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The conserved translation factor LepA is required for optimal synthesis of a porin family in Mycobacterium smegmatis.

ABSTRACT: The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to strains with lepA We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the lepA mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance.ImportanceThe mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in M. smegmatis LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.

SUBMITTER: Fishbein SRS

PROVIDER: S-EPMC8095456 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

Project description:Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG_5700 (rpfA), MSMEG_5439 (rpfB), MSMEG_4640 (rpfE2), and MSMEG_4643 (rpfE). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG_4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG_4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the ΔrpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules.IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis, a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis, encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance.

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The conserved translation factor LepA is required for optimal synthesis of a porin family in Mycobacterium smegmatis.

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