Biology subjects

Siegrist, M. S.

Publications and source records attributed to Siegrist, M. S..

17 recordsLinked to original sources

bioRxiv2026

Metabolic flexibility and an unusual route for peptidoglycan muramic acid recycling in mycobacteria

De novo biosynthesis of cell wall peptidoglycan is essential for bacterial viability under many growth conditions and is a well-validated antibiotic target. Although generally not essential for bacterial fitness under standard laboratory growth conditions, peptidoglycan recycling can aid bacterial survival under host or antibiotic stress. Peptidoglycan consists of alternating sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) cross-linked by peptides. Recycling of these sugars can proceed via GlcNAc and glucosamine intermediates (Escherichia coli-type) or, in the case of MurNAc, bypass these intermediates altogether (Pseudomonas-type). We serendipitously discovered that the pathogen Mycobacterium tuberculosis and model organism M. smegmatis assimilate 2-modified MurNAc probes into their peptidoglycan despite lacking the Pseudomonas-type machinery that is normally required for incorporation of these molecules. Our data suggest that unmodified and 2-modified MurNAc incorporate into M. smegmatis peptidoglycan via multiple pathways, the former preferentially via an E. coli-type route and the latter preferentially via a non-E. coli, non-Pseudomonas-type route with GlcNAc but not glucosamine intermediates. These findings reveal metabolic flexibility in mycobacterial cell wall recycling that encompasses a previously undescribed pathway. ImportanceCell wall biosynthesis is critical for bacterial replication under many conditions and is a successful drug target. Recycling of cell wall components is often dispensable under laboratory conditions but can promote bacterial survival under stress. We find that mycobacterial species can recycle a cell wall sugar via a mechanism distinct from those used by other bacteria. As pathogenic mycobacteria are subject to stress from the host environment and antibiotics, this mechanism is a potential novel vulnerability for these organisms.

microbiology↗

bioRxiv2026

Molecular Determinants Governing the Antitubercular Activity of Griselimycin

The emergence of drug-resistant strains of Mycobacterium tuberculosis makes anti-tubercular drug development a critical priority. Griselimycin is a cyclic peptide that targets the essential DNA sliding clamp of M. tuberculosis. While griselimycin is a promising antimycobacterial compound, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in M. tuberculosis. Incorporation of an N-terminal fluorophore enabled visualization of griselimycin accumulation inside of mycobacteria-infected macrophage cells. Derivatives containing altered cyclization chemistries, unnatural amino acids, expanded proline side chains, and alkyne handles were assessed, with a final design combining the most potent edits showing increased antibiotic activity. These findings present the comprehensive structure-activity investigation of griselimycin and can be used to rationally design future analogues.

pharmacology and toxicology↗

bioRxiv2025

Loss of Ag85A disrupts plasma membrane domains and promotes free mycolic acid accumulation in mycobacteria.

The mycomembrane of mycobacteria, composed primarily of long-chain mycolic acids, is critical for cell survival, structural integrity, and resistance to environmental stress, yet its underlying synthesis mechanisms remain incompletely understood. This study investigates the role of Ag85A, a key enzyme in mycomembrane synthesis, in regulating plasma membrane domains and cell envelope organization in Mycobacterium smegmatis. Using {Delta}Ag85A deletion mutants, we combined microscopy, biochemical assays, thin-layer chromatography, and lipid analysis to evaluate changes in membrane structure, chemical accumulation, and lipid composition. Ag85A deletion leads to altered plasma membrane domain organization, increased chemical accumulation, changes in cell envelope lipid composition. Unexpectedly, lipid analysis revealed accumulation--not depletion--of mycolic acids in the mutant, suggesting that increased permeability is not directly due to mycolic acid loss. These findings highlight a novel link between mycomembrane composition and plasma membrane domain stability. Our study not only advances understanding of mycobacterial cell envelope architecture but also identifies potential targets for enhancing drug penetration in resistant mycobacterial infections. SignificanceThe unique cell envelope of mycobacteria is central to survival, enabling it to resist immune defenses and antibiotic treatment. In this study, we reveal a novel function of Ag85A, a synthase of outmost layer of mycobacteria beyond its known role in mycolyltransferase activity: it is essential for the formation of plasma membrane domains that orchestrate cell envelope synthesis. By structuring plasma membrane domains, Ag85A contributes directly to the resilience of the cell envelope, reinforcing mycobacterial survival mechanisms under hostile conditions. Our findings provide a pivotal insight into mycobacterial cell biology, with broad implications for therapeutic development. Disrupting Ag85A-dependent membrane domain formation could weaken the protective cell envelope, offering a promising approach to enhance current treatments.

microbiology↗

bioRxiv2025

Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

Bacterial adaptation to stress involves changes in transcription and mRNA degradation rates. In Escherichia coli, the Nudix hydrolase RppH initiates mRNA degradation by removing pyrophosphate from mRNA 5-ends, converting 5-triphosphates to 5-monophosphates. We aimed to identify the RppH homolog in the globally important pathogen Mycobacterium tuberculosis (Mtb). We deleted each non-essential Nudix gene from Mtb to determine their impacts on mRNA phosphorylation states. Deletion of mutT4 (Rv3908) increased the relative abundance of 5-triphosphates on myriad mRNAs across the transcriptome. Purified MutT4 converted mRNA 5-triphosphates into monophosphates, and stimulated degradation by RNase E and RNase J. MutT4 has intrinsically disordered regions (IDRs), a common domain for biomolecular condensate formation. Microscopy showed that MutT4 forms condensates that dissociate upon addition of rifampicin, and that the N-terminal IDR is sufficient for condensate formation. These MutT4 condensates localize with RNase E and RNase J. Deletion of mutT4 in Mtb leads to a higher outer membrane permeability and resistance to oxidative stress. We conclude that MutT4 is the RppH homolog of Mtb, assembling in condensates that may act as degradation hubs. Our data indicate that MutT4 is unlikely to participate in DNA repair or nucleotide pool cleansing, and as such would more accurately be named RppH.

microbiology↗

bioRxiv2025

Identification of chemical features that influence mycomembrane permeation and antitubercular activity

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the deadliest single-agent infection worldwide. Current antibiotic treatment for TB takes a minimum of four months, underscoring the need for better therapeutics. The unique mycobacterial cell envelope, particularly the outermost mycomembrane, has long been thought to promote intrinsic antibiotic resistance by limiting compound entry into Mtb. Understanding chemical features that influence permeation across the mycomembrane may enable more accurate predictions of whole cell anti-Mtb activity, leading to development of more effective antibacterials. Here we query the mycomembrane permeation of over 1500 azide-tagged compounds in live Mtb with the bioorthogonal click chemistry-based assay PAC-MAN. We use cheminformatics and machine learning to identify chemical features associated with mycomembrane permeation and show that they have predictive value via systematic modification of two small molecule series. Additionally, we find that chemical features that influence mycomembrane permeation correlate with anti-Mtb activity in large compound libraries. These findings suggest that the mycomembrane is indeed a significant barrier to whole cell activity in Mtb and provide a rational framework for designing or modifying compounds to overcome this barrier.

microbiology↗

bioRxiv2025

Systematic Determination of the Impact of Structural Edits on Accumulation into Mycobacteria

Understanding the factors that influence the accumulation of molecules beyond the mycomembrane of Mycobacterium tuberculosis (Mtb) - the main barrier to accumulation - is essential for developing effective antimycobacterial agents. In this study, we investigated two design principles commonly observed in natural products and mammalian cell-permeable peptides: backbone N-alkylation and macrocyclization. To assess how these structural edits impact molecule accumulation beyond the mycomembrane, we utilized our recently developed Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings provide the first empirical evidence that peptide macrocyclization generally enhances accumulation in mycobacteria, while N-alkylation influences accumulation in a context-dependent manner. We examined these design principles in the context of two peptide antibiotics, tridecaptin A1 and griselimycin, which revealed the roles of N-alkylation and macrocyclization in improving both accumulation and antimicrobial activity against mycobacteria in specific contexts. Together, we present a working model for strategic structural modifications aimed at enhancing the accumulation of molecules past the mycomembrane. More broadly, our results also challenge the prevailing belief in the field that large and hydrophilic molecules, such as peptides, cannot readily traverse the mycomembrane.

microbiology↗

bioRxiv2025

The mycomembrane differentially and heterogeneously restricts antibiotic permeation

The recalcitrance of Mycobacterium tuberculosis to antibiotic treatment has been broadly attributed to the impermeability of the organisms outer mycomembrane. However, the studies that support this inference have been indirect and/or reliant on bulk population measurements. We previously developed the Peptidoglycan Accessibility Click-Mediated AssessmeNt (PAC-MAN) method to covalently trap azide-modified small molecules in the peptidoglycan cell wall of live mycobacteria, after they have traversed the mycomembrane. Using PAC-MAN we now show that the mycomembrane differentially restricts access of fluorophores and antibiotic derivatives. Mycomembranes of both M. tuberculosis and the model organism M. smegmatis discriminate between divergent classes of antibiotics as well as between antibiotics within a single family, the fluoroquinolones. By analyzing sub-populations of M. tuberculosis and M. smegmatis, we also found that some fluorophores and vancomycin are heterogeneously restricted by the mycomembrane. Our data indicate that the mycomembrane is a molecule- and cell-specific barrier to antibiotic permeation. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/630956v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4bdeb7org.highwire.dtl.DTLVardef@10ade47org.highwire.dtl.DTLVardef@872dceorg.highwire.dtl.DTLVardef@d04b59_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Trehalose dimycolate inhibits phagosome maturation and promotes intracellular M. tuberculosis growth via noncanonical SNARE interaction

Mycobacterial cell envelopes are rich in unusual lipids and glycans that play key roles during infection and vaccination. The most abundant envelope glycolipid is trehalose dimycolate (TDM). TDM compromises the host response to mycobacterial species via multiple mechanisms, including inhibition of phagosome maturation. The molecular mechanism by which TDM inhibits phagosome maturation has been elusive. We find that a clickable, photoaffinity TDM probe recapitulates key phenotypes of native TDM in macrophage host cells and binds several host SNARE proteins, including VTI1B, STX8, and VAMP2. VTI1B and STX8 normally promote endosome fusion by forming a complex with VAMP8. However, in the presence of Mycobacterium tuberculosis, VTI1B and STX8 complex with VAMP2, which in turn decreases VAMP8 binding. VAMP2 acts together with mycolate structure to inhibit phagosome maturation and promotes intracellular M. tuberculosis replication. Thus one mechanism by which TDM constrains the innate immune response to M. tuberculosis is via non-canonical SNARE complexation. Significance StatementGlycolipids from the Mycobacterium tuberculosis cell envelope, particularly trehalose dimycolate (TDM), play major roles in subverting the immune response to this intracellular pathogen. How subversion occurs is often unclear because glycans and lipids are technically challenging to study in cells. We discovered that a TDM-mimicking chemical probe interacts with three host SNARE proteins, including two that regulate endosome fusion and one that does not. The presence of TDM or M. tuberculosis triggers abnormal binding of these SNAREs, which in turn inhibits the fusion of M. tuberculosis-containing phagosomes with lysosomes and promotes M. tuberculosis replication. Our work provides an unusual example of a bacterial pathogen restricting the immune response via glycolipid-SNARE interactions.

microbiology↗

bioRxiv2022

Permeation Across the Mycomembrane in Live Mycobacteria

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most commonly ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. Nonetheless, there is limited information on the types of molecular scaffolds that can readily permeate past the mycomembrane of mycobacteria. To address this, we describe a novel assay that combines metabolic tagging of the peptidoglycan scaffold, which sits directly beneath the mycomembrane, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in Mycobacterium smegmatis and Mtb. A small panel of molecules was tested and we found a large range in the permeability profile of molecules. Interestingly, the general trend is similar across the two types of mycobacteria, with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria. The methods described, which do not require genetic manipulation, can be generally adopted to other species for which envelope permeability is treatment-limiting.

microbiology↗

bioRxiv2022

Genetic and lipidomic identification of tuberculostearic acid as a controller of mycobacterial membrane compartmentalization

Mycobacteria diverge in a basic way from other bacterial and eukaryotic cells based on their distinct membrane structures. Here we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. cfa, a gene that encodes a putative cyclopropane-fatty-acyl-phospholipid synthase, shows the most significant effect on recovery from a membrane destabilizer, dibucaine. Lipidomic analysis of cfa deletion mutants demonstrates an essential role of Cfa in the synthesis of specific membrane lipids containing a C19:0 monomethyl-branched stearic acid. This molecule, also known as tuberculostearic acid (TBSA), has been intensively studied for decades due to its high level and genus-specific expression in mycobacteria. The proposed Cfa-mediated conversion of an unsaturation to a methylation matched well with its proposed role in lateral membrane organization, so we used new tools to determine the non-redundant effects of Cfa and TBSA in mycobacterial cells. cfa expression regulated major classes of membrane lipids including phosphatidylinositols, phosphatidylethanolamines and phosphatidylinositol mannosides. Cfa localized within the intracellular membrane domain (IMD), where it controls both cellular growth and recovery from membrane fluidization by facilitating subpolar localization of the IMD. Overall, cfa controls lateral membrane partitioning but does not detectably alter orthogonal transmembrane permeability. More generally, these results support the proposed role of the subpolar IMD as a subcellular site of mycobacterial control of membrane function. SignificanceMycobacteria remain major causes of disease worldwide based in part on their unusual membrane structures, which interface with the host. Here we discover the long sought biosynthetic origin of tuberculostearic acid (TBSA), a major fatty acid found selectively in mycobacteria, as well as its role in mycobacterial cells. The lipid is produced by an enzyme called Cfa, whose loss causes a growth defect and slow reformation of a membrane domain near the pole of the rod-shaped cell. Thus, our study offers mechanistic insights to the intrinsic molecular factors critical for mycobacterial plasma membrane partitioning.

microbiology↗

bioRxiv2022

Inositol acylation of phosphatidylinositol mannosides: a rapid mass response to membrane fluidization in mycobacteria

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) rapidly remodel their structures upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a tri-acylated form: two acyl chains of PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol preexposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.

biochemistry↗

bioRxiv2022

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced by interactions with other membranes and solid supports. While cellular membranes can departition in response to various perturbations, including disruption of bilayer-extrinsic structures, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis can be spatially and biochemically departitioned by the fluidizing agent benzyl alcohol. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth upon fluidizer washout. PonA2s role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. We find that the cell wall polymer, but not active cell wall polymerization, is critical for membrane partitioning. Our work highlights a key initiating role for bilayer-extrinsic structures in patterning cellular membranes.

molecular biology↗

bioRxiv2021

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

The bacterial cell wall supports cell shape and prevents lysis due to internal turgor pressure. A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is comprised of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are often the target of antibiotics as they are essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study bacterial cell growth and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to build synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of a m-DAP analogue, selenolanthionine. We show that selenolanthionine is installed within the PG of live bacteria by the native cell wall crosslinking machinery in several mycobacteria species. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP containing organisms.

microbiology↗

bioRxiv2021

Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a non-redundant role for RodA in mycobacteria

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacteria from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA and a cognate class B PBP, and the class A PBPs. In contrast to laterally-growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps with that of the aPBP PonA1, and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies M. smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. ImportancePeptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover the vast majority of bacterial species are unlikely to fit a single paradigm because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under non-optimal conditions and in non-standard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multi-layered envelope. In this work we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.

microbiology↗

bioRxiv2019

Trehalose recycling promotes energy-efficient mycomembrane reorganization in nutrient-limited mycobacteria

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune and antibiotic insults. We find that there is mycomembrane remodeling along the periphery of nutrient-starved, non-replicating mycobacterial cells. Remodeling is supported by recycling of trehalose, a non-mammalian disaccharide that shuttles long-chain mycolate lipids to the mycomembrane. In the absence of trehalose recycling, mycomembrane synthesis continues but mycobacteria experience ATP depletion, enhanced respiration and redox stress. Redox stress from depletion of the trehalose pool is suppressed in a mutant that lacks the OtsAB de novo trehalose synthesis pathway. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling. Loss of trehalose salvage is known to attenuate M. tuberculosis during infection and render the bacterium more susceptible to a variety of drugs. Recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic stress.

microbiology↗

bioRxiv2018

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or sub-polar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

microbiology↗

bioRxiv2018

Sidewall cell envelope synthesis and remodeling in pole-growing mycobacteria

O_SCPLOWDC_SCPLOW-amino acid probes label cell wall peptidoglycan at both the poles and sidewall of pole-growing mycobacteria. Since peptidoglycan assembly along the cell periphery could provide a rapid, growth-independent means by which to edit the cell wall, we sought to clarify the precise metabolic fates of these probes. O_SCPLOWDC_SCPLOW-amino acid monopeptides were incorporated into peptidoglycan by O_SCPLOWLC_SCPLOWO_SCPCAP,C_SCPCAPO_SCPLOWDC_SCPLOW-transpeptidase remodeling enzymes to varying extents. Dipeptides were incorporated into cytoplasmic precursors. While dipeptide-marked peptidoglycan synthesis at the poles was associated with cell elongation, synthesis along the periphery was highly responsive to cell wall damage. Our observations suggest a post-expansion role for peptidoglycan assembly along the mycobacterial sidewall and provide a conceptual framework for understanding cell wall robustness in the face of polar growth.

microbiology↗