[R01] Identifying drug tolerance mechanisms in Mycobacterium tuberculosis glpK mutants to develop better treatments against drug tolerant persister bacilli.
Ente: National Institute of Allergy and Infectious Diseases
Scadenza: 2031-05-31
Importo max: 811.772 EUR
Paese: US
Descrizione
ABSTRACT
Treatment for tuberculosis (TB) requires adherence to complex, lengthy, and often toxic multi-drug regimens,
which predisposes to treatment failure and relapse. These adverse outcomes appear to be due in part to drug
tolerant populations of Mycobacterium tuberculosis (Mtb), the causative agent of TB. Studies of drug tolerant
Mtb have been difficult because this phenotype is transient and heterogeneously expressed in populations. We
recently discovered a form of drug tolerance caused by phase variation in the Mtb glycerol kinase gene, glpk.
Mtb glpK phase variants become drug tolerant via reversible slip strand mispairing of homopolymeric sequences
within glpK, causing frame shift mutations that reversibly inactivate the gene. These phase variants are multi-
drug tolerant in vitro, re-capitulate classic transcriptional alterations seen in drug tolerant Mtb, accumulate in
drug treated mice, have been found in the sputum of patients with multidrug resistant TB, and are associated
with decreased drug efficacy. Considerable new data suggests that this form of phase variation is selected for
in clinical Mtb strains and thus may be a common cause of clinical drug tolerance. We created an Mtb H37Rv
glpK knockout strain which presents us with a unique tool to study clinically relevant forms of drug tolerance.
These mutants have phenotypic and transcriptional characteristics similar to glpK phase variants but are
genetically more tractable because (unlike phenotypically or phase variant drug tolerant Mtb) these ∆glpK
mutants cannot revert to wild type and are thus stable drug tolerant strains, which can be studied in a variety of
conditions. We will use this glpK mutant strain to determine the genes and genetic pathways essential for drug
tolerance. ∆glpK mutants will be cultured in both standard and media and media modified with different carbon
sources including cholesterol to simulate intracellular or granuloma environments. We will then induce
macrophages into M0, M1, M2 and foamy states to study how specific host environments affect bacterial drug
tolerance and associated transcriptional programs within the bacteria. Together, these studies will identify within
pathogen, within host, and host-pathogen interactions that regulate Mtb survival and drug tolerance. Our results
will then be used to find potential drug leads that are particularly potent against drug tolerant Mtb in the following
aims: Aim 1: Identify the transcriptional and metabolic changes along with related genes essential for the
development of drug tolerance in Mtb cultured in physiologically relevant carbon sources. Aim 2: Determine how
the interactions between Mtb and the intracellular macrophage environment modulate drug tolerance. Aim 3:
Identify preliminary drug candidates with enhanced activity against drug tolerant Mtb.
Istituzione: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
PI: David Alland
Progetto: 1R01AI192623-01A1
Settori: National Institute of Allergy and Infectious Diseases
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