Outer Membrane Research Articles

Outer membrane barrier impairment by envC deletion reduces gut colonization of Crohn's disease pathobiont Escherichia coli.

Adherent-invasive Escherichia coli (AIEC) has been implicated in the aetiology of Crohn's disease (CD), a chronic inflammatory disorder of the gastrointestinal tract. The presence of Enterobacteriaceae, including AIEC, is heightened in the intestines of CD patients. Therefore, inhibiting AIEC colonization in the gastrointestinal tract could be a promising therapeutic intervention for CD. This study aims to assess the potential of EnvC as a novel therapeutic target, examining how disrupting EnvC activity through the deletion of the envC gene decreases AIEC gut colonization levels. EnvC serves as a catalyst for peptidoglycan (also called murein) amidases, facilitating bacterial cell division. An AIEC mutant lacking the envC gene exhibited impaired cell division. Furthermore, envC deletion led to a diminished outer membrane barrier, as seen in our finding that the envC mutant became susceptible to vancomycin. Finally, we found that the envC mutant is impaired in competitive gut colonization in a dysbiotic mouse model. The colonization defects might be attributable to reduced resistance to colonic bile acids, as evidenced by our finding that increased colonic levels of bile acids inhibited the colonization of the gastrointestinal tract by AIEC strains. The present findings suggest that targeting bacterial cell division through the inhibition of EnvC activity could represent a promising intervention for CD.

The outer membrane protein, OMP71, of Riemerella anatipestifer, mediates adhesion and virulence by binding to CD46 in ducks

The Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host’s respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC–MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δomp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.

Inhibition of BAK-mediated apoptosis by the BH3-only protein BNIP5.

BCL-2 family proteins regulate apoptosis by initiating mitochondrial outer membrane permeabilization (MOMP). Activation of the MOMP effectors BAX and BAK is controlled by the interplay of anti-apoptotic BCL-2 proteins (e.g., MCL-1) and pro-apoptotic BH3-only proteins (e.g., BIM). Using a genome-wide CRISPR-dCas9 transactivation screen we identified BNIP5 as an inhibitor of BAK-, but not BAX-induced apoptosis. BNIP5 blocked BAK activation in different cell types and in response to various cytotoxic therapies. The BH3 domain of BNIP5 was both necessary and sufficient to block BAK activation. Mechanistically, the BH3 domain of BNIP5 acts as a selective BAK activator, but a poor de-repressor of complexes between BAK and pro-survival BCL-2 family proteins. By promoting the binding of activated BAK to MCL-1 or BCL-xL, BNIP5 inhibits apoptosis when BAX is absent. Based on our observations, BNIP5 can act functionally as an anti-apoptotic BH3-only protein.

Complement-mediated killing of Escherichia coli by mechanical destabilization of the cell envelope.

Complement proteins eliminate Gram-negative bacteria in the blood via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. Initially, bacteria survived despite the formation of hundreds of MACs that were randomly distributed over the cell surface. This was followed by larger-scale disruption of the outer membrane, including propagating defects and fractures, and by an overall swelling and stiffening of the bacterial surface, which precede inner membrane permeation. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, leading to inner membrane permeation and cell death.

Appraisal of the causal effect of Chlamydia trachomatis infection on epithelial ovarian cancer risk: a two-sample Mendelian randomisation study.

History of Chlamydia trachomatis infection has previously been associated with epithelial ovarian cancer (EOC) in observational studies. We conducted a two-sample univariable Mendelian randomisation (MR) study to examine whether genetically predicted seropositivity to the C. trachomatis major outer membrane protein (momp) D is causally associated with EOC. MR analyses employed genetic associations derived from UK Biobank as proxies for momp D seropositivity in 25 509 EOC cases and 40 941 controls that participated in the Ovarian Cancer Association Consortium. Findings were replicated using a GWAS meta-analyses of global biobanks including the UK Biobank, FinnGen and BioBank Japan. Genetically predicted momp D seropositivity was associated with overall and high-grade serous EOC risk in inverse-variance weighted (IVW) and MR-Egger univariable MR analysis (odds ratio (OR) 1.06; 95% confidence interval (CI) 1.02-1.10, and OR 1.08; 95%CI 1.01-1.16, respectively). Replication yielded similar results for overall EOC (OR 1.11; 95%CI 1.01-1.22). This MR study supports a causative link between C. trachomatis infection and overall and high-grade serous EOC.

Intraperitoneal Injection of the Porphyromonas gingivalis Outer Membrane Vesicle (OMV) Stimulated Expressions of Neuroinflammatory Markers and Histopathological Changes in the Brains of Adult Zebrafish.

Porphyromonas gingivalis is the major pathogenic bacteria found in the subgingival plaque of patients with periodontitis, which leads to neuroinflammation. The bacteria destroy periodontal tissue through virulence factors, which are retained in the bacteria's outer membrane vesicles (OMV). This study aimed to determine the real-time effect of an intraperitoneal injection of P. gingivalis OMV on the production and expression of inflammatory markers and histopathological changes in adult zebrafishes' central nervous systems (CNS). Following the LD50 (21 µg of OMV), the zebrafish were injected intraperitoneally with 18 µg of OMVs, and the control group were injected with normal saline at seven different time points. Brains of experimental zebrafish were dissected at desired time points for colorimetric assays, ELISA, and histology. This study discovered that nitric oxide and PGE2 were significantly increased at 45 min, while IL-1β and IL-6 were expressed at subsequent 12 h and 24 h time points, respectively. Histopathological changes such as blood coagulation, astrocytosis, edema, spongiosis, and necrosis were observed between the 6hour and 24 h time points. The two apoptotic enzymes, caspases 3 and 9, were not expressed at any point. In summary, the OMV-induced neuroinflammatory responses and histopathological changes in adult zebrafish were time-point dependent. This study will enrich our understanding of the mechanism of P. gingivalis OMVs in neuroinflammation in a zebrafish model, most especially the timing of the expression of inflammatory mediators in relation to observable changes in brain tissues.

Outer Membrane Vesicles Formed by Clinical Proteus mirabilis Strains May Be Incorporated into the Outer Membrane of Other P. mirabilis Cells and Demonstrate Lytic Properties.

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient's cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs' interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis.

Voltage-Dependent Anion Channels in Male Reproductive Cells: Players in Healthy Fertility?

Male infertility affects nearly 50% of infertile couples, with various underlying causes, including endocrine disorders, testicular defects, and environmental factors. Spermatozoa rely on mitochondrial oxidative metabolism for motility and fertilization, with mitochondria playing a crucial role in sperm energy production, calcium regulation, and redox balance. Voltage-dependent anion channels (VDACs), located on the outer mitochondrial membrane, regulate energy and metabolite exchange, which are essential for sperm function. This review offers an updated analysis of VDACs in the male reproductive system, summarizing recent advances in understanding their expression patterns, molecular functions, and regulatory mechanisms. Although VDACs have been widely studied in other tissues, their specific roles in male reproductive physiology still remain underexplored. Special attention is given to the involvement of VDAC2/3 isoforms, which may influence mitochondrial function in sperm cells and could be implicated in male fertility disorders. This update provides a comprehensive framework for future research in reproductive biology, underscoring the significance of VDACs as a molecular link between mitochondrial function and male fertility.

Aspirin enhances radio/chemo-therapy sensitivity in C. elegans by inducing germ cell apoptosis and suppresses RAS overactivated tumorigenesis via mtROS-mediated DNA damage and MAPK pathway

Previous studies have demonstrated that combination therapy involving radiotherapy and aspirin decreases the survival rate of cancer cells. However, the mechanism by which aspirin exerts its radiation sensitization effect at the in vivo level remains largely unclear. In this study, we employed Caenorhabditis elegans (C. elegans) as a model organism to investigate the effect of aspirin combined with radio/chemo-therapy on tumors at the individual level. Here, we illustrate that high-dose aspirin increases the expression of genes involved in core apoptosis pathways (egl-1, ced-9, ced-4 and ced-3) and induces germ cell apoptosis in C. elegans through mitochondrial outer membrane permeabilization (MOMP) and elevation of reactive oxygen species (ROS) levels. Crucially, aspirin-induces ROS upregulates the expression of genes critical for DNA damage response (hus-1, clk-2 and cep-1) and genes involved in MAPK pathways (lin-45, mek-2, mpk-1, sek-1 and pmk-1), thereby mediating the enhanced sensitivity of radio/chemo-therapy by aspirin. Notably, aspirin fails to induce germ cell apoptosis and enhance radio/chemo-therapy in C. elegans lacking the expression of each of those genes. Furthermore, in a C. elegans tumor-like symptom model, aspirin enhances radio/chemo-therapy sensitivity through ROS induction. However, low-dose aspirin can diminish the apoptotic signal of reproductive cells in C. elegans and exert anti-inflammatory effects. Our research results suggest that the tumor-suppressive and radio/chemo-therapy sensitizing effects of aspirin provide robust experimental evidence for improving the clinical efficacy of tumor radio/chemo-therapy and deepening our understanding of aspirin's mechanism of action in cancer.

Breaking Barriers: Exploiting Envelope Biogenesis and Stress Responses to Develop Novel Antimicrobial Strategies in Gram-Negative Bacteria.

Antimicrobial resistance (AMR) has emerged as a global health threat, necessitating immediate actions to develop novel antimicrobial strategies and enforce strong stewardship of existing antibiotics to manage the emergence of drug-resistant strains. This issue is particularly concerning when it comes to Gram-negative bacteria, which possess an almost impenetrable outer membrane (OM) that acts as a formidable barrier to existing antimicrobial compounds. This OM is an asymmetric structure, composed of various components that confer stability, fluidity, and integrity to the bacterial cell. The maintenance and restoration of membrane integrity are regulated by envelope stress response systems (ESRs), which monitor its assembly and detect damages caused by external insults. Bacterial communities encounter a wide range of environmental niches to which they must respond and adapt for survival, sustenance, and virulence. ESRs play crucial roles in coordinating the expression of virulence factors, adaptive physiological behaviors, and antibiotic resistance determinants. Given their role in regulating bacterial cell physiology and maintaining membrane homeostasis, ESRs present promising targets for drug development. Considering numerous studies highlighting the involvement of ESRs in virulence, antibiotic resistance, and alternative resistance mechanisms in pathogens, this review aims to present these systems as potential drug targets, thereby encouraging further research in this direction.

Numerical assessments of geometry, proximity and multi-electrode effects on electroporation in mitochondria and the endoplasmic reticulum to nanosecond electric pulses

Most simulations of electric field driven bioeffects have considered spherical cellular geometries or probed symmetrical structures for simplicity. This work assesses cellular transmembrane potential build-up and electroporation in a Jurkat cell that includes the endoplasmic reticulum (ER) and mitochondria, both of which have complex shapes, in response to external nanosecond electric pulses. The simulations are based on a time-domain nodal analysis that incorporates membrane poration utilizing the Smoluchowski model with angular-dependent changes in membrane conductivity. Consistent with prior experimental reports, the simulations show that the ER requires the largest electric field for electroporation, while the inner mitochondrial membrane (IMM) is the easiest membrane to porate. Our results suggest that the experimentally observed increase in intracellular calcium could be due to a calcium induced calcium release (CICR) process that is initiated by outer cell membrane breakdown. Repeated pulsing and/or using multiple electrodes are shown to create a stronger poration. The role of mutual coupling, screening, and proximity effects in bringing about electric field modifications is also probed. Finally, while including greater geometric details might refine predictions, the qualitative trends are expected to remain.

Bovine Lactoferrin Impairs in-vitro Growth of Neonatal Sepsis-causingEscherichia Coli Clinical Isolates

Abstract Background E. coli is a dominant cause of neonatal sepsis with no prevention currently available. Lactoferrin (LF) is an iron-sequestering antibacterial protein that has been shown to decrease all-cause neonatal late-onset sepsis. However, the impact of LF on impairing growth of neonatal E. coli septicemia isolates carrying various iron acquisition virulence genes has not been investigated. We aimed to determine the effects of bovine lactoferrin (bLF) on in vitro growth of neonatal E. coli septicemia strains and to determine the presence of iron-acquisition virulence genes in the isolates. Methods We tested six clinical E. coli strains obtained from blood of septic neonates, including the archetypal RS218 strain. Isolates represent the most frequent phylogroups (B2 and D) and multi-locus sequence types (ST95, ST69, and ST131) among neonatal E. coli septicemia strains. The nonpathogenic strain DH5α was used as control. Strains were grown in media containing 0, 0.1, 1.0, and 10 mg/mL bLF. Optical density (OD) readings were obtained every 30 min. for 20 h. Experiments were repeated 3 times. OD area under the curve (AUC) values were compared with ANOVA. Whole genome sequencing (WGS) was obtained to determine the presence of iron-acquisition virulence genes using VirulenceFinder v. 2.0.3 (J Clin Micro 2020. 58 (10):e01269-20). Results For all strains, the growth decrease with 10 mg/mL bLF compared to zero bLF was highly significant (p < 0.001) (Fig.1). Growth was also significantly reduced at 1 mg/mL for most strains (p ≤ 0.02). Growth decrease at 0.1 mg/mL was only significant for isolate SCB31. Growth suppression at 10 mg/mL was greatest in DH5α. WGS analyses showed that all pathogenic strains carried several iron acquisition virulence genes, with chuA (hemin receptor), fyuA (siderophore receptor) in irp2 (protein 2 peptide synthetase) and sitA (iron transport protein) found in all (Fig. 2). DH5α had no iron acquisition virulence genes. Conclusion Bovine LF is effective in impairing growth of neonatal sepsis-causing E. coli regardless of the presence of iron acquisition genes known to contribute to virulence. Future studies will examine additional isolates and the effect of greater bLF concentrations. These data have the potential to inform dosing regimens for additional in vivo studies to further assess bLF’s protective effects against neonatal sepsis. Figure 1. Comparison of growth curve data among E. coli strains. Optical density AUC values of six neonatal septicemia E. coli isolates growth with increasing concentrations of bovine lactoferrin. DH5α is a nonpathogenic control strain. Bars depict means and standard deviations for each condition tested. Asterisks indicate statistically significant differences compared to the 0 mg/mL bLF condition (*p < 0.001, **p < 0.05). Figure 2. Presence of iron acquisition virulence genes in E. coli isolates. WGS analyses detected several genes, which are indicated in black. White indicates absence of gene. ST, Sequence type; chuA, Outer membrane hemin receptor; fyuA, Siderophore receptor (yersiniabactin); iroN, Siderophore receptor; irp2, High-molecular-weight protein 2 nonribosomal peptide synthetase; iucC, Aerobactin siderophore synthetase; iutA, Ferric aerobactin siderophore receptor; sitA, Iron transport protein. *Multi-drug resistant isolate.

Quantitative Biodistribution of OMVs Using SPECT/CT Imaging with HYNIC-Duramycin Radiolabeling.

Introduction: Bacterial outer membrane vesicles (OMVs) are emerging as important players in the host-microbiome interaction, while also proving to be a promising platform for vaccine development and targeted drug delivery. The available methods for measuring their biodistribution, however, are limited. We aimed to establish a high-efficiency radiolabeling method for the treatment of OMVs. Methods: 99mTc-HYNIC-duramycin was incubated with OMVs isolated from E. coli BL21(DE3) ΔnlpI ΔlpxM. Radiolabeling efficiency (RLE) and radiochemical purity (RCP) were measured with size-exclusion high-performance liquid chromatography. The biodistribution was quantitatively measured in mice using SPECT/CT imaging. Results: RLE was 81.84 ± 2.03% for undiluted OMV suspension and 56.17 ± 2.29% for 100× dilution. Postlabeling purification with a spin-desalting column results in 100% radioactivity in the OMV fraction according to HPLC, indicating 100% RCP of the final product. The biodistribution was found to be in line with previous data reported in the literature using other OMV tracking attempts. Conclusions: Our findings illustrate that using HYNIC-duramycin for labeling of the OMVs enhances efficiency and is easily implementable for in vivo imaging studies, significantly improving upon earlier methods.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice

ABSTRACT The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

Structural basis for synthase activation and cellulose modification in the E. coli Type II Bcs secretion system

Bacterial cellulosic polymers constitute a prevalent class of biofilm matrix exopolysaccharides that are synthesized by several types of bacterial cellulose secretion (Bcs) systems, which include conserved cyclic diguanylate (c-di-GMP)-dependent cellulose synthase modules together with diverse accessory subunits. In E. coli, the biogenesis of phosphoethanolamine (pEtN)-modified cellulose relies on the BcsRQABEFG macrocomplex, encompassing inner-membrane and cytosolic subunits, and an outer membrane porin, BcsC. Here, we use cryogenic electron microscopy to shed light on the molecular mechanisms of BcsA-dependent recruitment and stabilization of a trimeric BcsG pEtN-transferase for polymer modification, and a dimeric BcsF-dependent recruitment of an otherwise cytosolic BcsE2R2Q2 regulatory complex. We further demonstrate that BcsE, a secondary c-di-GMP sensor, can remain dinucleotide-bound and retain the essential-for-secretion BcsRQ partners onto the synthase even in the absence of direct c-di-GMP-synthase complexation, likely lowering the threshold for c-di-GMP-dependent synthase activation. Such activation-by-proxy mechanism could allow Bcs secretion system activity even in the absence of substantial intracellular c-di-GMP increase, and is reminiscent of other widespread synthase-dependent polysaccharide secretion systems where dinucleotide sensing and/or synthase stabilization are carried out by key co-polymerase subunits.

Unveiling the impact of Leptospira TolC efflux protein on host tissue adherence, complement evasion, and diagnostic potential.

The TolC family protein of Leptospira is a type I outer membrane efflux protein. Phylogenetic analysis revealed significant sequence conservation among pathogenic Leptospira species (83%-98% identity) compared with intermediate and saprophytic species. Structural modeling indicated a composition of six β-strands and 10 α-helices arranged in two repeats, resembling bacterial outer membrane efflux proteins. Recombinant TolC (rTolC), expressed in a heterologous host and purified via Ni-NTA chromatography, maintained its secondary structural integrity, as verified by circular dichroism spectroscopy. Polyclonal antibodies against rTolC detected native TolC expression in pathogenic Leptospira but not in nonpathogenic ones. Immunoassays and detergent fractionation assays indicated surface localization of TolC. The rTolC's recognition by sera from leptospirosis-infected hosts across species suggests its utility as a diagnostic marker. Notably, rTolC demonstrated binding affinity for various extracellular matrix components, including collagen and chondroitin sulfate A, as well as plasma proteins such as factor H, C3b, and plasminogen, indicating potential roles in tissue adhesion and immune evasion. Functional assays demonstrated that rTolC-bound FH retained cofactor activity for C3b cleavage, highlighting TolC's role in complement regulation. The rTolC protein inhibited both the alternative and the classical pathway-mediated membrane attack complex (MAC) deposition in vitro. Blocking surface-expressed TolC on leptospires using specific antibodies reduced FH acquisition by Leptospira and increased MAC deposition on the spirochete. These findings indicate that TolC contributes to leptospiral virulence by promoting host tissue colonization and evading the immune response, presenting it as a potential target for diagnostic and therapeutic strategies.

Development of an Intracellular Nitric Oxide-Donating Cell-Penetrating Polypeptide as an Immunogenic Cell Death Inducer.

Recently, nitric oxide (NO) has been shown to induce immunogenic cell death (ICD) in tumor cells through endoplasmic reticulum (ER) stress and mitochondrial outer membrane permeabilization (MOMP). However, NO is unstable, making direct delivery difficult. In this study, we developed a cell-penetrating polypeptide-based NO donor, poly(l-guanidine) (PLG). Given that the guanidine structure can be catalyzed by reactive oxygen species (ROS) to produce NO, helical PLG plays three roles: spontaneous cell penetration, intracellular ROS generation to produce NO, and induction of ICD. The results revealed that helical PLG generates NO inside the cell by self-inducible guanidine oxidation and that NO effectively elicits ICD by ER stress- and MOMP-dependent intertwined mechanisms.

Chicken GLUT4 function via enhancing mitochondrial oxidative phosphorylation and inhibiting ribosome pathway in skeletal muscle satellite cells

Glucose Transporter 4 (GLUT4) is a crucial protein facilitating glucose uptake and metabolism across cell membranes in mammals. However, information on GLUT4 in birds has historically been limited. In this study, we investigated the dynamic expression profile of chicken GLUT4 using real-time quantitative PCR (RT-qPCR) and examined its potential effects and mechanisms via GLUT4 overexpression and RNA sequencing (RNA-seq) in chicken primary skeletal muscle satellite cells (CP-SMSCs). Our results demonstrated that chicken GLUT4 is differentially expressed across tissues, with predominant expression in skeletal muscles, and across developmental stages of CP-SMSCs, with notable upregulation during the phases of cell proliferation and early differentiation. Notably, 0.1 μM insulin for 60 min significantly elevated the expression of GLUT4 in CP-SMSCs (P < 0.05). GLUT4 overexpression in CP-SMSCs promoted cell proliferation, as evidenced by Cell Counting Kit-8 (CCK-8) (P < 0.05) and 5-Ethynyl-2'-Deoxyuridine (EDU) assays (P < 0.05), and enhanced glucose consumption following 0.1 μM insulin treatment (P < 0.05). However, it inhibited glucose consumption 12 h after the addition of 5 g/L glucose (P < 0.05). After overexpressing GLUT4, we identified 302 differentially expressed genes (DEGs) in CP-SMSCs, with 134 upregulated and 168 downregulated. These DEGs are primarily enriched in pathways such as oxidative phosphorylation, ribosome, cardiac muscle contraction, ATP metabolic processes, and mitochondrial protein complexes. Specifically, in the enriched oxidative phosphorylation pathway, the upregulated DEGs (12) encode mitochondrial proteins, while the downregulated DEGs (6) are nuclear genome-derived. The ribosomal pathway is predominantly inhibited, accompanying with the downregulation of the translocase of outer mitochondrial membrane 7 (TOMM7)/translocase of inner mitochondrial membrane 8 (TIMM8A) complex responsible for mitochondrial protein transport, and a reduction in 28S (LOC121106978) and 18S (LOC112533601) ribosomal rRNAs. In conclusion, chicken GLUT4 is dynamically modulated during development and acts as an insulin responder that significantly regulates cellular glucose uptake and cell proliferation. This regulation occurs mainly through enhancing the mitochondrial oxidative phosphorylation and inhibiting ribosomal pathway.

Investigation of Citrobacter freundii clinical isolates in a Chinese hospital during 2020-2022 revealed genomic characterization of an extremely drug-resistant C. freundii ST257 clinical strain GMU8049 co-carrying blaNDM-1 and a novel blaCMY variant.

The emergence of multidrug-resistant Citrobacter freundii poses a significant threat to public health. C. freundii isolates were collected from clinical patients in a Chinese hospital during 2020-2022. An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two β-lactamase genes, including plasmid-mediated blaNDM-1 and chromosomal blaCMY encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to β-lactams. Conjugation experiment confirmed that the blaNDM-1 resistance gene located on pGMU8049 could be successfully transferred to Escherichia coli EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other C. freundii strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant C. freundii, highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.IMPORTANCEEmerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257 Citrobacter freundii clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of blaNDM-1 via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant C. freundii poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen.

Surface exclusion of IncC conjugative plasmids and their relatives.

The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.