The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria.
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ABSTRACT:
SUBMITTER: Belardinelli JM
PROVIDER: S-EPMC6656915 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR (Cell envelope Kinase and Regulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo, and identify genes that are directly and indirectly controlled by CenKR in Rb. sphaeroides. Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb. sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.
| S-EPMC9275681 | biostudies-literature
Project description:The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.
| S-EPMC5544280 | biostudies-literature
Project description:Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named "TMM transport factor A", TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosis IMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.
| S-EPMC6593404 | biostudies-literature
Project description:Mycobacterial cell envelope components have been a major focus of research due to their unique features that confer intrinsic resistance to antibiotics and chemicals apart from serving as a low-permeability barrier. The complex lipids secreted by Mycobacteria are known to evoke/repress host-immune response and thus contribute to its pathogenicity. This study focuses on the comparative genomics of the biosynthetic machinery of cell wall components across 21-mycobacterial genomes available in GenBank release 179.0. An insight into survival in varied environments could be attributed to its variation in the biosynthetic machinery. Gene-specific motifs like 'DLLAQPTPAW' of ufaA1 gene, novel functional linkages such as involvement of Rv0227c in mycolate biosynthesis; Rv2613c in LAM biosynthesis and Rv1209 in arabinogalactan peptidoglycan biosynthesis were detected in this study. These predictions correlate well with the available mutant and coexpression data from TBDB. It also helped to arrive at a minimal functional gene set for these biosynthetic pathways that complements findings using TraSH.
| S-EPMC3089613 | biostudies-literature
Project description:BackgroundBacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW.ResultsUsing a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsANg were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW.ConclusionsResults from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.
| S-EPMC5727935 | biostudies-literature
Project description:Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.
| S-EPMC3438161 | biostudies-literature
Project description:Background: Mtb's cell wall comprises peptidoglycan, arabinogalactan, and mycolic acids linked to capsule proteins and polysaccharides by noncovalent bonds. Cell division requires extensive remodeling by inserting cell wall-building subunits, which require multiple enzymes to ensure precision and accuracy during the addition and conjugation of biomolecules to the cell wall. Approximately 35% of division and cell wall cluster operon genes are involved in cell wall biosynthesis, 22% in cell division, and 43% are still unstudied. Results: Rv2166c was examined in M. smegmatis, a substitute for M. tuberculosis, using three strains: CRISPR-Cas12a-knockout ∆Ms_4236, complemented ∆Ms_4236::Rv2166c, and pALACE transformed Ms_Vec were grown at 370C in 7H9 or 7H10 to evaluate phenotypic, chemical stress, and antibiotic responses in normoxia and hypoxia. We also examined the whole transcriptome to identify genes associated with MSMEG_4236 deletion of the Rv2166c homolog under normoxia and hypoxia. Deletion and overexpression of Rv2166c affect mycobacterial biofilm formation, cell elongation, colony morphology, and sliding motility in normoxia but are alleviated in hypoxia. Overexpression showed resistance to nucleic acid-target antibiotics, but sensitivity to cell wall-target drugs, yet long-term expression arrests growth. According to transcriptome investigation, the deletion of MSMEG_4236 upregulated all division and cell wall cluster operon genes, several mycolic acids and arabinogalactan biosynthesis genes and downregulated numerous promoters outside of the dcw cluster operon through binding at AAAGTG[G/T] sequence motifs. Conclusion: This study shows that the mycobacterial Rv2166c represses the division and cell wall cluster operon as a transcriptional regulator. Thus, modulating this gene's expression affects many mycobacteria phenotypes and environmental stress resistance. Several AAAGTG[G/T] motifs containing promoters have been found, demonstrating that Rv2166c regulates genes other than its operon. These data suggest that the Rv2166c can be targeted as future antituberculosis medicines.
2023-12-15 | GSE250112 | GEO
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Project description:Mitotic progression is orchestrated by morphological and mechanical changes promoted by the coordinated activities of the microtubule (MT) cytoskeleton, the actin cytoskeleton and the plasma membrane (PM). MTs assemble the mitotic spindle, which assists sister chromatid separation, and contact the rigid and tensile actomyosin cortex rounded-up underneath the PM. Here, we highlight the dynamic crosstalk between MTs, actin and cell membranes during mitosis, and discuss the molecular connections between them. We also summarize recent views on how MT traction forces, the actomyosin cortex and membrane trafficking contribute to spindle positioning in isolated cells in culture and in epithelial sheets. Finally, we describe the emerging role of membrane trafficking in synchronizing actomyosin tension and cell shape changes with cell-substrate adhesion, cell-cell contacts and extracellular signalling events regulating proliferation.
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