Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda.

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Characteristics of a pseudolysogenic phage vB_YpM_HQ103 infecting Yersinia pestis.

The plague, caused by Yersinia pestis, is a natural focal disease and the presence of Y. pestis in the environment is a critical ecological concern worldwide. The role of Y. pestis phages in the ecological life cycle of the plague is crucial. Previously, a temperature-sensitive phage named vB_YpM_HQ103 was isolated from plague foci in Yunnan province, China. Upon infecting the EV76 strain of Y. pestis, vB_YpM_HQ103 exhibits lysogenic behavior at 21 °C and lytic behavior at 37 °C. Various methods including continuous passage lysogenic tests, in vitro lysis tests, comparative genomic assays, fluorescence quantitative PCR and receptor identification tests were employed to demonstrate that the lysogenic life cycle of this phage is applicable to wild Y. pestis strains; its lysogeny is pseudolysogenic (carrying but not integrating), allowing it to replicate and proliferate within Y. pestis. Furthermore, we have identified the outer membrane protein OmpA of Y. pestis as the receptor for phage infection. In conclusion, our research provides insight into the characteristics and receptors of a novel Y. pestis phage infection with a pseudolysogenic cycle. The findings of this study enhance our understanding of Y. pestis phages and plague microecology, offering valuable insights for future studies on the conservation and genetic evolution of Y. pestis in nature.

Evaluation of immunogenicity and protective efficacy of outer membrane vesicles from Salmonella Typhimurium and Salmonella Choleraesuis.

Outer membrane vesicles (OMVs) are membranous structures frequently observed in Gram-negative bacteria that contain bioactive substances. These vesicles are rich in bacterial antigens that can activate the host's immune system, making them a promising candidate vaccine to prevent and manage bacterial infections. The aim of this study was to assess the immunogenicity and protective efficacy of OMVs derived from Salmonella enterica serovar Typhimurium and S. Choleraesuis, while also focusing on enhancing OMV production. Initial experiments showed that OMVs from wild-type strains did not provide complete protection against homologous Salmonella challenge, possible due to the presence of flagella in the purified OMVs samples, which may elicit an unnecessary immune response. To address this, flagellin-deficient mutants of S. Typhimurium and S. Choleraesuis were constructed, designated rSC0196 and rSC0199, respectively. These mutants exhibited reduced cell motility and their OMVs were found to be flagellin-free. Immunization with non-flagellin OMVs derived from rSC0196 induced robust antibody responses and improved survival rates in mice, as compared to the OMVs derived from the wild-type UK-1. In order to enhance OMV production, deletions of ompA or tolR were introduced into rSC0196. The deletion of tolR not only increase the yield of OMVs, but also conferred complete protection against homologous S. Typhimurium challenge in mice. Collectively, these findings indicate that the flagellin-deficient OMVs with a tolR mutation have the potential to serve as a versatile vaccine platform, capable of inducing broad-spectrum protection against significant pathogens.

Phenotypic Investigation and Detection of Biofilm-Associated Genes in Acinetobacter baumannii Isolates, Obtained from Companion Animals.

Bacteria of the genus Acinetobacter, especially Acinetobacter baumannii (Ab), have emerged as pathogens of companion animals during the last two decades and are commonly associated with hospitalization and multidrug resistance. A critical factor for the distribution of relevant strains in healthcare facilities, including veterinary facilities, is their adherence to both biotic and abiotic surfaces and the production of biofilms. A group of 41 A. baumannii isolates obtained from canine and feline clinical samples in Greece was subjected to phenotypic investigation of their ability to produce biofilms using the tissue culture plate (TCP) method. All of them (100%) produced biofilms, while 23 isolates (56.1%) were classified as strong producers, 11 (26.8%) as moderate producers, and 7 (17.1%) as weak producers. A correlation between the MDR and XDR phenotypes and weak or moderate biofilm production was identified. Moreover, the presence of four biofilm-associated genes bap, blaPER, ompA, and csuE was examined by PCR, and they were detected in 100%, 65.9%, 97.6%, and 95.1% of the strains respectively. All isolates carried at least two of the investigated genes, whereas most of the strong biofilm producers carried all four genes. In conclusion, the spread and persistence of biofilm-producing Ab strains in veterinary facilities is a matter of concern, since they are regularly obtained from infected animals, indicating their potential as challenging pathogens for veterinarians due to multidrug resistance and tolerance in conventional eradication measures. Furthermore, considering that companion animals can act as reservoirs of relevant strains, public health concerns emerge.

Enantioselective synthesis, characterization, molecular docking simulation and ADMET profiling of α-alkylated carbonyl compounds as antimicrobial agents.

All living organisms produce only one enantiomer, so we found that all natural compounds are presented in enantiomerically pure form. Asymmetric synthesis is highly spread in medicinal chemistry because enantiomerically pure drugs are highly applicable. This study initially demonstrated the feasibility of a good idea for the asymmetric synthesis of α-alkylated carbonyl compounds with high enantiomeric purity ranging from 91 to 94% using different quinazolinone derivatives. The structure of all compounds was confirmed via elemental analysis and different spectroscopic data and the enantioselectivity was determined via HPLC using silica gel column. The synthesized compounds' mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-beta-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed that compound 6a has a broad biocidal activity and this in-vitro study was in line with the in-silico results. Overall, the formulated compound 6a can be employed as antimicrobial agent without any toxicity with high bioavailability in medical applications.

Anorectal lymphogranuloma venereum among men who have sex with men: a 3-year nationwide survey, France, 2020 to 2022.

BackgroundIn France, lymphogranuloma venereum (LGV) testing switched from universal to selective testing in 2016.AimTo investigate changes in LGV-affected populations, we performed a nationwide survey based on temporarily reinstated universal LGV testing from 2020 to 2022.MethodsEach year, during three consecutive months, laboratories voluntarily sent anorectal Chlamydia trachomatis-positive samples from men and women to the National Reference Centre for bacterial sexually transmitted infections. We collected patients' demographic, clinical and biological data. Genovars L of C. trachomatis were detected using real-time PCR. In LGV-positive samples, the ompA gene was sequenced.ResultsIn 2020, LGV positivity was 12.7% (146/1,147), 15.2% (138/907) in 2021 and 13.3% (151/1,137) in 2022 (p > 0.05). It occurred predominantly in men who have sex with men (MSM), with rare cases among transgender women. The proportion of HIV-negative individuals was higher than that of those living with HIV. Asymptomatic rectal LGV increased from 36.1% (44/122) in 2020 to 52.4% (66/126) in 2022 (p = 0.03). Among users of pre-exposure prophylaxis (PrEP), LGV positivity was 13.8% (49/354) in 2020, 15.6% (38/244) in 2021 and 10.9% (36/331) in 2022, and up to 50% reported no anorectal symptoms. Diversity of the LGV ompA genotypes in the Paris region increased during the survey period. An unexpectedly high number of ompA genotype L1 variant was reported in 2022.ConclusionIn rectal samples from MSM in France, LGV positivity was stable, but the proportion of asymptomatic cases increased in 2022. This underscores the need of universal LGV testing and the importance of continuous surveillance.

Investigation of Chlamydia pecorum in livestock from Switzerland reveals a high degree of diversity in bovine strains.

Chlamydia pecorum is a widespread veterinary chlamydial species causing endemic infections in livestock, such as ruminants and pigs, globally. However, there is limited contemporary knowledge on infecting strain diversity in various hosts. This study aimed to evaluate the genetic diversity of C. pecorum strains infecting Swiss livestock through C. pecorum genotyping and phylogenetic analyses in comparison to the global population, while also assessing chlamydial strains for plasmid carriage. A total of 263 C. pecorum positive samples from clinically healthy ruminant and pig herds (Bovines = 216, sheep = 25, pigs = 14) as well as placentae from eight C. pecorum positive ruminant abortion cases from other Swiss herds were investigated. The ompA and Multi-Locus sequence typing revealed novel C. pecorum genotypes, and bovine strains exhibited considerable genetic diversity, contrasting with lower diversity in sheep and pig strains. C. pecorum plasmid was detected in 100.0% of sheep (41/41) and pig (255/255) samples, and in 69.4% of bovine samples (150/216). In contrast, no plasmid was detected in the eight C. pecorum-positive ruminant abortion cases either representing plasmid-less strains or possibly escaping PCR detection due to autolysis of the placenta. This study supports the genetic diversity of C. pecorum strains, particularly in bovines, and identifies novel sequence types in Swiss livestock.

Bacteriophage LHE83 targeting OmpA as a receptor exhibited synergism with spectinomycin against Escherichia coli.

Understanding the characteristics of bacteriophages is crucial for the optimization of phage therapy. In this study, the biological and genomic characteristics of coliphage LHE83 were determined and its synergistic effects with different types of antibiotics against E. coli E82 were investigated. Phage LHE83 displayed a contractile tail morphology and had a titer of 3.02 × 109 pfu/mL at an optimal MOI of 0.01. Meanwhile, phage LHE83 exhibited good physical and chemical factors tolerance. The 1-step growth analysis revealed a latent period of approx. 10 min with a burst size of 87 pfu/infected cell. Phage LHE83 belongs to the genus Dhakavirus. Its genome consists of 170,464 bp with a 40% GC content, and a total of 268 Open Reading Frames (ORF) were predicted with no detected virulent or resistant genes. ORF 213 was predicted to encode the receptor binding protein (RBP) and confirmed by the antibody-blocking assay. Furthermore, a phage-resistant strain E. coli E82R was generated by co-culturing phage LHE83 with E. coli E82. Genomic analysis revealed that OmpA served as the receptor for phage LHE83, which was further confirmed by phage adsorption assay using E. coli BL21ΔOmpA, E. coli BL21ΔOmpA: OmpA and E. coli BL21:OmpA strains. Additionally, a synergistic effect was observed between phage LHE83 and spectinomycin against the drug-resistant strain E. coli E82. These results provide a theoretical basis for understanding the interactions between phages, antibiotics, and host bacteria, which can assist in the clinical application of phages and antibiotics against drug-resistant bacteria.

Unveiling the structure-emulsifying function relationship of truncated recombinant forms of the SA01-OmpA protein opens up a new vista in bioemulsifiers.

The emulsifying ability of SA01-OmpA (outer membrane protein A from Acinetobacter sp. SA01) was found to be constrained by challenges like low production efficiency and high costs associated with protein recovery from E. coli inclusion bodies, as described in our previous study. The present study sought to benefit from the advantages of the targeted truncating of SA01-OmpA protein, taking into account the reduced propensity of protein expression as inclusion bodies and cytotoxicity. Here, the structure and activity relationship of two truncated recombinant forms of SA01-OmpA protein was unraveled through a hybrid approach based on experimental data and computational methodologies, representing an innovative bioemulsifier with advantageous emulsifying activity. The recombinant truncated SA01-OmpA variants were cloned and heterologously expressed in E. coli host cells and subsequently purified. The results showed increased emulsifying activity of N-terminally truncated SA01-OmpA (NT-OmpA) compared to full-length SA01-OmpA. Molecular dynamics (MD) simulations analysis demonstrated a direct correlation between the C-terminally truncated SA01-OmpA (CT-OmpA) and its expression as inclusion bodies. Analysis of the structure-activity relationship of truncated variants of SA01-OmpA revealed that, compared to the full-length protein, deletion of the ß-barrel portion from the N-terminal of SA01-OmpA increased the emulsifying activity of NT-OmpA while lowering its expression as inclusion bodies. Contrary to the full-length protein, the N-terminally truncated SA01-OmpA was not as cytotoxic, according to the MTT assay, FCM analysis, and AO/EB staining. The findings of this extensive study advance our knowledge of SA01-OmpA at the molecular level as well as the design and development of efficient bioemulsifiers.IMPORTANCEPrevious research (Shahryari et al. 2021, mSystems 6: e01175-20) introduced and characterized the SA01-OmpA protein as a multifaceted protein with a variety of functions, including maintaining cellular homeostasis under oxidative stress conditions, biofilm formation, outer membrane vesicles (OMV) biogenesis, and beneficial emulsifying capacity. By truncating the SA01-OmpA protein, the current study presents a unique method for developing protein-type bioemulsifiers. The findings indicate that the N-terminally truncated SA01-OmpA (NT-OmpA) has the potential to fully replace full-length SA01-OmpA as a novel bioemulsifier with significant emulsifying activity. This study opens up a new frontier in bioemulsifiers, shedding light on a possible relationship between the structure and activity of SA01-OmpA truncated forms.

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.

Anti-OmpA antibodies as potential inhibitors of Acinetobacter baumannii biofilm formation, adherence to, and proliferation in A549 human alveolar epithelial cells.

Outer membrane protein A (OmpA) is a critical virulence factor in Acinetobacter baumannii, influencing adhesion, biofilm formation, host immune response, and host cell apoptosis. We investigated the invasion of A549 alveolar epithelial cells by A. baumannii and examined how anti-OmpA antibodies impact these interactions. OmpA was expressed and purified, inducing anti-OmpA antibodies in BALB/c mice. The potential toxicity of OmpA was evaluated in mice by analyzing histology from six organs. A549 cells were exposed to A. baumannii strains 19606 and a clinical isolate. Using cell culture and light microscopy, we scrutinized the effects of anti-OmpA sera on serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells. The viability of A549 cells was assessed upon exposure to live A. baumannii and anti-OmpA sera. OmpA-induced antibody demonstrated potent bactericidal effects on both strains of A. baumannii. Both strains formed biofilms, which were reduced by anti-OmpA serum, along with decreased bacterial adherence, internalization, and proliferation in A549 cells. Anti-OmpA serum improved the survival of A549 cells post-infection. Pre-treatment with cytochalasin D hindered bacterial internalization, highlighting the role of actin polymerization in invasion. Microscopic examination revealed varied interactions encompassing adherence, apoptosis, membrane alterations, vacuolization, and damage. A549 cells treated with anti-OmpA serum exhibited improved structures and reduced damage. The findings indicate that A. baumannii can adhere to and proliferate within epithelial cells with OmpA playing a pivotal role in these interactions, and the complex nature of these interactions shapes the intricate course of A. baumannii infection in host cells.

The effect of clinically relevant beta-lactam, aminoglycoside, and quinolone antibiotics on bacterial extracellular vesicle release from E. coli.

Sepsis, a leading cause of death in hospitals, can be defined as a dysregulated host inflammatory response to infection, which can lead to tissue damage, organ failure, and cardiovascular complications. Although there is no cure for sepsis, the condition is typically managed with broad spectrum antibiotics to eliminate any potential bacterial source of infection. However, a potential side-effect of antibiotic treatment is the enhanced release of bacterial extracellular vesicles (BEVs). BEVs are membrane-bound nanoparticles produced by a variety of mechanisms, one of which includes the pinching-off of the outer membrane (in Gram-negative bacteria) to enclose proteins and other biological molecules for transport and intercellular communication. Some of the Gram-negative EV cargo, including Peptidoglycan associated lipoprotein (Pal) and Outer membrane protein A (OmpA), have been shown to induce both acute and chronic inflammation in host tissue. We hypothesize that antibiotic concentration and its mechanism of action can have an effect on the amount of released BEVs, which could potentially exacerbate the host inflammatory response. In this study, we evaluated nine clinically relevant antibiotics for their effect on EV release from Escherichia coli. EVs were characterized using immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. Several beta-lactam antibiotics caused significantly more EV release, while quinolone and aminoglycosides caused relatively less vesiculation. Further study is warranted to corroborate the correlation between an antibiotic's mechanism of action and its effect on EV release, but these results underline the importance of antibiotic choice when treating sepsis patients.

Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

Epidemiology of Chlamydia sp. infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. RESULTS: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. CONCLUSION: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp.

An elevated OmpA expression during the production of a recombinant protein in Escherichia coli.

Escherichia coli cells rapidly respond to changes in the environment. Such response must be anticipated upon development of fermentation strategy for commercial purposes. The response may signal changes in cell physiology, which is critical for the cell growth and the level of the target protein production. One of the responses is the elevated expression of membrane proteins to tightly control the trafficking of molecules into and out from the cells. Normally, the expression level of the membrane protein is basal as the fermentation is carried out in physiological conditions. Here, we reported an elevated expression of the outer membrane protein A (OmpA) during a series of fermentation conduct, starting from the shake flask, 1-L to finally 10-L fermentor. The incidence led to a lower expression of the target protein and thereby resulting in lower process efficiency. OmpA expression was concomitant to the bacterial growth and already observed in the early exponential phase. Despite the drawback, this phenomenon actually inspires the observation of OmpA expression as one of the indicators for the E. coli cells response to the fermentation conditions. This auxiliary check would prevent the higher OmpA expression that led to the low expression of the target protein.

Molecular detection of Rickettsia africae from Amblyomma hebraeum ticks in Mafikeng city of North West Province, South Africa.

Rickettsia africae causes zoonotic African tick bite fever, which is a disease of "One Health" importance. There have been reported cases of tourists from Europe and Asia who have been bitten by ticks whilst visiting South Africa's nature reserves, and on their return to their countries, the display African Tick Bite Fever sickness. Hence, the aim of this study was to determine the occurrence of Rickettsia africae in Amblyomma hebraeum ticks infesting livestock in the North West Province. A total of 358 A. hebraeum ticks were collected from 60 ruminants (cattle, sheep and goats) in Mafikeng City of North West Province, South Africa. Ticks were identified morphologically and further confirmed by sequencing of their ITS2 gene. DNA was extracted from 60 pools of ticks which consisted of 5-6 adult ticks that were from the same ruminant host. Infections with Rickettsia spp. were found in 48%, 40%, and 32% of cattle, sheep, and goats, respectively, in amplification by PCR using the ompA gene. The ompA gene sequences showed that the Rickettsia spp. were identified as R. africae. Although the animals from whom the ticks were collected did not exhibit any clinical symptoms, it is well recognised that R. africae is a disease with significant zoonotic potential. Thus, it is important to use the "One Health" approach to formulate prevention and control measures for this pathogen for animal and human health as well as the tourism sector due to the ecotourism importance of the resultant disease.

Molecular docking and molecular dynamics studies of natural products unravel potential inhibitors against OmpA of Acinetobacter baumannii.

Emerging antimicrobial resistance has highlighted the need to design more effective antibiotics to treat deadly bacterial infections. Acinetobacter baumannii's outer membrane protein A (OmpA) is a critical virulence component involved in biofilm formation, immunomodulation, and antibiotic resistance, which characterizes it as a potential therapeutic target. The present study aimed to screen the natural product database (>1,00,000) to identify the potential inhibitor against OmpA. Molecular docking studies revealed that 10 compounds had good docking scores (≤ -7 kcal/mol) compared to the reported inhibitor epiestriol (-3.079). Further, these 10 compounds were subjected to ADME analysis and MMGBSA analysis. Based on MMGBSA results, we selected 5 compounds [NP-1 (MolPort-039-337-117), NP-5(MolPort-019-932-973), NP-6 (MolPort-005-948-336), NP-8(MolPort-042-673-978) and NP-9(MolPort-042-673-766)] with high binding affinity. Molecular dynamics simulation found that NP-5, NP-8, and NP-9 were stable after analysing their RMSD, RMSF, the radius of gyration, and hydrogen interactions of complexes. Our study revealed that NP-5, NP-8, and NP-9 bind perfectly with OmpA and can act as its potential inhibitors. The results of this study imply that the identified inhibitors have the potential for further investigation.Communicated by Ramaswamy H. Sarma.

Serum Level of Antibodies Against Novel Acinetobacter Baumannii OmpA-selected Peptides in ICU Staff: Promise for the Future of Vaccine Development.

Extensively drug-resistant Acinetobacter baumannii is considered one of the most dangerous threats to global health, requiring novel therapeutic interventions. The outer membrane protein A (OmpA) is an immunogenic agent that triggers immune responses. The current study evaluated serum antibody levels against previously determined immunogenic OmpA peptides from A baumannii in ICU staff. Serum samples were collected from 62 ICU staff members (representing the exposed group), healthy controls (representing the nonexposed group), and patients with systemic lupus erythematosus (SLE) (as controls for nonspecific antibody reactions). After excluding the cross-reactive antibodies via Escherichia coli lysate pretreatment, all the samples were assessed in the vicinity of A baumannii lysate by enzyme-linked immunosorbent assay (ELISA). All the positive samples were assessed for interaction with previously designed and selected peptides using ELISA. The protective potential of positive serum antibodies was surveyed in vitro using an opsonophagocytic study. The most antibody positive samples against one of the dominant peptides were determined in the ICU personnel (75%).  SLE serum samples did not react with candidate peptides. The strongest positive reaction was observed in serum treatment with one of the OmpA peptides (No. 5) with significant differences compared to other designed peptides. Our findings showed that ICU samples have substantially higher antibody levels than the nonexposed group; Positive samples show strong results in the opsonophagocytosiis assay. This study demonstrates A baumannii colonization at human mucosal surfaces, especially in exposed healthy workers. Novel OmpA-derived peptides could be used to identify immunogenic vaccine candidates. Therefore, more studies are needed  before this peptide and antibody levels are used in diagnosis, prevention, or treatment.

Outer membrane protein A of Acinetobacter baumannii regulates pulmonary inflammation through the TLR2-NF-κB pathway.

Acinetobacter baumannii (A. baumannii) is characterized by a high prevalence of drug resistance; how to effectively treat it is still a major clinical challenge. Our previous experiments confirmed that ompA, which is one of the most well-characterized virulence factors, may be dependent on the caspase-1 pathway-stimulated expression of NLRP3 inflammasome to enhance inflammation. TLRs (i.e., TLR2, etc.) is the initiating signal for NLRP3 inflammasome activation; how it relates to ompA in its underlying pathogenic mechanism is not clear. In this study, we proofed that ompA promoted NLRP3 inflammasome activation while the TLR2-NF-κB pathway was also activated after A. baumannii infection. Additionally, the expression of NLRP3 inflammasome-associated proteins and genes was inhibited by silencing TLR2 and NLRP3. This indicated that ompA might depend on the TLR2-NF-κB pathway to assemble and activate the NLRP3 inflammasome. OmpA promoted the assembly of the NLRP3 inflammasome through the TLR2-NF-κB pathway and inhibited the degradation of caspase-1 by the proteasome so that a large number of mature IL-1ß/IL-18 and other proinflammatory factors were released extracellularly to enhance the body's inflammatory response. Taken together, the results of the joint pre-study confirmed a novel TLR2-NF-κB/NLRP3/caspase-1-modulated mechanism underpinning ompA activity, the NLRP3 inflammasome pathway may be as a potential immunomodulatory target against A. baumannii infections.

Implication of the σE Regulon Members OmpO and σN in the ΔompA299-356-Mediated Decrease of Oxidative Stress Tolerance in Stenotrophomonas maltophilia.

Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299-356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299-356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299-356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299-356. KJΔOmpA299-356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299-356. Next, the involvement of the three σ factors in the ΔompA299-356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299-356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299-356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal ß-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to ß-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.