Protein OmpA Research Articles

PURIFICATION, CHARACTERIZATION AND EXPRESSION, OF RECOMBINANT OUTER MEMBRANE PROTEIN A (OMP A) OF ACINETOBACTER BAUMANNII LI311

Acinetobacter baumannii is a known hospital aquired pathogenic bacterium that increasingly resists antibiotics treatment. In order to characterize and produce a soluble OmpA protein that can be used to develop Acinetobacter vaccine, polymerase chain reaction (PCR) was used to produce the ompA gene, of A. baumannii strain LI311, which was cloned into the histidin taged pET19b expression plasmid. Immobilized metal affinity chromatography (IMAC) was utilized to purify the recombinant protein, and amino acid sequences for OmpA protein homologs were attained from the National Center for Biotechnology Information (NCBI) protein resource then analyzed using the blast tool and Jalview program. Protein topology prediction was done using NCBI tools and PRED-TMBB2. Analysis of amino acid sequence of OmpA of A. baumannii strain LI311 showed that it has homologies to other clinical Acinetobacter spices , including: A. pittii , A.nosocomialis , A.seifertii, A. calcoaceticus, and A. ursingii with identity percentages of 100%, 100%, 96%, 92%, and 91%. Protein topology prediction revealed two conserved domains belonging to OmpA family protein ,which are beta-barrel domain outer membrane protein (OMP_b-brl) and OmpA-C-like domain, and it is a 10-βeta -stranded transmembrane Outer Membrane Protein with a signal peptide at residues 1–22A. A recombinant Histidine tagged- OmpA (39.31kDa )was successfully expressed and purified in this study. In conclusion, OmpA protein of A.baumannii strain LI31 is highly conserved across clinical species of Acinetobacter, and the soluble recombinant OmpA created in this study can be used to develop a putative vaccine that may prevent infections caused by the clinical species of Acinetobacter.

The Outer Membrane Proteins OmpA, CarO, and OprD of Acinetobacter baumannii Confer a Two-Pronged Defense in Facilitating Its Success as a Potent Human Pathogen.

Of all the ESKAPE pathogens, carbapenem-resistant and multidrug-resistant Acinetobacter baumannii is the leading cause of hospital-acquired and ventilator-associated pneumonia. A. baumannii infections are notoriously hard to eradicate due to its propensity to rapidly acquire multitude of resistance determinants and the virulence factor cornucopia elucidated by the bacterium that help it fend off a wide range of adverse conditions imposed upon by host and environment. One such weapon in the arsenal of A. baumannii is the outer membrane protein (OMP) compendium. OMPs in A. baumannii play distinctive roles in facilitating the bacterial acclimatization to antibiotic- and host-induced stresses, albeit following entirely different mechanisms. OMPs are major immunogenic proteins in bacteria conferring bacteria host-fitness advantages including immune evasion, stress tolerance, and resistance to antibiotics and antibacterials. In this review, we summarize the current knowledge of major A. baumannii OMPs and discuss their versatile role in antibiotic resistance and virulence. Specifically, we explore how OmpA, CarO, and OprD-like porins mediate antibiotic and amino acid shuttle and host virulence.

Aeromonas veronii associated with red gill disease and its induced immune response in Macrobrachium nipponense

Aeromonas veronii is a freshwater bacterium associated with many diseases in aquatic animals. In this study, the pathogenic of A. veronii was isolated from diseased Macrobrachium nipponense showing signs of weakness, poor appetite and red gills in some farms in Jiangsu province, China. The pathogenicity of A. veronii to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. veronii NJ1 was pathogenic to M. nipponense, and the LD50 was 4.5 × 106 CFU/ml at 7 d post infection. Detection virulence factors of the A. veronii NJ1 showed that A. veronii NJ1 produced caseinase, lipase, amylase, lecithinase, gelatinase and haemolysin. Detection of virulence-related genes showed that A. veronii NJ1 was positive for aerolysin (aer), outer membrane protein (ompA), adhesin (aha), flagellin (flaA, flgH, flgM, flgN), lipoidase (lip), cytotonic enterotoxins (act, alt, ast), serine protease (ser), DNases (exu), haemolysin (hlyA), type IV pilus (tapA) and fimbrillin (fim). Antibiotic sensitivity testing indicated A. veronii NJ1 was susceptible to quinolones, but resistant to penicillins. Additionally, the immune-related gene expression in M. nipponense was evaluated, and the results showed that anti-lipopolysaccharide factor (ALF1, ALF2, ALF3), Cusrin1, Cusrin2, Cusrin3, Haemocyanin, i-lysozyme and Prophenoloxidase were significantly upregulated from 6 to 24 hr after A. veronii infection. The results of our study suggested that A. veronii was an aetiological element in the mass mortalities of M. nipponense and this study provided preliminary insights into the diversity in the immune response of M. nipponense to the A. veronii infection.

A New Design and Epitopes Analysis for Recombinant Vaccine against Salmonella typhi by In silico Analysis

Nowadays, foodborne diseases are one of the main problems of the world that infect humans due to consumption of contaminated water or food. Typhoid fever is one of the major causes of illness and death in the world caused by Salmonella typhi. Vaccination is one of the most effective approaches in order to reduction of the disease risk. The main goal of this study is designing and characterization of antigenic determinants of a fusion protein originated from S.typhi usable as an effective vaccine. In this study, the outer membrane proteins of salmonella have been considered as candidates conferring protection against typhoid. Considering the evidence, OmpA, OmpF and OmpC proteins of salmonella applied in a multivalent vaccine design. Conserved motives of these proteins were selected using the CLC software and then their extracellular regions of these peptides were identified with PRED-TMBB server. Appropriate motives were combined for design of final fusion protein. Finally epitops of designed protein with high antigenic properties were identified using BCPREDS, Ellipro, ABCpred, EpiJen, NetCTL-1.2, CTLpred, TAPpred, ProPred and VaxiJen servers. Predicted designed protein in this study reached a very high scores for antigenic indexes. Encoding Genetic construction of this fusion protein could be applied for production of the recombinant OmpA.OmpF.OmpC derived fusion protein with effective antigenic properties as a new vaccine against S.typhi. Laboratory experiments and animal challenging analyses is ongoing.

2005 – HSC AS A TRUE INNATE IMMUNE CELL?

Classically, hematopoietic stem cells (HSC) are seen as supplier of a sufficient number of mature cells, whereas the regulation of the immune response has been thought to be exclusively mediated by the differentiated immune effector cells. However, HSCs can directly sense both cytokines released during infection and inflammation, such as M-CSF, and pathogen associated molecular patterns such as TLR ligands. This direct sensing leads to instruction of myeloid differentiation in HSC thus changing dramatically both number and composition of the produced effector cells and contributing to an effective immune response. Here, we show that HSC can directly sense Brucela abortus, a gram-negative coccobacillus, through SLAM/CD150 receptor, nowadays the best marker for identification of the long-term HSC but so far of unknown function in HSC. Using an early myeloid commitment reporter mouse model, we demonstrate that HSC can sense directly the bacteria via a direct interaction of SLAM/CD150 with the bacteria outer membrane protein Omp25. This interaction induces a functional commitment of HSC in the bone marrow toward the myeloid lineage, leading to an increased production of downstream myeloid progenitor and mature cell. This provide the first demonstration of a direct recognition of a live pathogen by HSCs via SLAM/CD150. Moreover, we show that HSC can present an immune memory. HSC drive a more rapid and efficient immune response if they have previously been exposed to LPS. Acute immune stimulation with LPS induces persistent alterations in accessibility of specific myeloid lineage enhancers, as accessed by ATAC-seq on rare HSC, which increases responsiveness of associated immune genes to a secondary stimulation. Functionally, this is associated with increased myelopoiesis of pre-exposed HSC and improved innate immunity against the gram-negative bacterium P. aeruginosa. C/EBPbeta is the major actor of memory imprinting, describing a new function for this factor. HSC are not only at the top of hematopoietic development but also at the heart of the immune response. By sensing directly pathogens and inflammatory cytokines and presenting an innate immune memory, HSC participate much more than originally expected to a global efficient immune response. A better understanding of those newly discovered functions can undoubtedly have significant therapeutic implications.

Transcriptional control of the gonococcal ompA gene by the MisR/MisS two-component regulatory system

Neisseria gonorrhoeae, the causative agent of gonorrhea, is an exclusive human pathogen whose growing antibiotic resistance is causing worldwide concern. The increasing rise of antibiotic resistance expressed by gonococci highlights the need to find alternative approaches to current gonorrhea treatment such as vaccine development or novel therapeutics. The gonococcal OmpA protein was previously identified as a potential vaccine candidate due to its conservation and stable expression amongst strains of Neisseria gonorrhoeae. However, factors that might modulate levels of OmpA and therefore potential vaccine efficacy are unknown. Earlier work indicated that ompA is part of the MisR/MisS regulon and suggested that it was a MisR-activated gene. Herein, we confirmed MisR/MisS regulation of ompA and report that the MisR response regulator can bind upstream of the ompA translational start codon. Further, we describe the contribution of a DNA sequence upstream of the ompA promoter that is critical for MisR activation of ompA transcription. Our results provide a framework for understanding the transcription of gonococcal ompA through a regulatory system known to be important for survival of gonococci during experimental infection.

Microevolution in the major outer membrane protein OmpA of Acinetobacter baumannii.

Acinetobacter baumannii is nowadays a relevant nosocomial pathogen characterized by multidrug resistance (MDR) and concomitant difficulties to treat infections. OmpA is the most abundant A. baumannii outer membrane (OM) protein, and is involved in virulence, host-cell recognition, biofilm formation, regulation of OM stability, permeability and antibiotic resistance. OmpA members are two‐domain proteins with an N‐terminal eight‐stranded β‐barrel domain with four external loops (ELs) interacting with the environment, and a C‐terminal periplasmic domain binding non‐covalently to the peptidoglycan. Here, we combined data from genome sequencing, phylogenetic and multilocus sequence analyses from 975 strains/isolates of the Acinetobacter calcoaceticus / Acinetobacter baumannii complex (ACB), 946 from A. baumannii , to explore ompA microevolutionary divergence. Five major ompA variant groups were identified (V1 to V5) in A. baumannii , encompassing 52 different alleles coding for 23 different proteins. Polymorphisms were concentrated in five regions corresponding to the four ELs and the C‐terminal end, and provided evidence for intra‐genic recombination. ompA variants were not randomly distributed across the A . baumannii phylogeny, with the most frequent V1(lct)a1 allele found in most clonal complex 2 (CC2) strains and the second most frequent V2(lct)a1 allele in the majority of CC1 strains. Evidence was found for assortative exchanges of ompA alleles not only between separate A . baumannii lineages, but also different ACB species. The overall results have implications for A. baumannii evolution, epidemiology, virulence and vaccine design.

Immunoprotective evaluation of <I>Escherichia coli</I> outer membrane protein A against the main pathogens of animal mastitis

Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis.
 Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentation

OmpA Porin of Salmonella enterica serovar Enteritidis Possesses Aamyloid‐Forming Properties

The major focus of Salmonella enterica studies is the molecular determinants of the virulence and host‐specificity. Important virulence factors shared by the majority of pathogenic gram‐negative bacteria are the outer membrane proteins (OMP), many of which belong to the porins – beta barrel proteins serving as passive membrane pores. One of the most studied in terms of pathogenesis is the OmpA porin family. The proteins of the OmpA family are involved in the important virulence‐ and pathogenesis‐related processes including adhesion, penetration and damage of host tissues, as well as avoiding the immune response. Proteins of this family are highly conserved. For instance, the amino acid sequence similarity of OmpA proteins of Escherichia coli and S. enterica serovar Enteritidis is 94%. Considering ability of the E. coli OmpA porin to form amyloid‐like aggregates, we have produced recombinant OmpA protein from S. enterica serovar Enteritidis in vitro and have analyzed its amyloid properties. We have determined the typical fibrillary morphology of the obtained OmpA aggregates using transmission electron microscopy and have demonstrated that these aggregates exhibit yellow‐green birefringence being stained with amyloid‐specific dye Congo Red. We have also found that OmpA protein fused with YFP aggregates in yeast Saccharomyces cerevisiae while OmpA fibrils secreted in vivo at the surface of bacterial cells in the Curli‐Dependent Amyloid Generator system demonstrate properties typical for amyloids including unbranched morphology and birefringence upon binding with Congo Red. Taken together, these results suggest that the S. enterica serovar Enteritidis OmpA porin has amyloid‐forming properties.Support or Funding InformationThis work was supported by the Russian Science Foundation (Grant No 19‐76‐00026).

High-Resolution In Situ NMR Spectroscopy of Bacterial Envelope Proteins in Outer Membrane Vesicles.

The cell envelope of Gram-negative bacteria is an elaborate cellular environment, consisting of two lipid membranes separated by the aqueous periplasm. So far, efforts to mimic this environment under laboratory conditions have been limited by the complexity of the asymmetric bacterial outer membrane. To evade this impasse, we recently established a method to modify the protein composition of bacterial outer membrane vesicles (OMVs) released from Escherichia coli as a platform for biophysical studies of outer membrane proteins in their native membrane environment. Here, we apply protein-enriched OMVs to characterize the structure of three envelope proteins from E. coli using nuclear magnetic resonance (NMR) spectroscopy and expand the methodology to soluble periplasmic proteins. We obtain high-resolution in situ NMR spectra of the transmembrane protein OmpA as well as the periplasmic proteins CpxP and MalE. We find that our approach facilitates structural investigations of membrane-attached protein domains and is especially suited for soluble proteins within their native periplasmic environment. Thereby, the use of OMVs in solution NMR methods allows in situ analysis of the structure and dynamics of proteins twice the size compared to the current in-cell NMR methodology. We therefore expect our work to pave the way for more complex NMR studies of bacterial envelope proteins in the native environment of OMVs in the future.

A Novel pH-Regulated, Unusual 603 bp Overlapping Protein Coding Gene pop Is Encoded Antisense to ompA in Escherichia coli O157:H7 (EHEC).

Antisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. An active σ70 promoter, transcription start site (TSS), Shine-Dalgarno motif and rho-independent terminator were experimentally validated, providing evidence that this open reading frame has all the structural features of a functional gene. Furthermore, ribosomal profiling revealed translation of the mRNA, the protein was detected in Western blots and a pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene), thus adding another example to the growing list of overlapping, protein coding genes in bacteria.

Omp25-dependent engagement of SLAMF1 by Brucella abortus in dendritic cells limits acute inflammation and favours bacterial persistence in vivo.

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25-dependent engagement of SLAMF1 by B. abortus limits NF-κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro-inflammatory cytokine secretion and impairs DC activation. The Omp25-SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity.

Carbon fullerene acts as potential lead molecule against prospective molecular targets of biofilm-producing multidrug-resistant Acinetobacter baumanni and Pseudomonas aerugenosa: computational modeling and MD simulation studies

This study aimed to screen putative drug targets associated with biofilm formation of multidrug-resistant Acinetobacter baumannii and Pseudomonas areugenosa and prioritize carbon nano-fullerene as potential lead molecule by structure-based virtual screening. Based on the functional role, 36 and 83 genes that are involved in biofilm formation of A. baumannii and P. areugenosa respectively were selected and metabolic network was computationally constructed. The genes that lack three-dimensional structures were predicted and validated. Carbon nano-fullerene selected as lead molecule and their drug-likeliness and pharmacokinetics properties were computationally predicted. The binding potential of carbon nano-fullerene toward selected drug targets was modeled and compared with the binding of conventional drugs, doripenem, and polymyxin-B with their usual targets. The stabilities of four best-docked complexes were confirmed by molecular dynamic (MD) simulation. This study suggested that selected genes demonstrated relevant interactions in the constructed metabolic pathways. Carbon fullerene exhibited significant binding abilities to most of the prioritized targets in comparison with the binding of last-resort antibiotics and their usual target. The four best ligand–receptor interactions predicted by molecular docking revealed that stability throughout MD simulation. Notably, carbon fullerene exhibited profound binding with outer membrane protein (OmpA) and ribonuclease-HII (rnhB) of A. baumannii and 2-heptyl-4(1H)-quinolone synthase (pqsBC) and chemotaxis protein (wspA) of P. aeruginosa. Thus, the current study suggested that carbon fullerene was probably used as potential lead molecules toward selected targets of A. baumannii and P. aeruginosa and the applied aspects probably scaled up to design promising lead molecules toward these pathogens. Communicated by Ramaswamy H. Sarma.

Multifunctional magnetite nanoparticles to enable delivery of siRNA for the potential treatment of Alzheimer’s

Therapeutic drugs for Alzheimer’s disease have been extensively studied due to its recurrence and abundance among neurodegenerative diseases. It is thought that the accumulation of amyloid precursor protein (APP) products, a consequence of an up-regulation of the β-site APP-cleaving enzyme 1 (BACE1), is the main triggering mechanism during the early stages of the disease. This study aims to explore the ability of a multifunctional conjugate based on magnetite nanoparticles for the cellular delivery of siRNA against the expression of the BACE1 gene. We immobilized the siRNA strand on PEGylated magnetite nanoparticles and investigated the effects on biocompatibility and efficacy of the conjugation. Similarly, we co-immobilized the translocating protein OmpA on PEGylated nanoparticles to enhance cellular uptake and endosomal escape. BACE1 suppression was statistically significant in HFF-1 cells, without any presence of a cytotoxic effect. The delivery of the nanoconjugate was achieved through endocytosis pathways, where endosome formation was likely escaped due to the proton-sponge effect characteristic of PEGylated nanoparticles or mainly by direct translocation in the case of OmpA/PEGylated nanoparticles.

Evidence for accessory chemosensory cells in the adult human nasal cavity

The existence of functionally relevant accessory olfactory organs in humans is still a matter of controversy. A vomeronasal organ (VNO) with sensory and non-sensory epithelia exists only in macrosmatic mammals. A similar structure is regularly observed in humans during fetal development. The postnatal persistence of a VNO like epithelial duct has been described in about 10 %. Here we studied tissue samples of nasal mucosa from adults. In all individuals we found epithelial cells in the lower part of the nasal septum which exhibited morphological features of sensory neurons and which showed immunostaining for olfactory marker protein OMP. These cells were interposed by ciliated cells, goblet cells and small intraepithelial capillaries. Only occasionally we found such cells within a morphologically defined epithelial duct. A clear separation of sensory and non-sensory epithelia could not be observed. In most cases we found OMP positive groups of cells either in epithelial cavities or just embedded in respiratory epithelium. With RT-PCR we could confirm the presence of OMP encoding mRNA thus supporting the idea of intrinsic expression of this protein in the nasal mucosa. We conclude that accessory chemosensory structures are regularly conserved in adult humans in the approximate anatomical location of the VNO of microsmatic animals. Their functional importance is yet to be determined.

Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V.alginolyticus are poorly known. To gain insight into fish diseases caused by V.alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V.alginolyticus. V.alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V.alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V.alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V.alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V.alginolyticus strain.

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm−1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

In Vivo Cross-Linking MS Reveals Conservation in OmpA Linkage to Different Classes of β-Lactamase Enzymes.

Molecular interactions between two different classes of β-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A β-lactamase blaGES-11 and Class D β-lactamase Oxa23, responsible for hydrolysis of different types of β-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both β-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of β-lactamase enzymes in the periplasmic space.

Antibiofilm and antibacterial activities of repurposing auranofin against Bacteroides fragilis.

The aim of this study is to evaluate the antibiofilm and antibacterial effects of auranofin against WT-ETBF, rETBF, WT-NTBF and clinically isolated Bacteroides fragilis strains. The minimum inhibitory concentration and biofilm inhibitory concentration of 0.25μg/ml for auranofin against B. fragilis were determined, and the biofilm eradication concentration was 1μg/ml. At an auranofin concentration of 0.5μg/ml, little cellular metabolic activity was found. Confocal laser scanning microcopy results confirmed the inhibition of biofilm by auranofin. The effects of auranofin on the outer membrane protein (ompA) gene and the RND-type efflux pump (bmeB3) gene were investigated using quantitative real-time polymerase chain reaction (qRT-PCR). The qRT-PCR results showed that treatment with auranofin significantly reduced the gene expression compared to controls without auranofin. These data indicate the applicability of auranofin as a repurposed drug due to its inhibitory effect against biofilm formation of B. fragilis. Therefore, our study demonstrates that auranofin, already approved for human use, is a promising drug that has strong antibiofilm and antibacterial activity against B. fragilis.

Insights into the behavior of six rationally designed peptides based on Escherichia coli's OmpA at the water-dodecane interface.

The Escherichia coli’s membrane protein OmpA has been identified as a potential biosurfactant due to their amphiphilic nature, and their capacity to stabilize emulsions of dodecane in water. In this study, the influence of surfactant type, concentration, preservation time and droplet size on the crystallization of n-dodecane and water, in oil-in-water emulsions stabilized with six rationally designed Escherichia coli’s OmpA-based peptides was investigated. A differential scanning calorimetry (DSC) protocol was established using emulsions stabilized with Tween 20® and Tween 80®. A relationship between the surfactant concentration and the crystallization temperatures of n-dodecane and water was observed, where the crystallization temperatures seem to be dependent on the preservation time. A deconvolution analysis shows that the peak morphology possibly depends on the interactions at the interface because the enthalpic contributions of each Gaussian peak remained similar in emulsions stabilized with the same peptide. Adsorption results show that the main driver for adsorption and thus stabilization of emulsions is polar interactions (e.g. H-bonding) through the hydrophilic parts of the peptides. Those peptides with a preponderance of polar interaction groups distribution (i.e. NH2, COOH, imidazole) showed the highest interfacial activity under favorable pH conditions. This suggests that custom-made peptides whose hydrophilic/hydrophobic regions can be fine-tuned depending on the application can be easily produced with the additional advantage of their biodegradable nature.