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Principal Investigator: WILLIAM Robert JACOBS
Organization: ALBERT EINSTEIN COLLEGE OF MEDICINE
Fiscal Year: 2024
Award: $786,783
Funding agency: National Institute of Allergy and Infectious Diseases
Project Summary
Despite broad availability and extensive use of the vaccine Bacille Calmette Guerin (BCG) and sterilizing
chemotherapies, tuberculosis (TB) remains responsible for more than 1.5 million deaths annually. Although
COVID-19 caused more deaths than TB in 2020 and 2021, TB is expected to resume the position it held for most
of the last 100 years as the leading cause of death associated with an infectious agent. The mechanisms through
which Mycobacterium tuberculosis (Mtb) successfully evades vaccines and effective chemotherapies remain
unknown. We hypothesize that Mtb persister cells enter a physiological state that prevents them from
succumbing to death and sterilization. This proposal will use in vitro and in vivo models to dissect the
mechanisms enabling persistence, building on our preliminary data showing that persistence is a multifactorial
phenomenon involving amino acid metabolism, other metabolic pathways, interactions with the host immune
response, and effects on bacterial chromosome structure. Persisters were first identified as the 0.1% to 1% of
streptococci and staphylococci that survive treatment with the bacteriocidal antibiotic penicillin. We have
established similar, highly robust, reproducible models for isolating Mtb persisters using either isoniazid (INH) or
amikacin. In our INH model, 99% to 99.9% of Mtb cells are killed within 3 days of in vitro exposure. Microarray
analysis of surviving cells revealed a defined stress-associated signature. We compared the RNA sequencing
results for amikacin and INH persisters to those for Mtb under other persister-inducing conditions, including
hypoxia, nutrient starvation, and stationary phase. We found 25 commonly upregulated genes, which we
hypothesize are important genes for persister formation. We propose to construct deletion mutants for all 25
genes to define pathways leading to persistence. We further hypothesize that Mtb persisters evolved to prevent
sterilization by the adaptive immune response, supported by our studies of a new conditionally immune-sterilized
auxotrophic (CIMSAUX) Mtb mutant that contains deletions in methionine, leucine, and pantothenate
biosynthesis genes. The CIMSAUX mutant persists indefinitely in immunocompromised mice but is sterilized in
immunocompetent mice. The genetic factors contributing to the CIMSAUX mutant phenotype will be analyzed,
and additional CIMSAUX mutants will be isolated. Novel fluorescent reporter constructs will be engineered to
monitor the growth and persistence of the multiple mutants generated in this study. We present preliminary
evidence that deletion of the gene encoding the structural maintenance of the chromosome (SMC) protein in Mtb
results in a persister formation–defective mutant (Mtb Δsmc), suggesting that Mtb chromosome structure
contributes to persister formation. To test this hypothesis, we will delete genes with the potential to impact
chromosome structure in wild-type Mtb and Mtb Δsmc and test their persister capacities in vitro and in mouse
models. These studies will (i) provide new insights into the Mtb persister phenotype, (ii) generate valuable models
for investigating persistence, and (iii) lead to improved strategies for sterilizing Mtb infections.
Terms: <Affect><Amikacin><Amikin><Amiklin><Amino Acids><Amukin><Anabolism><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Antitubercular Drugs><Ascorbic Acid><BCG Live><Bacille Calmette-Guérin><Bacillus Calmette Guérin><Bacterial Chromosomes><Bacteriocidal Agents><Bacteriocides><Bar Codes><Biclin><Biklin><COVID-19><CV-19><Cause of Death><Cell Body><Cell Survival><Cell Viability><Cells><Cessation of life><Chromosomal Organization><Chromosomal Structure><Chromosome Organization><Chromosome Structures><Chromosomes><Coronavirus Infectious Disease 2019><Cysteine><Data><Death><Defect><Drugs><Engineering><Gene Deletion><Generalized Growth><Genes><Genetic><Genetic Determinism><Genetic analyses><Genus staphylococcus><Goals><Growth><Half-Cystine><Heritability><Hypoxia><Hypoxic><INH resistance><INH resistant><Immune><Immune response><Immunes><Immunocompetent><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunological response><Immunosuppressed Host><In Vitro><Infectious Agent><Isoniazid resistance><Isoniazid resistant><Isonicotinic Acid Hydrazide><L-Cysteine><Leucine><M tb><M tuberculosis><M tuberculosis infection><M. tb><M. tb infection><M. tuberculosis><M. tuberculosis infection><M.tb infection><M.tuberculosis infection><MTB infection><Maintenance><Mammalia><Mammals><Mediating><Medication><Metabolic><Metabolic Pathway><Metabolic Pathways of Other Amino Acids><Metabolism of Other Amino Acids - Reference Pathway><Methionine><Mice><Mice Mammals><Microarray Analysis><Microarray-Based Analysis><Miscellaneous Antibiotic><Modeling><Molecular Genetics><Monitor><Morbidity><Morbidity - disease rate><Murine><Mus><Mycobacterium tuberculosis><Mycobacterium tuberculosis (MTB) infection><Mycobacterium tuberculosis infection><Nutrient><Oxygen Deficiency><Pantothenic Acid><Pathway interactions><Penicillins><Pharmaceutical Preparations><Pharmacopoeias><Phase><Phenotype><Physiologic><Physiological><Play><Population><Position><Positioning Attribute><Predisposition><Principal Investigator><Proteins><RNA Seq><RNA sequencing><RNAseq><Rag1><Rag1 Mouse><Reporter><Reproducibility><Resistance><Role><Staphylococcus><Starvation><Sterilization><Streptococcus><Stress><Susceptibility><TB drugs><TB infection><TB therapy><TB treatment><Testing><Tissue Growth><Tuberculosis><VIT C><Vaccines><Vitamin C><Wild Type Mouse><adaptive immune response><adaptive immunity><amino acid metabolism><aminoacid><anti-TB drugs><anti-tuberculosis drugs><bactericidal><bactericide><barcode><biosynthesis><cell killing><chemotherapy><coronavirus disease 2019><coronavirus disease-19><coronavirus infectious disease-19><develop a vaccine><develop vaccines><development of a vaccine><disseminated TB><disseminated tuberculosis><drug/agent><gene deletion mutation><genetic analysis><genetic determinant><host response><hypoimmunity><immune competent><immune deficiency><immune system response><immunodeficiency><immunoresponse><immunosuppressed patient><improved><in vitro Model><in vivo><in vivo Model><infection due to Mycobacterium tuberculosis><infectious organism><insight><isoniazid><microarray analyses><microarray technology><mortality><mouse model><mtb><murine model><mutant><novel><ontogeny><overexpress><overexpression><pantothenate><pathway><prevent><preventing><resistance to isoniazid><resistant><resistant to isoniazid><social role><tool><transcriptome sequencing><transcriptomic sequencing><treat M. tuberculosis><treat Mtb><treat Mycobacterium tuberculosis><treat tb><treat tuberculosis><tuberculosis drugs><tuberculosis infection><tuberculosis therapy><tuberculosis treatment><tuberculous spondyloarthropathy><vaccine development><vaccine for immunotherapy><vaccine immunotherapy><vaccine-based immunotherapy><wildtype mouse>