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Mycolicibacterium smegmatis

ABSTRACT: Cellular variation in GatCAB determines rifampicin-specific phenotypic resistance

PROVIDER: PRJNA295683 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR2411920.fastq.gz	Fastqsanger.gz
	SRR2412132_1.fastq.gz	Fastqsanger.gz
	SRR2412132_2.fastq.gz	Fastqsanger.gz
	SRR2412463.fastq.gz	Fastqsanger.gz
	SRR2412134_1.fastq.gz	Fastqsanger.gz

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Project description:Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing posttranslational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of posttranslational phosphoarginine modifications in Mycolicibacterium smegmatis, a nonpathogenic surrogate of M. tuberculosis. Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identified arginine phosphosites on several functionally diverse targets within the M. smegmatis proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus. Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species. IMPORTANCE Mycobacteria that cause tuberculosis infections employ proteolytic pathways that modulate cellular behavior by destroying specific proteins in a highly regulated manner. Some proteolytic enzymes have emerged as novel antibacterial targets against drug-resistant tuberculosis infections. However, we have only a limited understanding of how these enzymes function in the cell and how they select proteins for destruction. Some proteolytic enzymes are capable of recognizing proteins that carry an unusual chemical modification, arginine phosphorylation. Here, we confirm the existence of arginine phosphorylation in mycobacterial proteins. Our work expands our understanding of a promising drug target in an important global pathogen.

Project description:The prevalence of drug-resistant Mycobacterium tuberculosis infections has prompted extensive efforts to exploit new drug targets in this globally important pathogen. ClpC1, the unfoldase component of the essential ClpC1P1P2 protease, has emerged as one particularly promising antibacterial target. However, efforts to identify and characterize compounds that impinge on ClpC1 activity are constrained by our limited knowledge of Clp protease function and regulation. To expand our understanding of ClpC1 physiology, we employed a coimmunoprecipitation and mass spectrometry workflow to identify proteins that interact with ClpC1 in Mycolicibacterium smegmatis, a surrogate for M. tuberculosis. We identify a diverse panel of interaction partners, many of which coimmunoprecipitate with both the regulatory N-terminal domain and the ATPase core of ClpC1. Notably, our interactome analysis establishes MSMEI_3879, a truncated gene product unique to M. smegmatis, as a novel proteolytic substrate. Degradation of MSMEI_3879 by ClpC1P1P2 in vitro requires exposure of its N-terminal sequence, reinforcing the idea that ClpC1 selectively recognizes disordered motifs on substrates. Fluorescent substrates incorporating MSMEI_3879 may be useful in screening for novel ClpC1-targeting antibiotics to help address the challenge of M. tuberculosis drug resistance. IMPORTANCE Drug-resistant tuberculosis infections are a major challenge to global public health. Much effort has been invested in identifying new drug targets in the causative pathogen, Mycobacterium tuberculosis. One such target is the ClpC1 unfoldase. Compounds have been identified that kill M. tuberculosis by disrupting ClpC1 activity, yet the physiological function of ClpC1 in cells has remained poorly defined. Here, we identify interaction partners of ClpC1 in a model mycobacterium. By building a broader understanding of the role of this prospective drug target, we can more effectively develop compounds that inhibit its essential cellular activities.

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Mycolicibacterium smegmatis

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