Biology subjects

Puffal, J.

Publications and source records attributed to Puffal, J..

6 recordsLinked to original sources

bioRxiv2026

The phage shock protein A (PspA) maintains membrane potential and supports NADH dehydrogenase function in mycobacteria

Maintenance of membrane integrity and proton motive force (PMF) is critical for bacterial survival. The phage shock protein (Psp) system, conserved across bacterial species, stabilizes the membrane, maintains PMF, and protects against envelope damage. However, how the conserved effector PspA contributes to PMF maintenance remains unclear. Here, using the mycobacterial Psp system as a genetically tractable model, we provide mechanistic insight into this process. We show that PspA and the accessory protein PspM jointly sustain membrane potential, with PspM required to maintain a ~70 kDa PspA isoform at the membrane during envelope stress. Loss of PspA increases susceptibility to thioridazine, which targets type II NADH dehydrogenase (NDH-2), and to Ro 48-8071, an inhibitor of menaquinone biosynthesis. Notably, hypersusceptibility to thioridazine is rescued by exogenous menaquinone. Consistent with these phenotypes, a pspA-deficient mutant exhibits impaired NADH dehydrogenase activity despite unchanged abundance of NDH-2 and menaquinone (MK-9). Together, these findings identify a functional link between PspA and NADH dehydrogenase-dependent respiration and suggest that PspA contributes to PMF maintenance by supporting respiratory electron transfer.

molecular biology↗

bioRxiv2024

Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in Mycobacterium tuberculosis

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins also bind to promoter region sequences and repress expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis, we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46, whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation, potentially in response to extracytoplasmic as well as intracellular signals. ImportanceIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here we show that antitoxin interactions with its cognate toxin, and with promoter DNA required for antitoxin and toxin expression, can be altered by Ser/Thr phosphorylation of the antitoxin, and thus affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

microbiology↗

bioRxiv2022

Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, 1,3-fucosyltransferase and fucosyl 4- O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface- exposed lipid virulence factors. IMPORTANCEM. tuberculosis remains an important public health threat, with more than one million deaths every year. The pathogens ability to survive in the human host for decades highlights the importance of understanding how this bacterium regulates and coordinates its metabolism, cell envelope elongation, and growth. The IMD is a membrane structure that associates with the subpolar growth zone of actively growing mycobacteria, but its existence is only known in a non- pathogenic model, M. smegmatis. Here, we demonstrated the presence of the IMD in M. tuberculosis, making the IMD an evolutionarily conserved plasma membrane compartment in mycobacteria. Furthermore, our study revealed that the IMD is the factory for synthesizing phenolic glycolipids, virulence factors produced by slow-growing pathogenic species.

microbiology↗

bioRxiv2021

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under non-growing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized envelope biosynthesis. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a handful of these have been verified by other experimental methods. Furthermore, we speculate that some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains each expressing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed fluorescence patterns similar to those of IMD proteins and, consistent with this co-localization, we had previously identified 20 of these using a proteomics approach. Of the nine remaining IMD candidate proteins, three were confirmed to be associated with the IMD while some others appear to be lipid droplet-associated. Taken together, our newly devised strategy is effective in verifying the IMD association of proteins found by proteomic analyses, while facilitating the discovery of additional IMD-associated proteins. ImportanceThe intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis, that likely contribute to the function of the IMD. How can we find more IMD-associated proteins that escaped proteomic detection? Here, as an alternative approach, fluorescence microscope images of 523 proteins were screened to identify IMD-associated proteins. We confirmed the IMD association of previously identified proteins and discovered three additional proteins associated with the IMD. Together, subcellular fractionation, proteomics, and fluorescence microscopy form a robust combination to more rigorously define IMD proteins, which will aid future investigations to decipher the synthesis, maintenance and functions of this membrane domain.

microbiology↗

bioRxiv2019

Membrane compartmentalization of mycobacterial peptidoglycan synthesis

Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. Different species have characteristic subcellular sites of peptidoglycan synthesis that they must carefully maintain for surface integrity and, ultimately, viability. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on a limited pool of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning is required for robust, orderly peptidoglycan synthesis, indicating that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself are essential for domain homeostasis. Thus, the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. We further show that our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, demonstrating that horizontal compartmentalization of precursors is a general feature of bacillary cell wall biogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗

bioRxiv2018

Demethylmenaquinone methyl transferase is a membrane domain-associated protein essential for menaquinone homeostasis in Mycobacterium smegmatis

The intracellular membrane domain (IMD) in mycobacteria is a spatially distinct region of the plasma membrane with diverse functions. Previous comparative proteomic analysis of the IMD suggested that menaquinone biosynthetic enzymes are associated with this domain. In the present study, we determined the subcellular site of these enzymes using sucrose density gradient fractionation. We found that the last two enzymes, the methyltransferase MenG, and the reductase MenJ, are associated with the IMD. MenA, the prenyltransferase that mediates the first membrane-associated step of the menaquinone biosynthesis, is associated with the conventional plasma membrane. For MenG, we additionally showed the polar enrichment of the fluorescent protein fusion colocalizing with an IMD marker protein in situ. To start dissecting the roles of IMD-associated enzymes, we further tested the physiological significance of MenG. The deletion of menG at the endogenous genomic loci was possible only when an extra copy of the gene was present, indicating that it is an essential gene in M. smegmatis. Using a tetracycline-inducible switch, we achieved gradual and partial depletion of MenG over three consecutive 24 hour subcultures. This partial MenG depletion resulted in progressive slowing of growth, which corroborated the observation that menG is an essential gene. Upon MenG depletion, there was a significant accumulation of MenG substrate, demethylmenaquinone, even though the cellular level of menaquinone, the reaction product, was unaffected. Furthermore, the growth retardation was coincided with a lower oxygen consumption rate and ATP accumulation. These results imply a previously unappreciated role of MenG in regulating menaquinone homeostasis within the complex spatial organization of mycobacterial plasma membrane.

microbiology↗