework for evaluating host-directed strategies to enhance sterilizing immunity without exacerbating tissue damage.

immunology↗
bioRxiv2026

Protection against disseminated disease following reinfection with Mycobacterium tuberculosis requires CD8+ lymphocytes

Concomitant immunity is protective adaptive immunity conferred by pre-existing infection against subsequent exposures. This phenomenon is effective in the context of human tuberculosis (TB) and has been successfully recapitulated in non-human primates (NHPs). We previously demonstrated that depletion of CD4 T cells prior to secondary challenge with Mycobacterium tuberculosis (Mtb) only partially abrogated protection, implicating a critical role for additional cell types. Here, we describe the effects of depleting CD8 expressing cells in this model. CD4+ T cells in the airway were unable to compensate for lost cytotoxicity due to CD8 depletion. Contrary to the results of CD4 depletion, CD8 depleted animals exhibited a unique inability to limit Mtb dissemination, with significant lymph node involvement despite a short 4-week infection. These data suggest that concomitant immunity relies on non-redundant roles played by CD8+ lymphocytes. Thus, future vaccines should be designed to induce effective responses by both CD4+ and CD8+ populations to maximize efficacy.

immunology↗
bioRxiv2026

Population-level genome sequencing reveals distinct Mycobacterium tuberculosis intrahost mutational trajectories in simian immunodeficiency virus co-infected and antiretroviral treated non-human primates

Whole genome sequencing of Mycobacterium tuberculosis (Mtb) populations from clinical samples has increasingly identified genes undergoing selection within and between hosts that drive differential infection and treatment outcomes. However, the intrahost Mtb mutational landscape--especially in the context of human immunodeficiency virus coinfection and antiretroviral therapy (ART)--remains less clear, as do the potential impacts of such mutations on Mtb infection dynamics. Here, we performed whole genome sequencing of Mtb populations isolated from 477 infected tissues across 20 non-human primates, including animals co-infected with simian immunodeficiency virus (SIV) with or without virological suppression by ART. We identified 116 mutations that emerged during infection, including those that are overrepresented within individual tissues and a subset that are shared across tissues during Mtb dissemination. We further find differential mutation trajectories across treatment groups, with higher mutation rate and bacterial outgrowth in SIV-infected hosts and increased prevalence of oxidative damage-associated mutations in coinfected animals on ART. Finally, we demonstrate a common pattern of mutation in Mtb lipid metabolism and polyketide synthase genes and identify a subset of NHP-derived mutations that have also independently arisen in human clinical isolates. Together, our population-based sequencing uncovers Mtb diversification during early infection, captures discrete bacterial dissemination events and infers differential immune pressures faced by Mtb in the setting of SIV-Mtb coinfection and ART suppression. ImportanceTuberculosis remains a leading cause of death worldwide, especially in people living with HIV. How HIV infection and antiretroviral therapy impacts Mycobacterium tuberculosis (Mtb) intrahost evolution remains unclear. Using whole genome sequencing from hundreds of infected tissues from non-human primates, we find that simian immunodeficiency virus co-infected hosts and those receiving antiretroviral therapy exact different immune pressures on Mtb leading to different mutation rates and types of DNA damage that are incurred. Mtb mutations were enriched in genes involved in lipid metabolisms and some of these are also seen in human TB strains. This work highlights the role of immune pressure to alter bacterial pathways that may enable Mtb adaptation to the host.

microbiology↗
bioRxiv2025

Protection against reinfection with Mycobacterium tuberculosis extends across heterologous Mtb lineages

Immunological memory elicited either through previous or ongoing M. tuberculosis (Mtb) infection provides a critical mechanism by which hosts protect against re-infection and disease progression upon Mtb re-exposure. Conversely, the uneven competition between distinct Mtb strains suggest certain bacterial clades have enhanced ability to spread across communities and circulate globally, potentially by evading memory responses gained by prior infection with genomically different strains. To address whether memory responses induced by one strain can protect against a genetically distinct strain, we conducted a heterologous reinfection study in cynomolgus macaques involving primary infection by a Lineage 4 Erdman Mtb strain and subsequent re-challenge by a Lineage 2 strain, HT-L2. Recent epidemiologic studies have shown that the clade to which HT-L2 belongs has been spreading successfully over the last decade in Lima, Peru. Here, through microbiologic, PET-CT imaging and sequencing of Mtb genomic barcodes, we show that reinfected animals developed fewer lung lesions and controlled both pulmonary and disseminated forms of infection better than naive animals without prior exposure to Mtb. Our data support that protection against reinfection is not limited by Mtb lineage, providing optimism that vaccines can be effective across populations and geographic locations.

immunology↗
bioRxiv2025

Characterizing PET CT patterns and bacterial dissemination features of tuberculosis relapse in the macaque model

Tuberculosis (TB) relapse after appropriate drug treatment is poorly understood but critical to developing shorter treatment regimens. Using a cynomolgus macaque model of human TB, macaques with active TB disease were treated with a short course of isoniazid and rifampin and subsequently infected with SIV. Serial clinical, microbiologic, immunologic and position emission and computed tomography (PET CT) assessments were performed to identify risk factors of relapse. Of the 12 animals, eight developed radiologically defined relapse including four that had clinical and/or microbiologic signs. Greater gross pathology and bacterial burden were observed in relapse animals. PET CT characteristics before, during, and at the end of treatment were similar amongst relapse and non-relapse animals. We show that complete sterilization or very low Mtb burden is protective against SIV-induced TB relapse but cannot be predicted by PET CT. Using bar-coded M. tuberculosis, we found that Mtb dissemination during relapse originated from both lung and thoracic lymph nodes, underscoring the importance of lymph nodes as a reservoir. By matching bar-coded Mtb and serial PET CT, we also demonstrate that not every site of persistent Mtb growth after drug treatment is capable of dissemination and relapse, underscoring the complex nature of drug treatment and relapse.

immunology↗
bioRxiv2024

CD4 T cells and CD8α+ lymphocytes are necessary for intravenous BCG-induced protection against tuberculosis in macaques

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide despite widespread intradermal (ID) BCG vaccination in newborns. We previously demonstrated that changing the route and dose of BCG vaccination from 5{xi}105 CFU ID to 5{xi}107 CFU intravenous (IV) resulted in prevention of infection and disease in a rigorous, highly susceptible non-human primate model of TB. Identifying the immune mechanisms of protection for IV BCG will facilitate development of more effective vaccines against TB. Here, we depleted select lymphocyte subsets in IV BCG vaccinated macaques prior to Mtb challenge to determine the cell types necessary for that protection. Depletion of CD4 T cells or all CD8 expressing lymphoycytes (both innate and adaptive) resulted in loss of protection in most macaques, concomitant with increased bacterial burdens ([~]4-5 log10 thoracic CFU) and dissemination of infection. In contrast, depletion of only adaptive CD8{beta}+ T cells did not significantly reduce protection against disease. Our results demonstrate that CD4 T cells and innate CD8+ lymphocytes are critical for IV BCG-induced protection, supporting investigation of how eliciting these cells and their functions can improve future TB vaccines. One Sentence SummaryAntibody depletion of lymphocytes in rhesus macques demonstrates key roles for CD4 T cells and innate-like CD8+ lymphocytes in conferring sterilizing immunity against tuberculosis following intravenous BCG vaccination.

immunology↗
bioRxiv2024

Markov Field network integration of multi-modal data predicts effects of immune system perturbations on intravenous BCG vaccination in macaques

Analysis of multi-modal datasets can identify multi-scale interactions underlying biological systems, but can be beset by spurious connections due to indirect impacts propagating through an unmapped biological network. For example, studies in macaques have shown that BCG vaccination by an intravenous route protects against tuberculosis, correlating with changes across various immune data modes. To eliminate spurious correlations and identify critical immune interactions in a public multi-modal dataset (systems serology, cytokines, cytometry) of vaccinated macaques, we applied Markov Fields (MF), a data-driven approach that explains vaccine efficacy and immune correlations via multivariate network paths, without requiring large numbers of samples (i.e. macaques) relative to multivariate features. Furthermore, we find that integrating multiple data modes with MFs helps to remove spurious connections. Finally, we used the MF to predict outcomes of perturbations at various immune nodes, including a B-cell depletion that induced network-wide shifts without reducing vaccine protection, which we validated experimentally.

systems biology↗
bioRxiv2024

Vaginal lactobacilli produce anti-inflammatory beta-carboline compounds.

The vaginal microbiome is a lactobacilli-dominant, human-adapted community consistently found in people around the world. Presence of lactobacilli-dominant vaginal microbial communities, apart from Lactobacillus iners, is associated with reduced vaginal inflammation and reduced levels of pro-inflammatory cytokines. Loss of lactobacilli-dominance is associated with inflammatory conditions such as bacterial vaginosis. It remains unclear if health-associated lactobacilli actively promote the anti-inflammatory immune homeostasis of the vaginal mucosa and if so, by which mechanisms. We have identified that Lactobacillus crispatus, a key vaginal bacterial species, secretes a family of {beta}-carboline compounds with anti-inflammatory activity. These compounds suppress NF{kappa}B and interferon signaling downstream of multiple pattern recognition receptors in human macrophages and significantly dampen type I interferon receptor activation. Topical vaginal application of an anti-inflammatory {beta}-carboline compound, perlolyrine, was sufficient to significantly reduce vaginal inflammation in a mouse model of genital herpes infection. Together, we identify a key family of compounds by which vaginal lactobacilli mediate host immune homeostasis and could highlight a new therapeutic avenue for vaginal inflammation.

microbiology↗
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↗
bioRxiv2023

Chemically-induced targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit this targeted protein degradation (TPD) approach for antibiotic development remains nascent due to our limited understanding of which bacterial proteins will be labile TPD targets. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the major human pathogen M. tuberculosis (Mtb). We find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex is sufficient to degrade many, but not all, endogenous Msm proteins, profoundly inhibiting bacterial growth for some targets. We also show that TPD can potentiate the effects of existing antibiotics targeting the same pathways and complexes. Together, our results identify specific endogenous mycobacterial proteins as attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

microbiology↗
bioRxiv2022

Tuberculosis treatment failure associated with evolution of antibiotic resilience

Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

microbiology↗