Biology subjects

Chase, M. R.

Publications and source records attributed to Chase, M. R..

8 recordsLinked to original sources

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↗

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↗

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

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases that are predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is an endoribonuclease hypothesized to play a major role in mRNA degradation due to its essentiality in mycobacteria and its role in mRNA degradation in gram- negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome- wide in the non-pathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in the degradation of at least 89% of protein-coding genes, with leadered transcripts generally being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in both M. smegmatis and M. tuberculosis, and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negatives. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming RNase E as a broad contributor to M. tuberculosis transcriptome structure.

molecular biology↗

bioRxiv2021

Arginine methylation helps SepIVA balance regulation of septation and elongation in Mycobacterium smegmatis

Growth of mycobacterial cells requires successful coordination between elongation and septation of the cell wall. However, it is not clear which factors directly mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of these methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG, and that this regulation may impact polar elongation. Finally, we map SepIVAs two regulatory functions to different sites on the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

microbiology↗

bioRxiv2021

Spontaneous control of SIV replication does not prevent immune dysregulation and bacterial dissemination in animals co-infected with M. tuberculosis

Individuals infected with both HIV and Mycobacterium tuberculosis (Mtb) are more likely to develop severe Tuberculosis (TB) disease than HIV-naive individuals. To understand how a chronic pre-existing Simian immunodeficiency virus (SIV) infection impairs the early immune response to Mtb, we used the Mauritian cynomolgus macaque (MCM) model of SIV/Mtb co-infection. We examined the relationship between peripheral viral control and Mtb burden, Mtb dissemination, and immunological function between SIV+ spontaneous controllers, SIV+ non-controllers, and SIV-naive MCM who were challenged with a barcoded Mtb Erdman strain and necropsied six weeks post infection. Mycobacterial burden was highest in the SIV+ non-controllers in all assessed tissues. In lung granulomas, we found the frequency of CD4+ T cells producing TNF was reduced in all SIV+ MCM, but CD4+ T cells producing IFN{gamma} were only lower in the SIV+ non-controllers. Further, while all SIV+ MCM had more PD1+ and TIGIT+ T cells in the lung granulomas relative to SIV-naive MCM, SIV+ controllers exhibited the highest frequency of cells expressing these markers. To measure the effect of SIV infection on within-host bacterial dissemination, we sequenced the molecular barcodes of Mtb present in each tissue and characterized the complexity of the Mtb populations. While Mtb population complexity was not associated with infection group, lymph nodes had increased complexity when compared to lung granulomas across all groups. These results provide evidence SIV+ animals, independent of viral control, exhibit dysregulated immune responses and enhanced dissemination of Mtb, likely contributing to the poor TB disease course across all SIV/Mtb co-infected animals. ImportanceHIV and TB remain significant global health issues, despite the availability of treatments. Individuals with HIV, including those who are virally suppressed, are at an increased risk to develop and succumb to severe TB disease when compared to HIV-naive individuals. Our study aims to understand the relationship between SIV replication, mycobacterial growth, and immunological function in the tissues of co-infected Mauritian cynomolgus macaques during the early phase of Mtb infection. Here we demonstrate that increased viral replication is associated with increased bacterial burden in the tissues and impaired immunologic responses, and that the damage attributed to virus infection is not fully eliminated when animals spontaneously control virus replication.

microbiology↗