The OprF porin as a potential target for the restoration of carbapenem susceptibility in Pseudomonas aeruginosa expressing acquired carbapenemases.

Carbapenem resistance in Pseudomonas aeruginosa is primarily due to the acquisition of carbapenemases and is often associated with a diminution of the membrane permeability. The outer membrane protein, OprD, is a well-known route, by which carbapenems, predominantly imipenem, can enter the cell, and its loss has been associated with reduced susceptibility to imipenem. In this study, we investigated the antibiotic susceptibility patterns of isogenic P. aeruginosa mutants containing various acquired ß-lactamases, including carbapenemases, in a porin-depleted background. We identified that the deletion of oprF was associated with some recovery of susceptibility to carbapenems.

Molecular epidemiology and carbapenem resistance mechanisms of Pseudomonas aeruginosa isolated from a hospital in Fujian, China.

The worldwide spread of Pseudomonas aeruginosa, especially carbapenem-resistant P. aeruginosa (CRPA), poses a serious threat to global public health. In this research, we collected and studied the clinical prevalence, molecular epidemiology, and resistance mechanisms of CRPA in Fujian, China. Among 167 non-duplicated P. aeruginosa isolates collected during 2019-2021, strains from respiratory specimens and wound secretions of older males in the intensive care unit dominated. Ninety-eight isolates (58.7 %) were resistant to at least one tested antibiotic, among which 70 strains were carbapenem-resistant. Moleclar typing of the CRPA isolates revealed they were highly divergent, belonging to 46 different sequence types. It is noteworthy that two previously reported high risk clones, ST1971 specific to China and the globally prevalent ST357, were found. Several carbapenem resistance-related characteristics were also explored in 70 CRPA isolates. Firstly, carbapenemase was phenotypically positive in 22.9 % of CRPA, genetically predominant by metallo-ß-lactamase (MBL) and co-carrige of different carbapenemase genes. Then, mutations of the carbapenem-specific porins oprD and opdP were commonly observed, with frequencies of 97.1% and 100.0%, respectively. Furthermore, the biofilm formation and relative transcription levels of 8 multidrug efflux pump genes were also found to be increased in 48.6 % and 72.9 % of CRPA isolates compared to the reference strain PAO1. These findings will help fill the data gaps in molecular characteristics of CRPA on the southeastern coast of China and emphasize the urgent need for data-based specific stewardship for antipseudomonal practices to prevent the dissemination of CRPA.

Bacterial Porins and Their Procoagulant Role: Implication in the Pathophysiology of Several Thrombotic Complications during Sepsis.

The association between sepsis and thrombotic complications is still not well known. Different mechanisms have been shown to be involved in the sepsis-induced prothrombotic state, but clinical scenarios may differ. In this review, we have summarized the role that bacterial products such as porins and toxins can have in the induction of the prothrombotic state during sepsis and the interaction that they can have with each other. Furthermore, the above-mentioned mechanisms might be involved in the pattern of the clinical presentation of thrombotic events during bacterial sepsis, which would secondarily explain the association between sepsis and venous thromboembolism, the association between sepsis and disseminated intravascular coagulation, and the association between sepsis and microangiopathic venous thromboembolism.

The Effect of Lipopolysaccharides on the Electrostatic Properties of Gram-Negative General Porins from Enterobacteriaceae.

We investigated, by using all-atom molecular dynamics simulations, the effect of the outer membrane of Gram-negative bacteria, composed in the outer leaflet by polar/charged lipopolysaccharides (LPS), on the electrostatic properties of general porins from the Enterobacteriaceae family. General porins constitute the main path for the facilitated diffusion of polar antibiotics through the outer membrane. As model system we selected OmpK36 from Klebsiella pneumoniae, the ortholog of OmpC from Escherichia coli. This species presents high variability of amino acid composition of porins, with the effect to increase its resistance to the penetration of antibiotics. The various properties we analyzed seem to indicate that LPS acts as an independent layer without affecting the internal electrostatic properties of OmpK36. The only apparent effect on the microsecond time scale we sampled is the appearance of calcium ions, when present at moderate concentration in solution, inside the pore. However, we noticed increased fluctuations of the polarization density and only minor changes on its average value.

What Contributes to the MIC? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: K. pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: MICs to ß-lactams and inhibitor combinations were determined by agar dilution or E-test. Outer membrane porin production was evaluated by western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by RT-qPCR. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. ESßLs and pAmpCs in addition to porin loss were sufficient to confer resistance to the third generation cephalosporins, pipercillin/tazobactam, ceftolozane/tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to pipercillin/tazobactam. DISCUSSION: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

BACKGROUND: One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. METHODS AND RESULTS: Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. CONCLUSIONS: Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

In vivo evaluation of new adjuvant systems based on combination of Salmonella Typhi porins with particulate systems: Liposomes versus polymeric particles.

Subunit vaccines that have weak immunogenic activity require adjuvant systems for enhancedcellular and long-acting humoral immune responses. Both lipid-based and polymeric-based particulate adjuvants have been widely investigated to induce the desired immune responses against the subunit vaccines. The adjuvant efficacy of these particulate adjuvants depends upon their physicochemical properties such as particle size, surface charge, shape and their composition. Previously, we showed in vitro effect of adjuvant systems based on combination of chitosan and Salmonella Typhi porins in microparticle or nanoparticle form, which were spherical with positive surface charge. In the present study, we have further developed an adjuvant system based on combination of porins with liposomes (cationic and neutral) and investigated the adjuvant effect of both the liposomal and polymeric systems in BALB/c mice using a model antigen, ovalbumin. Humoral immune responses were determined following priming and booster dose at 15-day intervals. In overall, IgM and IgG levels were induced in the presence of both the liposomal and polymeric adjuvant systems indicating the positive impact of combination with porins. The highest IgM levels were obtained on Day 8, and liposomal adjuvant systems were found to elicit significantly higher IgM levels compared to polymeric systems. IgG levels were increased significantly after booster, particularly more profound with the micro-sized polymeric system when compared to cationic liposomal system with nano-size. Our results demonstrated that the developed particulate systems are promising both as an adjuvant and delivery system, providing enhanced immune responses against subunit antigens, and have the potential for long-term protection.

Self-Assembling Peptide-Appended Metallomacrocycle Pores for Selective Water Translocation.

Artificial water channels selectively transport water, excluding all ions. Unimolecular channels have been synthesized via complex synthetic steps. Ideally, simpler compounds requesting less synthetic steps should efficiently lead to selective channels by self-assembly. Herein, we report a self-assembled peptide-bound Ni2+ metallomacrocycle, 1, in which rim-peptide-bound units are connected to a central macrocycle obtained via condensation in the presence of Ni2+ ions. Compound 1 achieves a single-channel permeability up to 107-108 water/s/channel and insignificant ion transport, which is 1 order of magnitude lower than those for aquaporins. Molecular simulations probe that spongelike aggregates can form to generate transient cluster water pathways through the bilayer. Altogether, adaptive metallosupramolecular self-assembly is an efficient and simple way to construct selective channel superstructures.

Polyamine as a microenvironment factor in resistance to antibiotics.

One of the main issues in modern medicine is the decrease in the efficacy of antibiotic therapy against resistant microorganisms. The advent of antimicrobial resistance has added significantly to the impact of infectious diseases, in number of infections, as well as added healthcare costs. The development of antibiotic tolerance and resistance is influenced by a variety of environmental variables, and it is important to identify these environmental factors as part of any strategy for combating antibiotic resistance. The review aims to emphasize that biogenic polyamines are one of such environmental cues that impacts the antibiotic resistance in bacteria. The biogenic polyamines can help bacteria acquire resistance to antibiotics either by regulating the level of number of porin channels in the outer membrane, by modifying the outer membrane liposaccharides or by protecting macromolecule from antibiotic stress. Thus, understanding the way polyamines function in bacteria can thus be beneficial while designing the drugs to combat diseases.

Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles.

Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

Effect of Non-Specific Porins from the Outer Membrane of Yersinia pseudotuberculosis on Mice Brain Cortex Tissues.

It was found that a single-dose immunization of mice with Yersinia pseudotuberculosis porins OmpF and OmpC causes development of pathological changes in the deep layers of cerebral cortex characterized by dystrophic changes in the cells against the background of the increasing titer of specific antibodies. At the same time, the increased level of caspase-3 expression is observed in the neurons, which indicates induction of proapoptotic signaling pathways. The obtained results indicate potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate development of degenerative changes in brain cells.

Revealing the single-channel characteristics of OprD (OccAB1) porins from hospital strains of Acinetobacter baumannii.

Nowadays, reports of antimicrobial resistance (AMR) against many antibiotics are increasing because of their misapplication. With this rise, there is a serious decrease in the discovery and development of new types of antibiotics amid an increase in multi-drug resistance. Unfermented Acinetobacter baumannii from gram-negative bacteria, which is one of the main causes of nosocomial infections and multi-drug resistance, has 4 main kinds of antibiotic resistance mechanism: inactivating antibiotics by enzymes, reduced numbers of porins and changing of their target or cellular functions due to mutations, and efflux pumps. In this study, characterization of the possible mutations in OprD (OccAB1) porins from hospital strains of A. baumannii were investigated using single channel electrophysiology and compared with the standard OprD isolated from wild type ATCC 19,606. For this aim, 5 A. baumannii bacteria samples were obtained from patients infected with A. baumannii, after which OprD porins were isolated from these A. baumannii strains. OprD porins were then inserted in an artificial lipid bilayer and the current-voltage curves were obtained using electrical recordings through a pair of Ag/AgCl electrodes. We observed that each porin has a characteristic conductance and single channel recording, which then leads to differences in channel diameter. Finally, the single channel data have been compared with the gene sequences of each porin. It was interesting to find out that each porin isolated has a unique porin diameter and decreased anion selectivity compared to the wild type.

Evaluation of copper-induced biomolecular changes in different porin mutants of Escherichia coli W3110 by infrared spectroscopy.

Copper (Cu), one of the heavy metals, plays a vital role in many complex biochemical reactions as a trace element. However, it often becomes toxic when its concentration in the cell exceeds a certain level. Homeostasis of metals in the cell is primarily related to regulating metal transport into and out of the cell. Therefore, it is thought that porin proteins, which have a role in membrane permeability, may also play a role in developing Cu resistance. This study identified the differences between the molecular profiles of wild-type Escherichia coli W3110 and its seven different porin mutants exposed to Cu ions using attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. The results showed that the absence of porin genes elicits global changes in the structure and composition of membrane lipids and proteins, in both the absence and presence of Cu. The lack of porin genes significantly elevated the amounts of fatty acids and phospholipids. When the alterations in protein secondary structures were compared, the quantity of amide I proteins was diminished by the presence of Cu. However, the amount of amide II proteins increased in porin mutant groups independent of Cu presence or absence. The DNAs are transformed from B- and Z-form to A-form due to porin mutations and the presence of Cu ions. The lack of porin genes increased polysaccharide content independent of Cu presence. This study can help characterize Cu detoxification efficiency and guide for obtaining active living cells to be used in bioremediation.

Computational prediction of extracellular loops of the Por39 outer membrane porin of Rhodospirillum rubrum suitable for epitope surface display.

Outer membrane porins from Gram-negative bacteria are established vehicles for the production of vaccines. Typically, one or more of the extracellular loops of a porin are replaced by a peptide encoding a foreign epitope, and recombinant porin is then used as a vaccine. However, many host strains are potentially pathogenic, and also produce toxic lipopolysaccharide (LPS), both of which are undesirable for safety reasons. In contrast, the outer membrane porins from photosynthetic, purple bacteria have no known human pathology and produce only weakly toxic LPS. The purple bacterium Rhodospirillum rubrum is well-suited for large-scale biotechnology, and expresses a major porin, Por39, which is a candidate for a vaccine platform. Unfortunately, the atomic structure of Por39 could not be determined so far, and Por39 shows only a weak homology to other porins of known structure, making the assignment of external loops difficult. Here, we construct a knowledge-based model of Por39 using secondary structure constraints from both the low sequence homology to the 2POR porin from Rhodobacter capsulatus, for which the X-ray structure is known, as well as those obtained using secondary structure prediction packages. The secondary structure predictions were used to constrain a three-dimensional model created using the I-TASSER package. The modelling procedure was validated by predicting the structure of 2POR using the same strategy, but excluding the 2POR X-ray structure from the I-TASSER database. The final Por39 model allows three external loops to be defined precisely, and could also be used to obtain an initial model for the closely related Por41 using molecular modelling. These structures provide a good starting point for the insertion of epitopes with vaccine potential.

The Small RNA NcS25 Regulates Biological Amine-Transporting Outer Membrane Porin BCAL3473 in Burkholderia cenocepacia.

Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia cenocepacia, and this study aimed to characterize the biological role of the conserved small RNA NcS25 and its cognate target, outer membrane protein BCAL3473. The B. cenocepacia genome carries a large number of genes encoding porins with yet-uncharacterized functions. Expression of the porin BCAL3473 is strongly repressed by NcS25 and activated by other factors, such as a LysR-type regulator and nitrogen-depleted growth conditions. The porin is involved in transport of arginine, tyrosine, tyramine, and putrescine across the outer membrane. Porin BCAL3473, with NcS25 as a major regulator, plays an important role in the nitrogen metabolism of B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is a Gram-negative bacterium which causes infections in immunocompromised individuals and in people with cystic fibrosis. A low outer membrane permeability is one of the factors giving it a high level of innate resistance to antibiotics. Porins provide selective permeability for nutrients, and antibiotics can also traverse the outer membrane by this means. Knowing the properties and specificities of porin channels is therefore important for understanding resistance mechanisms and for developing new antibiotics and could help in overcoming permeability issues in antibiotic treatment.

Penetration through Outer Membrane and Efflux Potential in Pseudomonas aeruginosa of Bulgecin A as an Adjuvant to ß-Lactam Antibiotics.

The treatment of infections by Gram-negative bacteria remains a difficult clinical challenge. In the light of the dearth of discovery of novel antibiotics, one strategy that is being explored is the use of adjuvants to enhance antibacterial activities of existing antibiotics. One such adjuvant is bulgecin A, which allows for the lowering of minimal-inhibitory concentrations for ß-lactam antibiotics. We have shown that bulgecin A inhibits three of the pseudomonal lytic transglycosylases in its mode of action, yet high concentrations are needed for potentiation activity. Herein, we document that bulgecin A is not a substrate for pseudomonal efflux pumps, whose functions could have been a culprit in the need for high concentrations. We present evidence that the penetration barrier into the periplasm is at the root of the need for high concentrations of bulgecin A in its potentiation of ß-lactam antibiotics.

The SDBC is active in quenching oxidative conditions and bridges the cell envelope layers in Deinococcus radiodurans.

Deinococcus radiodurans is known for its remarkable ability to withstand harsh stressful conditions. The outermost layer of its cell envelope is a proteinaceous coat, the S-layer, essential for resistance to and interactions with the environment. The S-layer Deinoxanthin-binding complex (SDBC), one of the main units of the characteristic multilayered cell envelope of this bacterium, protects against environmental stressors and allows exchanges with the environment. So far, specific regions of this complex, the collar and the stalk, remained unassigned. Here, these regions are resolved by cryo-EM and locally refined. The resulting 3D map shows that the collar region of this multiprotein complex is a trimer of the protein DR_0644, a Cu-only superoxide dismutase (SOD) identified here to be efficient in quenching reactive oxygen species. The same data also showed that the stalk region consists of a coiled coil that extends into the cell envelope for ∼280 Å, reaching the inner membrane. Finally, the orientation and localization of the complex are defined by in situ cryo-electron crystallography. The structural organization of the SDBC couples fundamental UV antenna properties with the presence of a Cu-only SOD, showing here coexisting photoprotective and chemoprotective functions. These features suggests how the SDBC and similar protein complexes, might have played a primary role as evolutive templates for the origin of photoautotrophic processes by combining primary protective needs with more independent energetic strategies.

Multidrug-Resistant Acinetobacter baumannii: An Emerging Aspect of New Drug Discovery.

BACKGROUND: Acinetobacter baumannii is an opportunistic multidrugresistant, aerobic, glucose non-fermentative, and oxidative-negative coccobacilli bacteria. This life-threatening nosocomial infection is associated with immunocompromised patients. OBJECTIVE: This review aims to investigate the multiple drug resistance mechanisms and new emerging diagnostics & treatments for Acinetobacter baumannii. METHODS: All the articles that were most relevant to A. baumannii virulence and drug resistance mechanisms were founded by a literature search on PubMed. Google Patents were used to find discoveries related to diagnostics and treatment. RESULTS: Efflux pumps, ß-lactamases, aminoglycosides, outer membrane proteins, and alteration of the target sites were identified in the Acinetobacter baumannii pathogen as the most prevalent drug resistance mechanisms. Gene detection, peptide detection, and antigen-antibody-associated detection were the latest diagnostics. Novel antimicrobial peptides, sterilization techniques using blue light, and combination therapies are being developed to effectively treat A. baumannii infections. CONCLUSION: This review concludes that new drugs and formulations with high efficiency, low cytotoxicity, and no nephrotoxicity are in absolute need. In the near future, we can expect omics technology to play a significant role in discovering new drugs and potential targets.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective ß-Barrel Porin Requirements for Polymer Secretion.

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM ß-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 ß-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize ß-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning ß-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.

Development of multistage recombinant protein vaccine formulations against toxoplasmosis using a new chitosan and porin based adjuvant system.

Toxoplasmosis is a global health problem affecting both human and animal populations. The lack of effective treatment makes the development of a vaccine against toxoplasmosis one of the main goals in the management of this disease. In our study, vaccine formulations containing the multistage recombinant antigens, rBAG1 + rGRA1 were developed with a combined adjuvant system consisting of chitosan and Salmonella Typhi porins in micro (MicroAS) and nanoparticulate (NanoAS) forms. BALB/c mice were immunized intraperitoneally with vaccine formulations two times at three-week intervals. Three weeks after the second vaccination, mice were challenged with 7-8 live tissue cysts of the virulent T. gondii PRU strain by oral gavage. Higher cellular uptake by macrophages and enhanced cellular (IFN-γ and I-4 in stimulated spleen cells) and humoral (IgG, IgG1, IgG2a) responses were obtained with the adjuvanted formulation, higher with microsystem when compared to that of nanosystem. Microsystem was found to stimulate Th1-polarized immune responses, whereasnon-adjuvanted antigens stimulated Th2-polarized immune response. The highest survival rate and reduction in cysts numbers and T. gondii DNA were obtained with the adjuvanted antigens.Our study showed that adjuvanted multistage recombinant vaccine systems increase theimmune response with strong protection againstT. gondii, more profoundly in microparticulate form.