Biology subjects

Kado, T.

Publications and source records attributed to Kado, T..

9 recordsLinked to original sources

bioRxiv2026

PonA2?s contributions to biofilm and colony formation independent of its catalytic domains in Mycobacterium smegmatis.

Biofilm and colony growth creates microenvironments that require coordinated localization and function of cell surface molecules within the membrane. However, the mechanisms by which membrane organization regulates these surface molecules during these biofilm-associated growth are poorly understood, limiting our understanding of how bacteria adapt and survive in multicellular communities. Mycobacterium smegmatis contains an inner membrane domain (IMD) at the subpolar regions of the cell that helps mediate cell envelope synthesis. Prior research has identified ponA2 as critical for de novo formation of distinct plasma membrane domains in planktonic growth. PonA2 is a penicillin-binding protein that catalyzes peptidoglycan synthesis by transglycosylase (TG) and transpeptidase (TP) activities. To investigate the role of PonA2 in membrane domain organization in biofilm and colony growth, wild-type, {Delta}ponA2, the complement strain (cponA2), and catalytic inactive variants of PonA2 (TG-, TP-, and TG-/TP-) were analyzed. The IMD subpolar localization in wild-type was preserved in biofilm and colony growth, indicating that IMD localization is not exclusive to planktonic growth. Both biofilm and colony growth of {Delta}ponA2 showed significant structural deformities compared to wild-type. In contrast, the catalytic inactive mutants produced biofilm and colony structures that resembled wild-type, suggesting that PonA2 has additional noncatalytic functions during multicellular growth. The IMD localization of the catalytic inactive mutants was minimally impacted, suggesting that neither catalytic domain is required for IMD localization in biofilm and colony growth. Together, these findings advance our understanding of the complex mycobacterial membrane biology. ImportancePlanktonic, pellicle biofilm, and colony growth expose mycobacteria to distinct environmental conditions that can affect cell-envelope organization and survival. Prior research has shown that mycobacteria form subpolar plasma membrane domains that support polar cell elongation in planktonic growth, but it remains unclear whether this organization is conserved in multicellular biofilm and colony growth, where cells experience nutrient and oxygen gradients and altered cell-to-cell interactions. Our current study analyzed the plasma membrane domain formation across the two growth states and showed that membrane domain localization is conserved, while the mechanisms required to maintain these domains differ depending on growth conditions. These findings suggest that mycobacteria use growth state specific mechanisms to coordinate membrane organization.

microbiology↗

bioRxiv2025

Phenotypic Characterization of Signature-Tagged Mutants Identifies Physiological Determinants of Vibrio vulnificus Fitness

Vibrio vulnificus is an opportunistic marine pathogen that causes severe wound-associated and systemic infections. Following entry into the host, the bacterium must rapidly adapt to host-associated stresses that differ substantially from those encountered in aquatic environments. However, the physiological functions supporting bacterial fitness during infection remain incompletely understood. Previously, we applied signature-tagged mutagenesis (STM) to identify genes required for V. vulnificus survival during host infection, revealing numerous loci that did not correspond to classical toxin-encoding genes. In the present study, we extended this genome-wide screen by linking STM-identified mutations to observable fitness-related phenotypes. Functional annotation revealed enrichment of genes associated with chemotaxis, flagellar motility, regulation, metabolism, and poorly characterized functions. Phenotypic analyses showed that many STM-derived mutants exhibited defects in swimming motility and altered colony surface properties. Bioluminescence imaging further revealed distinct patterns of impaired persistence and dissemination within host tissues, while several mutants displayed increased susceptibility to phagocytic stress in an HL-60-derived neutrophil model. Notably, some regulatory mutants affecting global signaling pathways exhibited impaired tissue dissemination despite retaining resistance to phagocytic stress, indicating the presence of fitness determinants that operate independently of classical surface-associated or cytotoxic traits. Together, these findings demonstrate that V. vulnificus fitness during infection depends on diverse physiological pathways beyond classical virulence factors, highlighting the value of phenotype-centered analyses for understanding bacterial adaptation in host-associated environments. ImportanceVibrio vulnificus causes rapidly progressive wound infections and septicemia, yet the bacterial functions that support fitness within host environments remain incompletely defined. While substantial effort has focused on canonical virulence factors and regulation, increasing evidence suggests that successful infection also depends on broader physiological adaptation. In this study, we link signature-tagged mutagenesis with systematic phenotypic analyses to define physiological determinants of V. vulnificus fitness during host-associated infection. Our results demonstrate that genes involved in motility, regulatory signaling, metabolism, and stress tolerance collectively shape bacterial persistence and dissemination in host tissues. Notably, we identify regulatory and metabolic determinants that influence fitness independently of classical surface-associated or cytotoxic traits, highlighting noncanonical pathways that contribute to pathogenic success. By integrating genome-wide screening with phenotype-centered analyses, this work advances understanding of how physiological adaptation underpins V. vulnificus infection and provides a framework for studying bacterial fitness alongside established concepts of bacterial virulence.

microbiology↗

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

Transcriptional Regulator, Metabolic, and Pilus Biosynthesis Genes as Candidate Virulence Markers in High-Virulence Mycobacterium abscessus

Mycobacterium abscessus is a rapidly growing non-tuberculosis mycobacterium that opportunistically causes pulmonary infections and is notable for its high resistance to antibiotics. While ubiquitous in environmental reservoirs such as soil, water, and biofilms, the genetic factors that enable certain strains to invade and persist in human hosts have remained elusive. To address this gap, we compared whole-genome sequences from 45 environmental isolates-- primarily collected on Hawaii Island--with a globally sourced set of clinical isolates retrieved from NCBI RefSeq. Our phylogenetic reconstruction delineated environmental and clinical lineages, and pangenome profiling revealed a conserved core genome of approximately 4,800 genes alongside a large accessory genome. Crucially, we found 20 genes conserved in clinical isolates, but not environmental isolates. Clinical isolates were found to have conserved genes coding for erm(41), three transcriptional regulators, a pilus synthesis gene, an NADP-dependent oxidoreductase, a carbonic anhydrase, a probable L-ectoine synthase, a ribonuclease P protein component, and 11 hypothetical genes, whereas environmental isolated did not conserve any of these genes. This indicates that clinical isolates have undergone broad changes relating to metabolism, pilus biosynthesis, and transcriptional regulators, which may be necessary for pathogenicity. The candidate virulence markers uncovered here lay the groundwork for experimental validation, rapid diagnostics for non-tuberculous mycobacteria, and targeted surveillance of environmental reservoirs to mitigate the emergence of clinically significant strains. ImportanceMycobacterium abscessus is an emerging pathogen that causes pulmonary infections in susceptible individuals. Though currently an opportunistic pathogen, M. abscessus may be undergoing evolutionary changes to become an obligate pathogen. Because of this, there is the need to identify markers of virulence in M. abscessus in order to better screen isolates and assess their risk of causing infection. This also provides the opportunity to study a similar pathogenesis to what Mycobacterium tuberculosis may have undergone. This study revealed that clinical isolates of M. abscessus have conserved genes relating to pilus biosynthesis, metabolism, and transcriptional regulators that environmental isolates do not have. These conserved genes may be required for M. abscessus to acquire virulence, and these genes have potential use as biomarkers to screen isolates for possible pathogenicity.

microbiology↗

bioRxiv2023

Isoxanthohumol improves obesity and glucose metabolism via inhibiting intestinal lipid absorption with a bloom of Akkermansia muciniphila in mice

AimsDysbiosis is an important factor that leads to metabolic disorders by disrupting energy balance and insulin sensitivity. A decrease in Akkermansia muciniphila is a phenotype of obesity-induced dysbiosis. Although interventions to increase A. muciniphila are expected to improve glucose metabolism, the underlying mechanism has not been fully understood. MethodsIsoxanthohumol (IX), a prenylated flavonoid found in beer hops was administered to high fat diet-fed mice. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice. ResultsThe administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration improved the gut barrier function and reduced metabolic endotoxemia. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of A. muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis. ConclusionOur study confirmed a direct role of A. muciniphila in energy metabolism, which was induced by microbial actions of IX. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

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

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↗