Cell Envelope Research Articles (Page 1)

The cell envelope of diderm bacteria: a unified scaffold, not a stack of layers.

Pseudomonas aeruginosa is a Gram-negative bacterium that poses a serious threat to patients with weakened immunity, cystic fibrosis, severe burns, or those in hospitals. Its ability to resist antibiotics comes largely from its outer membrane, which blocks drug entry. This means higher doses are often needed, raising the risk of side effects. To design new treatments, researchers need drugs that not only bind strongly to bacterial targets but also cross this tough membrane. Unfortunately, there are few reliable methods to directly measure how easily drugs pass through the Pseudomonas aeruginosa cell envelope. Recent advances, such as electrophysiology-based flux studies, have started to reveal how different antibiotics particularly β-lactams move through porin channels. These studies show large differences in permeability, but the findings remain scattered. What is missing is a unified dataset that captures permeability under varied conditions. Such a resource would clarify how porin structures influence drug entry and help chemists design better compounds. This review brings together current knowledge on drug permeability in Pseudomonas aeruginosa, with a focus on electrophysiological and related methods. This review highlights the need for standardized approaches that generate consistent and comparable data. A comprehensive “permeability atlas” could guide the development of new antibiotics by fine-tuning molecular properties like size, charge, and lipophilicity, ultimately improving porin passage and restoring treatment effectiveness against this challenging pathogen.

The Tol-Pal system is essential for maintaining outer membrane (OM) stability during cell division in Gram-negative bacteria. The inner membrane complex TolQRA harnesses proton motive force (PMF) to establish transient interactions within the periplasm, thereby coordinating cell envelope remodeling and facilitating OM invagination at division sites. However, the precise mechanism remains unclear. Here, we present cryo-electron microscopy structures of Escherichia coli TolQRA in multiple conformational states at 2.92–3.52 Å resolution, revealing rotary dynamics within the complex. Computational simulations reveal a proton-conductive channel comprising the putative proton-accepting residue Asp23 and the conserved polar residues Thr145 and Thr178, with monitored inter-residue distances providing support for a proton-driven rotary mechanism. Site-directed mutagenesis combined with functional assays validates the AlphaFold-predicted structure of the periplasmic domains of TolR and TolA, and further pinpoints critical residues required for complex function. Together, these findings advance our understanding of TolQRA-mediated proton transduction and offer new avenues for antibiotic drug development.

Engineering MtrAB two-component system enhances protein and glutamate export in corynebacterium glutamicum through cell wall remodeling.

Investigating inhibitory effect of sterol targeting compounds against B. anthracis: Membrane Microdomain a probable target.

Klebsiella pneumoniae (K. pneumoniae), a common nosocomial pathogen causing severe pulmonary infections, is often complicated by coinfections. Bacterial ghosts, which are empty bacterial cell envelopes, hold significant promise as vaccine adjuvants. This study aims to develop and evaluate a novel combination vaccine platform utilizing K. pneumoniae ghosts (KP ghosts) to explore their intrinsic immunogenic properties as both vaccine and natural adjuvants. In this study, we showed that KP ghosts enhanced maturation and activation of bone marrow-derived dendritic cells, increasing surface markers (CD40, CD80, CD86, and MHC II) and cytokine secretion (IL-1β, TNF-α, and IL-12p70). The KP ghost-based vaccine provided strong immune protection in mice, significantly improving survival rates and reducing bacterial loads in organs after bacterial challenge. Additionally, to assess the adjuvant potential of KP ghosts, C57BL/6 mice were co-immunized with KP ghosts and a model antigen, ovalbumin (OVA). In comparison to OVA alone, the combination of OVA and KP ghosts elicited higher levels of specific IgG antibodies. Furthermore, OVA combined with KP ghosts increased the expression of the early activation marker CD69 on T cells after in vitro antigen stimulation and raised the frequencies of central memory T cells (Tcm) and CD4+ IFN-γ+ T cells. In conclusion, KP ghosts are effective as both vaccine and adjuvant components, enhancing the innate immune response of dendritic cells and the antigen-specific response of T cells. These findings highlight KP ghosts as a dual-purpose vaccine/adjuvant platform for broader antibacterial vaccine development.

Polymyxin antibiotics target lipopolysaccharides (LPS) in Gram-negative bacteria, but persistence and mcr-mediated resistance increasingly compromise their therapeutic efficacy. Here, we use super-resolution localisation microscopy to investigate the nanoscale organisation of LPS and membrane lipids in colistin-persistent Escherichia coli and Pseudomonas aeruginosa. We find that persister cells exhibit heterogeneous LPS and membrane lipid distributions, with localised LPS clustering along the cell envelope, compared to susceptible cells. Unexpectedly, mcr-1-positive E. coli displays a similar LPS clustering phenotype to that of persisters, without altering membrane organisation. These findings suggest that both persistence and resistance involve envelope reorganisation, with increased LPS clustering and remodelling observed in both E. coli and P. aeruginosa. This work reveals a morphological signature of persistence and identifies a shared feature between persistence and resistance in Gram-negative bacteria.

Microbial manufacturing offers a sustainable and environmentally friendly approach for chemical production. However, the inherent toxicity of certain high-value chemicals to microbial cell factories presents a significant challenge, severely constraining production efficiency. To enhance microbial tolerance, extensive synthetic biology strategies have been developed. The cell envelope serves as the primary natural barrier in microorganisms, and both its intrinsic composition, including membrane lipids, membrane proteins, and cell wall components, and the regulation of these components play crucial roles in modulating cellular responses to environmental stress. Engineering strategies targeting intracellular components, such as transcription factors and repair pathways, have demonstrated effectiveness in enhancing microbial tolerance to toxic end-products and intermediates. Additionally, recent advances have focused on extracellular engineering, including biofilm formation and the modulation of intercellular interactions, which have garnered significant scientific interest. This review aims to provide a systematic overview of these strategies and offers insights to facilitate the industrial translation and commercialization of microbial production of toxic end-products and intermediates.

Tuberculosis (TB) remains the world's deadliest bacterial infection, with 8.2 million newly notified cases and an estimated 1.25 million deaths in 2023. Alarmingly, ∼19% of multidrug- or rifampicin-resistant (MDR/RR) strains already meet the World Health Organization (WHO) definition of pre-XDR-TB because they are resistant to at least one fluoroquinolone (FQ). Although gyrA/gyrB target-site mutations dominate clinical FQ resistance, Mycobacteria also rely on transcriptional networks that help them withstand the oxidative and DNA strand-breaking stress caused by these drugs. Central to this response is the heterodimeric transcription factor pafBC, whose WYL domain binds to single-stranded DNA and redirects RNA polymerase to a dedicated promoter set, thereby orchestrating a LexA-independent DNA-damage response (DDR). Up-regulation of pafBC has been linked to enhanced intracellular survival of M. tuberculosis and nontuberculous mycobacteria after FQ exposure, yet the downstream phenotypes and their connection to drug or phage resistance have remained unclear. Here, we demonstrate that deletion of pafBC in Mycobacterium smegmatis profoundly remodels the cell envelope, as evidenced by altered colony rugosity, reduced sliding motility, enhanced aggregation, and a three- to 5-fold decline in quantitative biofilm biomass. Untargeted lipid profiling revealed the selective depletion of long-chain trehalose polyphosphates and other apolar glycolipids that normally decorate the outer membrane─lipid classes that have recently been shown in other studies to serve as essential receptors for therapeutic mycobacteriophages such as BPs and Muddy. Consistent with this lipid deficit, the pafBC mutant exhibited markedly reduced phage adsorption and plaque formation; ectopic expression of RecA restored adsorption efficiency, implicating DDR envelope crosstalk in antiphage defense. Complementation with wild-type pafBC rescued lipid composition, biofilm mass, and phage resistance, whereas a WYL-domain mutant that cannot bind single-stranded DNA failed to do so, underscoring the necessity of canonical pafBC activation for envelope homeostasis. Immunoprofiling in THP-1 macrophages further showed that pafBC-proficient bacilli induce significantly higher secretion of IL-1β, TNF-α, and IL-6 compared to their isogenic mutant. This effect correlated with the presence of intact surface glycolipids, molecules known to interact with scavenger and Toll-like receptors on phagocytes and to enhance opsonizing antibody deposition at the host-pathogen interface. Overall, our findings connect the molecular mechanisms of the pafBC DDR with observable phenotypes such as fluoroquinolone tolerance, biofilm structure, phage resistance, and host immune recognition, by highlighting cell-envelope remodeling as the central factor.

Bacterial membrane vesicles (MVs) are a heterogeneous group of lipid-bound structures produced by bacteria. Antibiotic stress aggravates the secretion of MVs that contributes to the development of bacterial antibiotic resistance. This review provides a focused, resistance-oriented perspective on the interplay between MVs and antibiotic resistance. We outline MV biogenesis, emphasizing the distinct formation mechanisms of Gram-negative and Gram-positive bacteria. We further focus on the secretion of MVs under antibiotic stress, highlighting pathways such as bacterial envelope stress, SOS response, and cell wall disruption. The pivotal role of MVs in bacterial antibiotic resistance is also elucidated, including neutralizing antibiotics, absorbing phages, and facilitating drug efflux, biofilm formation, and horizontal gene transfer. Current challenges and future prospects for elucidating MV-mediated mechanisms in antibiotic resistance are discussed.

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane (OM) and peptidoglycan (PG) layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking OM and PG homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in PG and incorporate d-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates PG precursor genes (e.g., dadA and alr), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a ∆ldtJ ∆mla double mutant, implicating the OM lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the ∆ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between PG remodeling and OM lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through PG modification but also by influencing broader regulatory and metabolic networks.IMPORTANCEAcinetobacter baumannii is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan (PG) layer and outer membrane (OM). While LD-transpeptidases are traditionally known for reinforcing PG structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of ldtJ results in pronounced growth defects and abnormal cell morphology-phenotypes that are fully suppressed by disrupting the OM lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between PG remodeling and OM lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria.

ABSTRACTMultidrug‐resistant (MDR) bacterial infections constitute one of the top global public health threats. This study investigated the potential of gallium liquid metal nanoparticles (GaLM NPs) as a new agent against MDR pathogens. GaLM NPs was bactericidal against methicillin‐resistant Staphylococcus aureus (MRSA) isolates and a vancomycin‐intermediate S. aureus reference strain (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values between 39 and 156 μg/mL). The bactericidal activity of GaLM NPs was supported by transmission electron microscopy showing marked ultrastructural changes in the cell envelope of MRSA USA300. GaLM NPs were bacteriostatic against selected Gram‐negative (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) reference strains and isolates (MICs between 39 and 625 μg/mL). Furthermore, GaLM NPs demonstrated additive and bacteriostatic activity when combined with sub‐inhibitory concentrations of colistin against P. aeruginosa isolates. Additionally, GaLM NPs showed anti‐biofilm activity against MRSA USA300 (minimum biofilm eradication concentration of 625 μg/mL); morphology changes of GaLM NPs‐treated cells was demonstrated by scanning electron microscopy. Finally, GaLM NPs was safe to human epidermal keratinocyte cell line at 1024 µg/mL (6.5 × MIC). We conclude that GaLM NPs warrant further exploration for the effective treatment of Gram‐positive or Gram‐negative infections both alone and in combination with antimicrobial drugs.

Soil bacterial communities maintain membrane fluidity in response to variations in temperature and pH through modifications in lipid composition, branching degree and chain length. Recent studies have shown that analyses of bacterial membrane lipids, in particular 3-hydroxy fatty acids (3-OH FAs), can be used for reconstruction of environmental conditions. However, the link between bulk changes in membrane lipid composition in environmental bacterial isolates and the structure of the cell envelope in which they are found remains poorly characterized. In this study, we use transmission electron microscopy to correlate such changes in response to temperature and pH variations using three previously investigated environmental isolates of Gram-negative soil bacteria from the Bacteriodota phylum. We show structural changes in the cell envelope and other structures under the influence of pH and most importantly temperature. While a previous study showed temperature to also significantly affect 3-OH FA composition in these strains, no causal link to structure was found. However, this remains a limited study, highlighting the necessity for expanded research involving an increased diversity and number of samples to see if correlations exist in other bacteria between molecular analyses of lipid composition and cell structure observations in response to changing environmental conditions.

Pathways of intrinsic resistance in bacteria are promising targets for novel antibiotics and resistance breakers. Here, we used a genome-wide screen to identify single gene knockouts of Escherichia coli that were hypersusceptible to trimethoprim and chloramphenicol, two chemically diverse broad-spectrum antibiotics. Among the hits from our screen, knockouts of acrB, an efflux pump, and rfaG or lpxM, both involved in cell envelope biogenesis, were hypersensitive to multiple antimicrobials and could sensitize genetically resistant E. coli strains to antibiotics. Using experimental evolution under trimethoprim pressure, we show that high drug selection regimes drove these knockouts to extinction more frequently than wild type. Among them, ΔacrB was most compromised in its ability to evolve resistance, establishing it as a promising target for “resistance proofing.” At a sub-inhibitory trimethoprim concentration, however, all three knockouts adapted to the antibiotic and consequently recovered from hypersensitivity, albeit to different extents. This recovery was driven by mutations in drug-specific resistance pathways, rather than compensatory evolution, frequently involving upregulation of the drug target. Notably, resistance-conferring mutations could by-pass defects in cell wall biosynthesis more effectively than efflux even though resistant mutations did not directly engage either pathway. Since inhibiting drug-efflux emerged as a better strategy, we tested the ability of chlorpromazine, an efflux pump inhibitor (EPI), to resistance proof E. coli against trimethoprim. While qualitatively similar in the short term, genetic and pharmacological inhibition differed dramatically on an evolutionary time scale due to evolution of resistance to the EPI. Further, adaptation to the EPI-antibiotic pair also led to multidrug adaptation. The lack of concordance between genetic and pharmacological inhibition revealed a crucial lacuna in our understanding of the mutational repertoires that facilitate adaptation to antibiotics in bacteria. We propose that while intrinsic resistance mechanisms are effective targets for antibiotic sensitization, rapid evolutionary recovery may significantly limit their utility.

Abstract Listeria monocytogenes is a foodborne pathogen that can cause severe disease in immunocompromised persons, and its ability to survive stressors encountered in food production environments (FPEs) makes it difficult to eliminate from the food chain. Previous transcriptomic analysis revealed that in response to lactic acid exposure L. monocytogenes significantly upregulates Rli47, a noncoding RNA that has previously been shown to interact with the ilvA transcript and suppress growth of L. monocytogenes in the absence of isoleucine. We show that at logarithmic phase, an rli47 deletion mutant had a higher survival compared to the parent strain after exposure to lactic acid. Flow cytometry indicated that lactic acid exposure did not differentially affect the proportion of metabolically active cells in the deletion mutant and wild type. Transcriptomic analysis and in silico target prediction suggested that Rli47 could affect pathways involved with cell envelope structure; due to the link between cell envelope integrity and organic acid stress, it is possible that in the absence of rli47 the cell envelope of logarithmic phase L. monocytogenes cells is more resistant to lactic acid exposure. These results suggest that Rli47 functionality may vary due to factors including temperature and nutrient availability.

Upon reacting with cellular components, Hg(II) ions elicit the production of reactive oxygen species (ROS). While the ROS-promoted cytotoxic and genotoxic effects induced by Hg(II) have been widely described in eukaryotes, such effects have been less studied in bacteria. In this work, the prokaryotic environmental model Bacillus subtilis was employed to evaluate the cytotoxic and genotoxic impact of Hg(II) over strains proficient or deficient in SOS, general stress and antioxidant responses, as well as the global transcriptional response elicited by this ion. The exposure to HgCl2 significantly increased the mutation frequency to rifampicin resistance (RifR) in WT and mutant strains, suggesting a major contribution of these pathways in counteracting the genotoxic effects of Hg(II). Detection of A → T and C → G transversion mutations in the rpoB gene of Hg(II)-exposed cells suggested the generation of 8-oxo-guanines (8-OxoGs) and other oxidized DNA bases. The RNA-seq study revealed upregulation of genes involved in efflux and/or reduction of metal ions, synthesis of sulfur-containing molecules, and downregulation of genes implicated in iron metabolism and cell envelope stress. Therefore, our results indicate that metal extrusion and scavenging of Hg(II) by thiol-rich molecules may constitute a line of defense of B. subtilis that counteracts the noxious effects of ROS resulting from an imbalance in iron metabolism elicited by this ion.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that thrives in diverse environments. This pathogen causes a range of infections, including microbial keratitis (MK), a sight-threatening corneal infection. Central to its virulence are six specialized protein secretion systems (Types 1-6) that span the complex cell envelope. One-step systems (Types 1, 3, 4, 6) directly translocate effectors across both of its cell membranes, while two-step systems (Types 2, 5) first export substrates into the periplasm via the Sec or Tat pathways before outer-membrane release. These molecular machines deliver toxins, enzymes and competitive effectors that facilitate tissue damage, immune evasion, nutrient acquisition, biofilm formation and interbacterial killing. Their expression and activity are often coordinated by quorum-sensing networks (Las, Rhl, Pqs, Iqs). Although various secretion systems and their effectors have been characterized, the specific contributions of each system to corneal infection have yet to be comprehensively reviewed. PubMed and Google Scholar were searched to synthesize current knowledge of the structure, regulation, and substrates of these secretion systems, to highlight their contributions to keratitis pathogenesis, and to evaluate emerging anti-virulence strategies targeting these pathways as novel therapeutics. The T3SS system and its effectors ExoU and ExoS play dominant roles in keratitis severity while other secretion systems further enhance virulence by facilitating toxin release, biofilm development, and interbacterial competition. Regulatory interactions with quorum-sensing pathways amplify their impact during infection. Understanding the functional roles of all six secretion pathways and their regulatory mechanisms will be critical for identifying and developing novel anti-virulence therapeutics for MK.

BackgroundEscherichia coli, Staphylococcus aureus, and Salmonella pullorum are significant pathogens that threaten livestock and poultry health. Although antibiotics and synthetic antimicrobial agents can combat these pathogens, antibiotic resistance remains a major concern. Recent decades have seen growing interest in antibiotic alternatives. Juglone, a natural naphthoquinone compound from Juglandaceae plant, exhibits strong antimicrobial activity against S. aureus. However, its antimicrobial mechanism is not yet fully understood. This study investigated the antimicrobial mechanism of juglone from the perspectives of cell biology, cell morphology, and transcriptomics.ResultsJuglone had potent antimicrobial effects against E. coli, S. aureus, and S. pullorum. The minimum inhibitory concentration (MIC) of juglone against all three bacterial strains was 15.6 µg/mL. Treatment with juglone decreased bacterial metabolic activity, reduced the intracellular DNA and RNA fluorescence intensity, resulted in the leakage of intracellular alkaline phosphatase (AKP) and ions, and caused a decline in ATP content and ATPase activity. Scanning electron microscopy (SEM) revealed significant membrane damage in each of the three bacterial species following juglone treatment. Transcriptomic sequencing and Gene Ontology (GO) enrichment analysis of S. pullorum revealed that juglone treatment resulted in a significant upregulation of GO terms related to translation, while those terms associated with transport, localization, and membrane functions were significantly downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the pathways related to oxidative phosphorylation and the citrate cycle were significantly upregulated, whereas those pathways related to ABC transporters and quorum sensing were significantly downregulated.ConclusionThese findings suggest that juglone compromises the permeability and integrity of the cell envelope in E. coli, S. aureus, and S. pullorum, resulting in cytoplasmic leakage and metabolic impairment. Additionally, juglone alters the gene expression of transporters, interferes with the energy metabolism, protein synthesis and transport, quorum sensing, and biofilm formation of S. pullorum, thereby exerting antimicrobial effects.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-025-04354-0.

Campylobacter hepaticus is the etiological agent of Spotty Liver Disease (SLD), a newly emerging bacterial disease of laying hens resulting in significant mortality and production losses primarily in free-range systems. Although its economic impact continues to grow, the molecular basis of C. hepaticus pathogenesis remains poorly understood. In this study, we conducted transcriptomic profiling of C. hepaticus in three host-relevant conditions, exposure to chicken bile, infection of a chicken liver hepatocellular carcinoma (LMH) cell line, and isolation from liver lesions of naturally infected chickens. Through RNA-seq analysis, we found unique gene expression signatures in each environment. In the bile, C. hepaticus exhibited differential expression of 412 genes, with upregulation of genes related to motility, cell envelope remodeling, glycosylation, nitrate respiration, and multidrug efflux systems, indicating a stress-adaptive, metabolically active lifestyle. In LMH, on the other hand, 125 genes were differentially expressed, primarily reflecting downregulation of motility, oxidative stress response, chaperones, and core metabolic processes, suggesting that these cells adopt a less active, intracellular dormant lifestyle. Transcriptomic analysis of C. hepaticus isolated from the liver identified 26 differentially expressed genes, featuring selective upregulation of genes associated with nitrate respiration, sulfur metabolism, and pyridoxal 5’ phosphate homeostasis, alongside downregulation of the major outer membrane porin (momp), stress response chaperones (dnaK, groL), and genes involved in oxidative stress defense and energy production. Furthermore, the immune evasion-related gene cmeA and a glycosyltransferase gene were found to be highly upregulated. This study presents the first in-depth transcriptomic exploration of C. hepaticus in multiple host relevant niches. Our findings reveal niche-specific gene expression profiles and highlight metabolic and structural adaptations that enable C. hepaticus to survive during bile exposure, persist within host cells, and contribute to liver pathology. These insights provide a basis for identifying novel virulence determinants and may inform the development of targeted interventions, including vaccines or antimicrobial therapy, to control SLD in commercial poultry operations.

Arabinogalactans (AG) from the Mycobacterium tuberculosis (Mtb) cell wall represent potential therapeutic agents against the notorious disease tuberculosis (TB). However, the synthetic access to these long, highly branched, and complex arabinogalactans remains a challenging task, hindering structure-activity relationship studies. Here, we report the chemical synthesis of arabinogalactan 92-mer 1 and shorter sequences 14-mer 2, 30-mer 3, and 50-mer 4 from M. tuberculosis cell envelope via an orthogonal one-pot glycosylation strategy based on glycosyl ortho-(1-phenylvinyl)benzoates, which avoids such issues as aglycone transfer inherent to one-pot assemblies based on thioglycosides. The synthetic route also features the following characteristics: 1) highly stereoselective construction of eight 1,2-cis-Araf-(1→2) linkages via hydrogen-bond-mediated aglycone delivery strategy; 2) effective one-pot assembly of several linear and branched glycans by strategic utilizations of glycosyl N-phenyltrifluoroacetimidates, ortho-alkynylbenzoates, and ortho-(1-phenylvinyl)benzoates; 3) a one-pot and convergent [(7×2+7)×2+50] assembly of arabinogalactan 92-mer with the simultaneous formations of six furanosidic bonds. Conformational analysis using molecular dynamics simulations and NMR spectroscopy, as well as immunological studies of synthetic arabinogalactans 1-4 in human cell models, revealed that the surface-exposed 30-mer 3 epitope induced only a modest NF-κB activation while preserving cell viability.