Cell Envelope Structure Research Articles

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α−1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.

The phage shock protein (PSP) envelope stress response: discovery of novel partners and evolutionary history.

Bacterial phage shock protein (PSP) systems stabilize the bacterial cell membrane and protect against envelope stress. These systems have been associated with virulence, but despite their critical roles, PSP components are not well characterized outside proteobacteria. Using comparative genomics and protein sequence-structure-function analyses, we systematically identified and analyzed PSP homologs, phyletic patterns, domain architectures, and gene neighborhoods. This approach underscored the evolutionary significance of the system, revealing that its core protein PspA (Snf7 in ESCRT outside bacteria) was present in the last universal common ancestor and that this ancestral functionality has since diversified into multiple novel, distinct PSP systems across life. Several novel partners of the PSP system were identified: (i) the Toastrack domain, likely facilitating assembly of sub-membrane stress-sensing and signaling complexes, (ii) the newly defined HTH-associated α-helical signaling domain-PadR-like transcriptional regulator pair system, and (iii) multiple independent associations with ATPase, CesT/Tir-like chaperone, and Band-7 domains in proteins thought to mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and other domains, such as novel variants of SHOCT-like domains, suggesting roles in assembling membrane-associated complexes of proteins with disparate biochemical functions. Results are available at our interactive web app, https://jravilab.org/psp.IMPORTANCEPhage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in Proteobacteria and Firmicutes. We now show that a minimal PSP system was present in the last universal common ancestor that evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate the use of domain architecture and genetic context for discovery in comparative genomics.

Inhibition of iron oxidation in Acidithiobacillus ferrooxidans by low-molecular-weight organic acids: Evaluation of performance and elucidation of mechanisms

The catalytic role of Acidithiobacillus ferrooxidans (A. ferrooxidans) in iron biooxidation is pivotal in the formation of Acid Mine Drainage (AMD), which poses a significant threat to the environment. To control AMD generation, treatments with low-molecular-weight organic acids are being studied, yet their exact mechanisms are unclear. In this study, AMD materials, organic acids, and molecular methods were employed to gain a deeper understanding of the inhibitory effects of low-molecular-weight organic acids on the biooxidation of iron by A. ferrooxidans. The inhibition experiments of A. ferrooxidans on the oxidation of Fe2+ showed that to attain a 90 % inhibition efficacy within 72 h, the minimum concentrations required for formic acid, acetic acid, propionic acid, and lactic acid are 0.5, 6, 4, and 10 mmol/L, respectively. Bacterial imaging illustrated the detrimental effects of these organic acids on the cell envelope structure. This includes severe damage to the outer membrane, particularly from formic and acetic acids, which also caused cell wall damage. Coupled with alterations in the types and quantities of protein, carbohydrate, and nucleic acid content in extracellular polymeric substances (EPS), indicate the mechanisms underlying these inhibitory treatments. Transcriptomic analysis revealed interference of these organic acids with crucial metabolic pathways, particularly those related to energy metabolism. These findings establish a comprehensive theoretical basis for understanding the inhibition of A. ferrooxidans' biooxidation by low-molecular-weight organic acids, offering a novel opportunity to effectively mitigate the generation of AMD at its source.

Structure of the Bacterial Cell Envelope and Interactions with Antimicrobials: Insights from Molecular Dynamics Simulations.

Bacteria have evolved over 3 billion years, shaping our intrinsic and symbiotic coexistence with these single-celled organisms. With rising populations of drug-resistant strains, the search for novel antimicrobials is an ongoing area of research. Advances in high-performance computing platforms have led to a variety of molecular dynamics simulation strategies to study the interactions of antimicrobial molecules with different compartments of the bacterial cell envelope of both Gram-positive and Gram-negative species. In this review, we begin with a detailed description of the structural aspects of the bacterial cell envelope. Simulations concerned with the transport and associated free energy of small molecules and ions through the outer membrane, peptidoglycan, inner membrane and outer membrane porins are discussed. Since surfactants are widely used as antimicrobials, a section is devoted to the interactions of surfactants with the cell wall and inner membranes. The review ends with a discussion on antimicrobial peptides and the insights gained from the molecular simulations on the free energy of translocation. Challenges involved in developing accurate molecular models and coarse-grained strategies that provide a trade-off between atomic details with a gain in sampling time are highlighted. The need for efficient sampling strategies to obtain accurate free energies of translocation is also discussed. Molecular dynamics simulations have evolved as a powerful tool that can potentially be used to design and develop novel antimicrobials and strategies to effectively treat bacterial infections.

Photoacoustic based evaluation of viscoelastic properties of Gram-negative and Gram-positive bacterial colonies

Mechanical properties of bacterial colonies are crucial considering both addressing their pathogenic effects and exploring their potential applications. Viscoelasticity is a key mechanical property with major impacts on the cell shapes and functions, which reflects the information about the cell envelope constituents. Hereby, we have proposed the application of photoacoustic viscoelasticity (PAVE) for studying the rheological properties of bacterial colonies. In this regard, we employed an intensity-modulated laser beam as the excitation source followed by the phase delay measurement between the generated PA signal and the reference for the characterization of colonies of two different types of Gram-positive and Gram-negative bacteria. The results of our study show that the colony of Staphylococcus aureus as Gram-positive bacteria has a significantly higher viscoelasticity ratio compared to that value for Acinetobacter baumannii as Gram-negative bacteria (77% difference). This may be due to the differing cell envelope structure between the two species, but we cannot rule out effects of biofilm formation in the colonies. Furthermore, a lumped model has been provided for the mechanical properties of bacterial colonies.

New Strategies to Kill Metabolically-Dormant Cells Directly Bypassing the Need for Active Cellular Processes.

Antibiotic therapy failure is often caused by the presence of persister cells, which are metabolically-dormant bacteria capable of surviving exposure to antimicrobials. Under favorable conditions, persisters can resume growth leading to recurrent infections. Moreover, several studies have indicated that persisters may promote the evolution of antimicrobial resistance and facilitate the selection of specific resistant mutants; therefore, in light of the increasing numbers of multidrug-resistant infections worldwide, developing efficient strategies against dormant cells is of paramount importance. In this review, we present and discuss the efficacy of various agents whose antimicrobial activity is independent of the metabolic status of the bacteria as they target cell envelope structures. Since the biofilm-environment is favorable for the formation of dormant subpopulations, anti-persister strategies should also include agents that destroy the biofilm matrix or inhibit biofilm development. This article reviews examples of selected cell wall hydrolases, polysaccharide depolymerases and antimicrobial peptides. Their combination with standard antibiotics seems to be the most promising approach in combating persistent infections.

Antimicrobial mechanisms of ZnO nanoparticles to phytopathogen Pseudomonas syringae: Damage of cell envelope, suppression of metabolism, biofilm and motility, and stimulation of stomatal immunity on host plant

Nanoparticles have recently been employed as a new strategy to act as bactericides in agricultural applications. However, the effects and mechanisms of foliar deposition of nanoparticles on bacterial pathogens, plant physiology and particularly plant immunity have not been sufficiently understood. Here, we investigated the effects and mechanisms of ZnO NPs in controlling of tobacco wildfire caused by Pseudomonas syringae pv. tabaci, through the comprehensive analysis of biological changes of both bacteria and plants. The global gene expression changes of Pseudomonas syringae pv. tabaci supported that the functions of “protein secretion”, “membrane part”, “signal transducer activity”, “locomotion”, “chemotaxis” and “taxis” in bacteria, as well as the metabolic pathways of “bacterial chemotaxis”, “two-component system”, “biofilm formation”, “ABC transporters” and “valine, leucine and isoleucine degradation” were significantly down-regulated by ZnO NPs. Correspondingly, we reconfirmed that the cell envelope structure, biofilm and motility of Pseudomonas syringae pv. tabaci were directly disrupted or suppressed by ZnO NPs. Different from completely killing Pseudomonas syringae pv. tabaci, ZnO NPs (0.5 mg/mL) potentially improved plant growth and immunity through enzymatic activity and global molecular response analysis. Furthermore, the changes of gene expression in ABA signaling pathway, ABA concentration and stomatal aperture all supported that ZnO NPs can specifically stimulate stomatal immunity, which is important to defend bacterial infection. Taken together, we proposed that both the inhibition or damage of motility, biofilm, metabolisms, virulence and cell envelope on P. syringae pv. tabaci, and the activation of the stomatal immunity formed two-layered antibacterial mechanisms of ZnO NPs on phytopathogenic bacteria.

The Brucella Cell Envelope.

The cell envelope is a multilayered structure that insulates the interior of bacterial cells from an often chaotic outside world. Common features define the envelope across the bacterial kingdom, but the molecular mechanisms by which cells build and regulate this critical barrier are diverse and reflect the evolutionary histories of bacterial lineages. Intracellular pathogens of the genus Brucella exhibit marked differences in cell envelope structure, regulation, and biogenesis when compared to more commonly studied gram-negative bacteria and therefore provide an excellent comparative model for study of the gram-negative envelope. We review distinct features of the Brucella envelope, highlighting a conserved regulatory system that links cell cycle progression to envelope biogenesis and cell division. We further discuss recently discovered structural features of the Brucella envelope that ensure envelope integrity and that facilitate cell survival in the face of host immune stressors.

Cationised Fibre-Based Cellulose Multi-Layer Membranes for Sterile and High-Flow Bacteria Retention and Inactivation.

Low-cost, readily available, or even disposable membranes in water purification or downstream biopharma processes are becoming attractive alternatives to expensive polymeric columns or filters. In this article, the potential of microfiltration membranes prepared from differently orientated viscose fibre slivers, infused with ultrafine quaternised (qCNF) and amino-hydrophobised (aCNF) cellulose nanofibrils, were investigated for capturing and deactivating the bacteria from water during vacuum filtration. The morphology and capturing mechanism of the single- and multi-layer structured membranes were evaluated using microscopic imaging and colloidal particles. They were assessed for antibacterial efficacy and the retention of selected bacterial species (Escherichia coli, Staphylococcus aureus, Micrococcus luteus), differing in the cell envelope structure, hydrodynamic biovolume (shape and size) and their clustering. The aCNF increased biocidal efficacy significantly when compared to qCNF-integrated membrane, although the latter retained bacteria equally effectively by a thicker multi-layer structured membrane. The retention of bacterial cells occurred through electrostatic and hydrophobic interactions, as well as via interfibrous pore diffusion, depending on their physicochemical properties. For all bacterial strains, the highest retention (up to 100% or log 6 reduction) at >50 L/h∗bar∗m2 flow rate was achieved with a 4-layer gradient-structured membrane containing different aCNF content, thereby matching the performance of industrial polymeric filters used for removing bacteria.

Light-induced antifungal activity of nanoparticles with an encapsulated porphyrin photosensitizer

The strong antifungal effect of sulfonated polystyrene nanoparticles (NPs) with an encapsulated tetraphenylporphyrin (TPP) photosensitizer is reported here. TPP is activated by visible light, resulting in the generation of singlet oxygen. Its antifungal action is potentiated in the presence of potassium iodide, yielding I2/I3⁻, another antifungal species. The NPs exhibit no dark toxicity, but a broad spectrum of antifungal photodynamic effects. The efficiency of this rapid killing (on the order of minutes) depends on the concentration of TPP NPs, potassium iodide, yeast species and temperature. A strong antifungal activity of TPP NPs is demonstrated on eleven pathogenic and opportunistic pathogenic yeast species (six Candida species and other yeast species, including melanized Hortaea werneckii). The composition and architecture of yeast cell envelope structures clearly influence the efficacy of photodynamic therapy. Candida krusei is the most sensitive to photodynamic therapy. Despite expectations, melanin does not provide Hortaea cells with marked resistance compared to white yeast species. The kinetics of the interaction of NPs with yeast cells is also described. This study may inspire and promote the fabrication of a new type of antiseptic for various skin injuries in clinical medicine.

Small protein Cgl2215 enhances phenolic tolerance by promoting MytA activity in Corynebacterium glutamicum

Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments. Among the identified proteins encoded by genes within the C. glutamicum genome, nearly 400 are still functionally unknown. Based on previous transcriptome analysis, we found that the hypothetical protein gene cgl2215 was highly upregulated in response to phenol, ferulic acid, and vanillin stress. The cgl2215 deletion mutant was shown to be more sensitive than the parental strain to phenolic compounds as well as other environmental factors such as heat, ethanol, and oxidative stresses. Cgl2215 interacts with C. glutamicum mycoloyltransferase A (MytA) and enhances its in vitro esterase activity. Sensitivity assays of the ΔmytA and Δcgl2215ΔmytA mutants in response to phenolic stress established that the role of Cgl2215 in phenolic tolerance was mediated by MytA. Furthermore, transmission electron microscopy (TEM) results showed that cgl2215 and mytA deletion both led to defects in the cell envelope structure of C. glutamicum, especially in the outer layer (OL) and electron-transparent layer (ETL). Collectively, these results indicate that Cgl2215 can enhance MytA activity and affect the cell envelope structure by directly interacting with MytA, thus playing an important role in resisting phenolic and other environmental stresses.

Selfish, promiscuous and sometimes useful: how mobile genetic elements drive horizontal gene transfer in microbial populations.

Horizontal gene transfer (HGT) drives microbial adaptation but is often under the control of mobile genetic elements (MGEs) whose interests are not necessarily aligned with those of their hosts. In general, transfer is costly to the donor cell while potentially beneficial to the recipients. The diversity and plasticity of cell–MGEs interactions, and those among MGEs, result in complex evolutionary processes where the source, or even the existence of selection for maintaining a function in the genome, is often unclear. For example, MGE-driven HGT depends on cell envelope structures and defense systems, but many of these are transferred by MGEs themselves. MGEs can spur periods of intense gene transfer by increasing their own rates of horizontal transmission upon communicating, eavesdropping, or sensing the environment and the host physiology. This may result in high-frequency transfer of host genes unrelated to the MGE. Here, we review how MGEs drive HGT and how their transfer mechanisms, selective pressures and genomic traits affect gene flow, and therefore adaptation, in microbial populations. The encoding of many adaptive niche-defining microbial traits in MGEs means that intragenomic conflicts and alliances between cells and their MGEs are key to microbial functional diversification.This article is part of a discussion meeting issue ‘Genomic population structures of microbial pathogens’.

Cryopreservation of stool samples altered the microbial viability quantitively and compositionally.

Stool is the most commonly used sample for gut microbiota analysis in humans and animals. Cryopreservation of stool at -80°C is a feasible and simple method in clinics and researches, especially in large-scale cohort studies. However, the viability of bacteria in stool after freezing has yet well-demonstrated quantitatively and compositionally. This study determined the viable microbiota of samples under cryopreservation at -80°C, relative to fresh samples and that stored at ambient. Stool samples were collected from three healthy adults. Propidium monoazide treatment combined with quantitative PCR and 16S rRNA gene sequencing was performed to target viable microbiota. After freezing, the number of viable bacteria decreased, though inter-individual difference existed. Notably, the alpha diversity of viable microbiota after freezing did not change significantly, while its composition changed. Freezing significantly reduced the viable bacteria in Gram-negative genera of Bacteroidetes and Firmicutes, and proportionally increased Gram-positive bacteria in genera of Actinobacteria and Firmicutes, including Bifidobacterium, Collinsella and Blautia, implying that the cell envelope structure associated with the bacterial sensitivity to freezing. On the contrary, the room temperature storage not only decreased the number of viable bacteria, but also decreased the microbial alpha diversity, and remarkably enriched facultative anaerobes of Escherichia-Shigella, Enterococcus and Lactococcus, some of which are opportunistic pathogens. Our findings suggested that changes in viable microbiota in stool samples caused by cryopreservation should be paid enough attention for downstream utilization.

Synergistic gentamicin-photodynamic therapy against resistant bacteria in burn wound infections

BackgroundMulti-resistant bacteria, partially a result of the abuse of antibiotics, have greatly diminished the effectiveness of antibiotics. The combination of antibiotics with other therapies like antimicrobial photodynamic therapy (aPDT) may provide a useful strategy for fighting resistant bacteria. Here, the synergistic bactericidal effects of toluidine blue (TB)-aPDT and gentamicin (GEN) were evaluated in vitro and in vivo. MethodsThe Post-antibacterial effects were measured at 600 nm (OD600) by a microplate reader. The bacterial envelope and biofilm were observed by a field emission scanning electron microscope. The expression of oxidative stress and Agr system-related genes was analyzed by qRT-PCR after GEN combined with TB-aPDT (GEN&aPDT). Besides, the burn infection model was established to investigate the cloning efficiency of immobilized bacteria, wound healing and inflammatory factors in the lesions. ResultsGEN&aPDT could inhibit the growth of Staphylococcus aureus (S. aureus) and multidrug-resistant S. aureus (MDR S. aureus) for up to 15 h, and destroyed the cell envelope and biofilm structure of S. aureus and MDR S. aureus. During the process, ROS played an important role, inducing oxidative stress and downregulating the expression of AgrA, AgrB and PSM in the Agr system, resulting in decreased bacterial virulence and infectivity. In addition, GEN&aPDT cotreatment could effectively promoted wound healing in burn-infected mice by reducing the numbers of bacterial colonization in the wound, decreasing the content of inflammatory factors, and increasing the expression of growth factors. ConclusionThe present study confirmed a bactericidal synergy between GEN and aPDT in vitro and in vivo, therein, the oxidative stress exhibited an important role in decreasing bacterial virulence and infectivity, which may bring new ideas for the treatment of bacterial resistance.

Disruption of sncRNA Improves the Protective Efficacy of Outer Membrane Vesicles against Helicobacter pylori Infection in a Mouse Model.

The outer membrane vesicles (OMVs) secreted by Helicobacter pylori contain various bacterial components, such as proteins, phospholipids, toxins, and nucleic acids, including small noncoding RNA (sncRNA), which have regulatory functions in cell envelope structure, metabolism, bacterial communication, biofilm formation, and virulence. We previously showed that knocking out sncRNAs sR-989262 and sR-2509025 at the cellular level increased interleukin 8 (IL-8) levels in mice exposed to OMVs. In this study, we show that immunization with ΔsR-989262 and ΔsR-2509025 OMVs intragastrically significantly increased immunoglobulin G (IgG) and secreted IgA levels in mice compared to wild-type OMVs and without weight changes, which indicated that sncRNA-deficient OMVs are relatively safe to immunize mice. The detection of IgG subtypes IgG1 and IgG2c showed that the sncRNA-deficient OMVs primarily stimulate the T helper 2 (Th2)-mediated immune response. Moreover, levels of the cytokines IL-4, IL-13, gamma interferon (IFN-γ), IL-12 (p40), IL-8, and IL-17 indicate that ΔsR-989262 and ΔsR-2509025 OMVs trigger the Th2-type immune response but primarily trigger a Th1-mediated and Th17-mediated immune response. These findings show that OMV-encapsulated sncRNA plays an important role in regulating the immune response in hosts infected by H. pylori at the animal level. Moreover, they show that knocking out of sR-989262 and sR-2509025 improves the immunogenicity and protective efficacy of OMVs, and this may be beneficial to the design of OMV-based H. pylori vaccines.

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

SUMMARYLipid A, the membrane-anchored portion of lipopolysaccharide (LPS), is an essential component of the outer membrane (OM) of nearly all Gram-negative bacteria. Here we identify regulatory and structural factors that together render lipid A nonessential in Caulobacter crescentus. Mutations in the ferric uptake regulator fur allow Caulobacter to survive in the absence of either LpxC, which catalyzes an early step of lipid A synthesis, or CtpA, a tyrosine phosphatase homolog we find is needed for wild-type lipid A structure and abundance. Alterations in Fur-regulated processes, rather than iron status per se, underlie the ability to survive when lipid A synthesis is blocked. Fitness of lipid A-deficient Caulobacter requires an anionic sphingolipid, ceramide phosphoglycerate (CPG), which also mediates sensitivity to the antibiotic colistin. Our results demonstrate that, in an altered regulatory landscape, anionic sphingolipids can support the integrity of a lipid A-deficient OM.

Peptidoglycan from Akkermansia muciniphila MucT: chemical structure and immunostimulatory properties of muropeptides.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835), analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N-acetylglucosamine. Some of the N-acetylmuramic acid (MurNAc) subunits were O-acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non-N-acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host-microbiome interactions.

One small step toward all-atom modeling and simulation of the mycobacteria cell envelope

Mycobacteria are generally non-motile bacteria covered by an protective envelope consisting of a mycobacterial outer membrane (MOM), a peptidoglycan (PG) layer and a mycobacterial inner membrane (MIM). This cell envelope provides the bacterial cell with structural integrity and protects the cell from hostile environments while allowing the transport of solutes and proteins. Despite its importance, our understanding on this cell envelope is rudimentary at best. Because of technical difficulties in deciphering these complex cell envelopes by experiments, all-atom modeling and simulation should be able to provide an unprecedented understanding of the structure and dynamics of the mycobacterial cell envelope.

Immobilization of Polyethyleneimine (PEI) on Flat Surfaces and Nanoparticles Affects Its Ability to Disrupt Bacterial Membranes.

Interactions between a widely used polycationic polymer, polyethyleneimine (PEI), and a Gram-negative bacteria, E. coli, are investigated using atomic force microscopy (AFM) quantitative imaging. The effect of PEI, a known membrane permeabilizer, is characterized by probing both the structure and elasticity of the bacterial cell envelope. At low concentrations, PEI induced nanoscale membrane perturbations all over the bacterial surface. Despite these structural changes, no change in cellular mechanics (Young’s modulus) was detected and the growth of E. coli is barely affected. However, at high PEI concentrations, dramatic changes in both structure and cell mechanics are observed. When immobilized on a flat surface, the ability of PEI to alter the membrane structure and reduce bacterial elasticity is diminished. We further probe this immobilization-induced effect by covalently attaching the polymer to the surface of polydopamine nanoparticles (PDNP). The nanoparticle-immobilized PEI (PDNP-PEI), though not able to induce major structural changes on the outer membrane of E. coli (in contrast to the flat surface), was able to bind to and reduce the Young’s modulus of the bacteria. Taken together, our data demonstrate that the state of polycationic polymers, whether bound or free—which greatly dictates their overall configuration—plays a major role on how they interact with and disrupt bacterial membranes.

MapZ deficiency leads to defects in the envelope structure and changes stress tolerance of Streptococcus mutans.

Cell division is a central process in bacteria and a prerequisite for pathogenicity. Several proteins are involved in this process to ensure the accurate localization and proper function of the division machinery. In Streptococcus mutans, MapZ marks the division sites and position of the Z-ring to regulate cell division; however, whether MapZ deficiency can impair the cariogenic virulence of S. mutans remains unclear. Here, using a phenotypic assay and RNA-seq, we investigated the role of MapZ in cell envelope maintenance, biofilm formation, and stress tolerance in S. mutans. The results show that MapZ is important for normal cell shape and envelope structure, and its deletion causes abnormal septum structure and a thin cell wall. Subsequently, we found that the absence of MapZ leads to a greater level of cell death within 12 h biofilms, but it does not seem to affect biofilm architecture and accumulation. mapZ deletion also results in a decreased acid and osmotic stress tolerance. Furthermore, RNA-seq data reveal that MapZ deficiency causes changes in the expression levels of genes involved in transport systems, sugar metabolism, nature competence, and bacteriocin synthesis. Interestingly, we found that mapZ mutation renders S. mutans more sensitive to chlorhexidine. Taken together, our study suggests that MapZ plays a role in maintaining cell envelope structure and stress tolerance in S. mutans, showing a potential application as a drug target for caries prevention.