Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

Native ß-barrel substrates pass through two shared intermediates during folding on the BAM complex.

The assembly of ß-barrel proteins into membranes is mediated by the evolutionarily conserved ß-barrel assembly machine (BAM) complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of ß-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in the substrate ß-strands, we can compare the residence time of each substrate strand within the BamA lumen. We validated this method using two defective, slow-folding substrates. We used this method to characterize stable intermediates which occur during folding of two structurally different native substrates. Strikingly, these intermediates occur during identical stages of folding for both substrates: soon after folding has begun and just before folding is completed. We suggest that these intermediates arise due to barriers to folding that are common between ß-barrel substrates, and that the BAM catalyst is able to fold so many different substrates because it addresses these common challenges.

A minimum functional form of the Escherichia coli BAM complex constituted by BamADE assembles outer membrane proteins in vitro.

The biogenesis of outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM), which is a heteropentomeric complex composed of five proteins named BamA-E in Escherichia coli. Despite great progress in the BAM structural analysis, the molecular details of BAM-mediated processes as well as the exact function of each BAM component during OMP assembly are still not fully understood. To enable a distinguishment of the function of each BAM component, it is the aim of the present work to examine and identify the effective minimum form of the E. coli BAM complex by use of a well-defined reconstitution strategy based on a previously developed versatile assay. Our data demonstrate that BamADE is the core BAM component and constitutes a minimum functional form for OMP assembly in E. coli, which can be stimulated by BamB and BamC. While BamB and BamC have a redundant function based on the minimum form, both together seem to cooperate with each other to substitute for the function of the missing BamD or BamE. Moreover, the BamAE470K mutant also requires the function of BamD and BamE to assemble OMPs in vitro, which vice verse suggests that BamADE are the effective minimum functional form of the E. coli BAM complex.

Prevalence of Slam-dependent hemophilins in Gram-negative bacteria.

Iron acquisition systems are crucial for pathogen growth and survival in iron-limiting host environments. To overcome nutritional immunity, bacterial pathogens evolved to use diverse mechanisms to acquire iron. Here, we examine a heme acquisition system that utilizes hemophores called hemophilins which are also referred to as HphAs in several Gram-negative bacteria. In this study, we report three new HphA structures from Stenotrophomonas maltophilia, Vibrio harveyi, and Haemophilus parainfluenzae. Structural determination of HphAs revealed an N-terminal clamp-like domain that binds heme and a C-terminal eight-stranded ß-barrel domain that shares the same architecture as the Slam-dependent Neisserial surface lipoproteins. The genetic organization of HphAs consists of genes encoding a Slam homolog and a TonB-dependent receptor (TBDR). We investigated the Slam-HphA system in the native organism or the reconstituted system in Escherichia coli cells and found that the efficient secretion of HphA depends on Slam. The TBDR also played an important role in heme uptake and conferred specificity for its cognate HphA. Furthermore, bioinformatic analysis of HphA homologs revealed that HphAs are conserved in the alpha, beta, and gammaproteobacteria. Together, these results show that the Slam-dependent HphA-type hemophores are prevalent in Gram-negative bacteria and further expand the role of Slams in transporting soluble proteins. IMPORTANCE: This paper describes the structure and function of a family of Slam (Type IX secretion System) secreted hemophores that bacteria use to uptake heme (iron) while establishing an infection. Using structure-based bioinformatics analysis to define the diversity and prevalence of this heme acquisition pathway, we discovered that a large portion of gammaproteobacterial harbors this system. As organisms, including Acinetobacter baumannii, utilize this system to facilitate survival during host invasion, the identification of this heme acquisition system in bacteria species is valuable information and may represent a target for antimicrobials.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins.

Toll-like receptors (TLRs) are essential components of innate immunity that serves as the first line of defense against the invaded microorganisms. However, successful infectious pathogens subvert TLR signaling to suppress the activation of innate and adaptive responses. Brucella species are infectious intracellular bacterial pathogens causing the worldwide zoonotic disease, brucellosis, that impacts economic growth of many countries. Brucella species are considered as stealthy bacterial pathogens as they efficiently evade or suppress host innate and adaptive immune responses for their chronic persistence. However, the bacterial effectors and their host targets for modulating the immune responses remain obscure. Brucella encodes various outer membrane proteins (Omps) that facilitate their invasion, intracellular replication, and immunomodulation. Outer membrane protein 25 (Omp25) of Brucella plays an important role in the immune modulation through suppression of proinflammatory cytokines. However, the mechanism and the signaling pathways that are targeted by Omp25 to attenuate the production of proinflammatory cytokines remain obscure. Here, we report that Omp25 and its variants, viz. Omp25b, Omp25c, and Omp25d, suppress production of proinflammatory cytokines that are mediated by various TLRs. Furthermore, we demonstrate that Omp25 and its variants promote enhanced ubiquitination and degradation of TLRs and their adaptor proteins to attenuate the expression of proinflammatory cytokines. Targeting multiple TLRs and adaptor proteins enables Omp25 to effectively suppress the expression of proinflammatory cytokines that are induced by diverse pathogen-associated molecular patterns. This can contribute to the defective adaptive immune response and the chronic persistence of Brucella in the host.

Outer membrane ß-barrel structure prediction through the lens of AlphaFold2.

Most proteins found in the outer membrane of gram-negative bacteria share a common domain: the transmembrane ß-barrel. These outer membrane ß-barrels (OMBBs) occur in multiple sizes and different families with a wide range of functions evolved independently by amplification from a pool of homologous ancestral ßß-hairpins. This is part of the reason why predicting their three-dimensional (3D) structure, especially by homology modeling, is a major challenge. Recently, DeepMind's AlphaFold v2 (AF2) became the first structure prediction method to reach close-to-experimental atomic accuracy in CASP even for difficult targets. However, membrane proteins, especially OMBBs, were not abundant during their training, raising the question of how accurate the predictions are for these families. In this study, we assessed the performance of AF2 in the prediction of OMBBs and OMBB-like folds of various topologies using an in-house-developed tool for the analysis of OMBB 3D structures, and barrOs. In agreement with previous studies on other membrane protein classes, our results indicate that AF2 predicts transmembrane ß-barrel structures at high accuracy independently of the use of templates, even for novel topologies absent from the training set. These results provide confidence on the models generated by AF2 and open the door to the structural elucidation of novel transmembrane ß-barrel topologies identified in high-throughput OMBB annotation studies or designed de novo.

Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles.

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Diversity and antigenic potentials of Mycoplasmopsis bovis secreted and outer membrane proteins within a core genome of strains isolated from North American bison and cattle.

Mycoplasmopsis bovis is a worldwide economically important pathogen of cattle that can cause or indirectly contribute to bovine respiratory disease. M. bovis is also a primary etiological agent of respiratory disease in bison with high mortality rates. A major challenge in the development of an efficacious M. bovis vaccine is the design of antigens that contain both MHC-1 and MHC-2 T-cell epitopes, and that account for population level diversity within the species. Publicly available genomes and sequence read archive libraries of 381 M. bovis strains isolated from cattle (n = 202) and bison (n = 179) in North America were used to identify a core genome of 575 genes, including 38 that encode either known or predicted secreted or outer membrane proteins. The antigenic potentials of the proteins were characterized by the presence and strength of their T-cell epitopes, and their protein variant diversity at the population-level. The proteins had surprisingly low diversity and varying predictive levels of T-cell antigenicity. These results provide a reference for the selection or design of antigens for vaccine testing against strains infecting North American cattle and bison.

OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus.

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

Salicylic acids and pathogenic bacteria: new perspectives on an old compound.

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid-immune modulation in response to microbial infection-was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines.

Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus.

Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va's drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.

Escherichia coli Activate Extraintestinal Antibody Response and Provide Anti-Infective Immunity.

The effects of intestinal microflora on extraintestinal immune response by intestinal cytokines and metabolites have been documented, but whether intestinal microbes stimulate serum antibody generation is unknown. Here, serum antibodies against 69 outer membrane proteins of Escherichia coli, a dominant bacterium in the human intestine, are detected in 141 healthy individuals of varying ages. Antibodies against E. coli outer membrane proteins are determined in all serum samples tested, and frequencies of antibodies to five outer membrane proteins (OmpA, OmpX, TsX, HlpA, and FepA) are close to 100%. Serum antibodies against E. coli outer membrane proteins are further validated by Western blot and bacterial pull-down. Moreover, the present study shows that OstA, HlpA, Tsx, NlpB, OmpC, YfcU, and OmpA provide specific immune protection against pathogenic E. coli, while HlpA and OmpA also exhibit cross-protection against Staphylococcus aureus infection. These finding indicate that intestinal E. coli activate extraintestinal antibody responses and provide anti-infective immunity.

Phenotypic Changes in Phage Survivors of Multidrug-Resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) infections have become a major global issue in the healthcare sector. Alternative viable tactics for combating bacterial infections, such as the use of bacteriophages, can be considered. One of the major challenges in phage therapy is the emergence of phage-resistant bacteria. This study isolated bacteriophages from water and soil samples against MDR-KP isolates. Susceptible bacterial hosts were exposed to phages at different concentrations and prolonged durations of time to obtain phage-resistant survivors. Phenotypic changes such as changes in growth rates, biofilm formation ability, antibiotic sensitivity patterns, and outer membrane proteins (OMPs) profiling of the survivors were studied. Our findings indicate that the phage ØKp11 and ØKp26 survivors had reduced growth rates and biofilm formation ability, altered antibiotic sensitivity patterns, and reduced OMPs expression compared with the parent MDR-KP002 isolate. These results suggest that the alternations in the bacterial envelope result in phenotypic phage resistance among MDR bacterial isolates. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01217-6.

The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNß, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNß and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNß-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNß can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, ß, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

NlpE Is an OmpA-Associated Outer Membrane Sensor of the Cpx Envelope Stress Response.

Gram-negative bacteria utilize several envelope stress responses (ESRs) to sense and respond to diverse signals within a multilayered cell envelope. The CpxRA ESR responds to multiple stresses that perturb envelope protein homeostasis. Signaling in the Cpx response is regulated by auxiliary factors, such as the outer membrane (OM) lipoprotein NlpE, an activator of the response. NlpE communicates surface adhesion to the Cpx response; however, the mechanism by which NlpE accomplishes this remains unknown. In this study, we report a novel interaction between NlpE and the major OM protein OmpA. Both NlpE and OmpA are required to activate the Cpx response in surface-adhered cells. Furthermore, NlpE senses OmpA overexpression and the NlpE C-terminal domain transduces this signal to the Cpx response, revealing a novel signaling function for this domain. Mutation of OmpA peptidoglycan-binding residues abrogates signaling during OmpA overexpression, suggesting that NlpE signaling from the OM through the cell wall is coordinated via OmpA. Overall, these findings reveal NlpE to be a versatile envelope sensor that takes advantage of its structure, localization, and cooperation with other envelope proteins to initiate adaptation to diverse signals. IMPORTANCE The envelope is not only a barrier that protects bacteria from the environment but also a crucial site for the transduction of signals critical for colonization and pathogenesis. The discovery of novel complexes between NlpE and OmpA contributes to an emerging understanding of the key contribution of OM ß-barrel protein and lipoprotein complexes to envelope stress signaling. Overall, our findings provide mechanistic insight into how the Cpx response senses signals relevant to surface adhesion and biofilm growth to facilitate bacterial adaptation.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1): a novel modulator of mitochondrial autophagy.

A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.

The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane-proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel ß-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel ß-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent-in stark contrast to the primarily positive surface charge of the left-handed parallel ß-roll "type" protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left-handed parallel ß-roll structures may differ depending on their position in the structure.

DH5α Outer Membrane-Coated Biomimetic Nanocapsules Deliver Drugs to Brain Metastases but not Normal Brain Cells via Targeting GRP94.

Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.

Outer membrane proteins and vesicles as promising vaccine candidates against Vibrio spp. infections.

Indiscriminate use of antibiotics to treat bacterial infections has brought unmanageable antibiotic-resistant strains into existence. Vibrio spp. represents one such gram-negative enteric pathogenic group with more than 100 species, infecting humans and fish. The Vibrio spp. is demarcated into two groups, one that causes cholera and the other producing non-cholera or vibriosis infections. People who encounter contaminated water are at risk, but young children and pregnant women are the most vulnerable. Though controllable, Vibrio infection still necessitates the development of preventative measures, such as vaccinations, that can lessen the severity of the infection and reduce reliance on antibiotic use. With emerging multi-drug resistant strains, efforts are needed to develop newer vaccines, such as subunit-based or outer membrane vesicle-based. Thus, this review strives to bring together available information about Vibrio spp. outer membrane proteins and vesicles, encompassing their structure, function, and immunoprotective role.