Proteomic Landscape of a Drug-Tolerant Persister Subpopulation of Mycobacterium tuberculosis

Abstract

Persisters are a subpopulation of bacteria that resist killing by antibiotics, even though they are genetically similar to their drug-susceptible counterpart. Like in several other bacteria, persisters are also reported in the human pathogen Mycobacterium tuberculosis (Mtb). Stochastic formation of Mtb persisters with a high level of antimicrobial tolerance set the stage for subsequent multidrug-resistant mutations. Despite significant advancement in our understanding, much remains to be learnt about the biology of this drug-recalcitrant bacterial subpopulation. Most of the information pertaining to the metabolic evolution required for emergence of drug tolerance in tuberculosis (TB) pathogens has come from transcriptional, metabolomic, and mutagenesis studies. Since proteins are the key functional molecules regulating the majority of metabolic activities in the cell, investigation of the whole-cell protein expression profile will further provide valuable insights into the physiology of Mtb persisters. We performed a quantitative proteomic analysis of Mtb H37Rv cultured under an in vitro persistence model to identify the proteomic profile of the phenotypic drug-tolerant bacterial population. Our study reveals that proteins related to intermediary metabolism and respiration, cell-wall and cell processes, lipid metabolism, information pathways, and virulence, detoxification and adaptation functional categories are primarily modulated in the persister subpopulation. Further, we demonstrate that various surface-localized mycobacterial membrane protein large (MmpL) proteins, which exhibit a high level of expression in Mtb persisters, are crucial for the mycobacterial survival during persistent growth state. A drug-induced persister subpopulation of Mtb exhibit various differentially regulated proteins that might be critical in mitigating the antimicrobial effect of drugs and can be further explored to develop novel anti-TB agents. The peptide identifications and tandem mass spectra (MS/MS) have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013621.

HtpG Is a Metal-Dependent Chaperone Which Assists the DnaK/DnaJ/GrpE Chaperone System of Mycobacterium tuberculosis via Direct Association with DnaJ2

“…104, Table 2 ) (see Fig. S4A at the URL mentioned above) ( 50 ) and then confirmed by Southern blotting with the help of labeled probe (see Fig. S4B at the URL mentioned above).…”

“…Although mHtpG is maintained by M. tuberculosis throughout the course of genetic evolution, we have yet to understand how M. tuberculosis is able to endure the loss of this protein. Hence, to enlighten the pathogen’s response to the loss of mHtpG and to further understand its effect on bacterial physiology, quantitative proteomics by isobaric tags for relative and absolute quantitation (iTRAQ) was performed with the whole-cell lysates (WCLs) of the wild-type and Δ htpG strains of M. tuberculosis H37Rv, typically as described previously ( 50 , 51 ). We adopted an 8-plex iTRAQ-based quantitative proteomics approach to identify proteins that exhibit a consistent pattern of expression across 4 biological replicates ( Fig.…”

“…Absence of mHtpG expression in the mutant strain was confirmed by Western blotting using anti-mHtpG antibodies (see Fig. S4C at the URL mentioned above), as described previously ( 50 ).…”

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene

“…(2) The transcriptomic/proteomic data analyzed was collected in the absence of drug stimulation so we could hone in on native physiological and biochemical mechanisms/states that underlie drug resistance. Our study, therefore, does not address drug-induced changes in expression that may lead to phenotypic resistance in intrinsically drug sensitive isolates 23,24,40 . Emerging evidence that drug-induced proteomic changes can lead to drug tolerance/resistance in drug sensitive isolates is an important consideration that requires further investigation, particularly given the growing momentum in the field for widespread application of molecular testing.…”

Proteomics in antibiotic resistance and tolerance research: Mapping the resistome and the tolerome of bacterial pathogens

“…To map the proteomic landscape of M. tuberculosis persisters, Sharma et al. has recently used iTRAQ‐based quantitative proteomics to study the H37Rv strain under ciprofloxacin and isoniazid treatment [77]. They observed that the persisters subpopulation has differential expression of proteins related to intermediary metabolism and respiration, cell wall processes, lipid metabolism, information pathways, virulence, detoxification, and adaptation.…”