Acinetobacter Baumannii Outer Membrane Protein Research Articles

Epitope mapping of Acinetobacter baumannii outer membrane protein W (OmpW) and laboratory study of an OmpW-derivative peptide

Outer membrane protein W (OmpW) is a less-known A. baumannii antigen with potential immunogenic properties. The epitopes of this protein are not well-identified yet. Therefore, in the present study, B- and T-cell epitopes of A. baumannii OmpW were found using comprehensive in silico and partially in vitro studies. The T-cell (both class-I and class-II) and B-cell (both linear and conformational) epitopes were predicted and screened through many bioinformatics approaches including the prediction of IFN-γ production, immunogenicity, toxicity, allergenicity, human similarity, and clustering. A single 15-mer epitopic peptide containing a linear B-cell and both classes of T-cell epitopes were found and used for further assays. For in vitro assays, patient- and healthy control-derived peripheral blood mononuclear cells were stimulated with the 15-mer peptide, Phytohemagglutinin, or medium alone, and cell proliferation and IFN-γ production assays were performed. The bioinformatics studies led to mapping OmpW epitopes and introducing a 15-mer peptide. In vitro assays to some extent showed its potency in cell proliferation but not in IFN-γ induction, although the responses were not very expressive and faced some questions/limitations. In general, in the current study, we mapped the most immunogenic epitopes of OmpW that may be used for future studies and also assayed one of these epitopes in vitro, which was shown to have an immunogenicity potential. However, the induced immune responses were not strong which suggests that the present peptide needs a series of biotechnological manipulations to be used as a potential vaccine candidate. More studies in this field are recommended.

Two peptides derivate from Acinetobacter baumannii outer membrane protein K as vaccine candidates: a comprehensive in silico study

BackgroundThe lack of appropriate vaccines is an obstacle to the effective management of A. baumannii infections. Peptide vaccines offer an attractive and promising preventive strategy against A. baumannii.ObjectiveIn this study, we identified specific T cell epitopes of A. baumannii outer membrane protein K (OMPK) using comprehensive bioinformatics and detailed molecular docking analysis.MethodsBoth class-I and class-II T cell epitopes of A. baumannii OMPK were predicted by three tools namely IEDB, SYFPEITHI, and ProPred. The predicted epitopes were shortlisted based on several analyses including prediction scoring, clustering, exclusion of human similarity, considering immunogenicity and cytokine production, and removal of toxic and/or allergen epitopes. The epitopic peptides with high prediction scores and appropriate properties containing both class-I and class-II T cell epitopes were selected. Two of these class I/II epitopic peptides were chosen for molecular docking studies and assessing their physicochemical properties as vaccine candidates.ResultsThe results showed many T-cell epitopes of OMPK that could be evaluated for possible immunogenicity. Two of these epitopes (containing both class-I and II epitopes) had high prediction scores, were predicted by several tools, attached to several HLAs, and had the best docking score. They had different physicochemical properties and were conserved among Acinetobacter species.DiscussionWe identified the A. baumannii OMPK high immunogenic class-I and class-II T cell epitopes and introduced two promising high immunogenic peptides as vaccine candidates. It is recommended to perform in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.

In Silico Design and Evaluation of Acinetobacter baumannii Outer Membrane Protein a Antigenic Peptides As Vaccine Candidate in Immunized Mice.

Acinetobacter baumannii is a Gram-negative bacterium that has recently been identified as a leading nosocomial pathogen. Infections by this pathogen result in significant mortality due to antibiotic resistance. An effective vaccine would help alleviate the burden of disease incurred by this pathogen; however, there are currently no licensed vaccines offering protection against Acinetobacter baumannii infection. In this study, considering the fact that outer membrane protein A is one of the most promising vaccine candidates, we predicted T cell and B cell epitopes on this protein using sequence-based epitope prediction tools and determined whether or not mice immunized with these peptides induce an immune response. We selected consensus epitopes including five peptides in different tools with the highest score. 48 female C5BL/6 SPF injected subcutaneously with the peptides (peptide1 to peptide 5 separately) in 100 μL of the solution and sham groups received adjuvant and PBS alone on the same schedule: on day 0 (primary dose) and two booster doses were administered on days 14 and 28. At the end of time, animals euthanized by Isoflurane, and collected sera for assessment of specific antibodies against each peptide by ELISA (Enzyme-linked immunosorbent assay). Immunization of mice showed one of the novel synthetic peptides (peptide 1 (24-50 amino acids)) elicited immune responses. We conclude to combine theoretical methods of epitope prediction and evaluating the potential of immunogenicity for developing vaccines is important.

Distinct role of outer membrane protein A in the intrinsic resistance of Acinetobacter baumannii and Acinetobacter nosocomialis

Acinetobacter baumannii outer membrane protein A (AbOmpA) contributes to the intrinsic resistance of A. baumannii through the OmpA-like domain. The present study investigated the role of Acinetobacter nosocomialis OmpA (AnOmpA) in the intrinsic resistance of A. nosocomialis and compared it with the role of AbOmpA. The minimal inhibitory concentrations (MICs) of antimicrobial agents against wild-type A. nosocomialis ATCC 17903, ∆AnompA mutant, and single-copy AnompA-complemented strains were determined by performing E-test or agar dilution. Single-copy ompA cross-complemented strains were constructed by cross-inserting AnompA and AbompA open reading frames (ORFs) along with their native promoters into ∆AbompA and ∆AnompA mutant strains, respectively, and the MICs of antimicrobial agents against these strains were determined. The ∆AnompA mutant of A. nosocomialis was more susceptible to colistin (20.0-fold) and gentamicin (4.8-fold) than the wild-type strain. The MICs of gentamicin and tetracycline against the ∆AnompA mutant did not decrease in the presence of an efflux pump inhibitor. The MIC of trimethoprim against the ∆AnompA mutant harbouring PAbompA and AbompA ORF increased by >4.0-fold compared with that against the wild-type strain. However, the MICs of all the tested antimicrobial agents were similar against the wild-type A. baumannii ATCC 17978 and ∆AbompA mutant harbouring PAnompA and AnompA ORF. These results indicate that AnOmpA contributes to the intrinsic resistance of A. nosocomialis similar to AbOmpA. However, AbOmpA and AnOmpA perform different roles in the intrinsic resistance of trimethoprim.

Bioinformatics analysis and epitope screening of a potential vaccine antigen TolB from Acinetobacter baumannii outer membrane protein

The clinical isolation rate of multidrug-resistant or pan-resistant Acinetobacter baumannii (A. baumannii) is increasing, resulting that optional antibiotics are very limited in clinical practice. To deal with such a dilemma in treatment, the development of effective vaccines serves as a good strategy. Outer membrane proteins (Omp) often contain potential excellent vaccine antigens, and NCBI has published >300 Omp sequences of A. baumannii (including the duplicates). To accurately screen out the potential excellent antigen molecules from a large number of sequences, and avoid repetitive experimental processes is of great significance. In this study, we used the bioinformatics software to give extensive predictions of TolB protein. Results suggest it is a potential vaccine antigen. We then cloned the TolB gene fragments and confirmed it was highly conserved among the strains. Finally, we designed a good recombinant epitopes and conducted experimental verification. These findings provided grounds for animal immunology experiments in the future, and showed an orientation for the efficient development of A. baumannii vaccine.

Outer membrane protein A contributes to antimicrobial resistance of Acinetobacter baumannii through the OmpA-like domain.

Acinetobacter baumannii outer membrane protein A (AbOmpA) is involved in bacterial pathogenesis. However, the role of AbOmpA in the antimicrobial resistance of A. baumannii has not been fully elucidated. This study aimed to investigate the role of the OmpA-like domain of AbOmpA in the antimicrobial resistance of A. baumannii. The MICs of antimicrobial agents for the WT A. baumannii ATCC 17978, ΔompA mutant, OmpA-like domain-deleted (amino acids 223-356) AbOmpA mutant and single-copy ompA-complemented strain were determined by the Etest method. The MICs of antimicrobial agents for MDR strain 1656-2 and its ΔompA mutant strains were also determined. The ΔompA mutant strain of ATCC 17978 was more susceptible to trimethoprim (>5.3-fold) and other antimicrobial agents tested (<2.0-fold), except tigecycline, than the WT strain. The ΔompA mutant strain of 1656-2 was more susceptible to trimethoprim (>4.0-fold), tetracycline (2.3-fold) and other antimicrobial agents (<2.0-fold), including tigecycline, colistin and imipenem, than the WT strain. The MICs of gentamicin, imipenem and nalidixic acid for the WT ATCC 17978 and ΔompA mutant strains were decreased in the presence of an efflux pump inhibitor. A mutant strain of ATCC 17978 with the OmpA-like domain of AbOmpA deleted was more susceptible (≥2.0-fold) to substrates of the resistance-nodulation-division efflux pumps, including aztreonam, gentamicin, imipenem and trimethoprim, than the WT strain. This study demonstrates that AbOmpA contributes to the antimicrobial resistance of A. baumannii through the OmpA-like domain.

Immunoprotective Efficacy of Acinetobacter baumannii Outer Membrane Protein, FilF, Predicted In silico as a Potential Vaccine Candidate.

Acinetobacter baumannii is emerging as a serious nosocomial pathogen with multidrug resistance that has made it difficult to cure and development of efficacious treatment against this pathogen is direly needed. This has led to investigate vaccine approach to prevent and treat A. baumannii infections. In this work, an outer membrane putative pilus assembly protein, FilF, was predicted as vaccine candidate by in silico analysis of A. baumannii proteome and was found to be conserved among the A. baumannii strains. It was cloned and expressed in E. coli BL21(DE3) and purified by Ni-NTA chromatography. Immunization with FilF generated high antibody titer (>64,000) and provided 50% protection against a standardized lethal dose (108 CFU) of A. baumannii in murine pneumonia model. FilF immunization reduced the bacterial load in lungs by 2 and 4 log cycles, 12 and 24 h post infection as compared to adjuvant control; reduced the levels of pro-inflammatory cytokines TNF-α, IL-6, IL-33, IFN-γ, and IL-1β significantly and histology of lung tissue supported the data by showing considerably reduced damage and infiltration of neutrophils in lungs. These results demonstrate the in vivo validation of immunoprotective efficacy of a protein predicted as a vaccine candidate by in silico proteomic analysis and open the possibilities for exploration of a large array of uncharacterized proteins.

Immunostimulatory Activity of Dendritic cells pulsed with carbonic anhydrase IX and Acinetobacter baumannii outer membrane protein A

Dendritic cell (DC)-based immunotherapy is a potent therapeutic modality for treating renal cell carcinoma (RCC), but development of antigens specific for tumor-targeting and anti-tumor immunity is of great interest for clinical trials. The present study investigated the ability of DCs pulsed with a combination of carbonic anhydrase IX (CA9) as an RCC-specific biomarker and Acinetobacter baumannii outer membrane protein A (AbOmpA) as an immunoadjuvant to induce anti-tumor immunity against murine renal cell carcinoma (RENCA) in a murine model. Murine bone-marrow-derived DCs pulsed with a combination of RENCA lysates and AbOmpA were tested for their capacity to induce DC maturation and T cell responses in vitro. A combination of RENCA lysates and AbOmpA up-regulated the surface expression of co-stimulatory molecules, CD80 and CD86, and the antigen presenting molecules, major histocompatibility (MHC) class I and class II, in DCs. A combination of RENCA lysates and AbOmpA also induced interleukin-12 (IL-12) production in DCs. Next, the immunostimulatory activity of DCs pulsed with a combination of CA9 and AbOmpA was determined. A combination of CA9 and AbOmpA up-regulated the surface expression of co-stimulatory molecules and antigen presenting molecules in DCs. DCs pulsed with a combination of CA9 and AbOmpA effectively secreted IL-12 but not IL-10. These cells interacted with T cells and formed clusters. DCs pulsed with CA9 and AbOmpA elicited the secretion of interferon-γ and IL-2 in T cells. In conclusion, a combination of CA9 and AbOmpA enhanced the immunostimulatory activity of DCs, which may effectively induce anti-tumor immunity against human RCC.

Acinetobacter baumannii outer membrane protein a induces dendritic cell death through mitochondrial targeting

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a potential virulence factor that induces epithelial cell death, but its pathologic effects on the immune system have yet to be determined. The present study investigated the pathologic events occurring in dendritic cells (DCs) exposed to a cytotoxic concentration of AbOmpA. AbOmpA induced early-onset apoptosis and delayed-onset necrosis in DCs. AbOmpA targeted the mitochondria and induced the production of reactive oxygen species (ROS). ROS were directly responsible for both apoptosis and necrosis of AbOmpA-treated DCs. These results demonstrate that the AbOmpA secreted from A. baumannii induces DC death, which may impair T cell biology to induce adaptive immune responses against A. baumannii.

Nuclear translocation and DNAse I-like enzymatic activity ofAcinetobacter baumanniiouter membrane protein A

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a potential virulence factor that induces host cell death. Based on previous findings that AbOmpA translocated into the nuclei of host cells, the cell-death mechanism of AbOmpA through the nuclear targeting was investigated. Acinetobacter baumannii secreted AbOmpA in in vitro culture. The recombinant AbOmpA (rAbOmpA) was internalized by the host cells. The intracellular rAbOmpA was degraded into several forms of subfragments in the cytosol and then two subfragments of rAbOmpA translocated into the nuclei. The rAbOmpA exhibited the divalent cation-dependent endonuclease activity. In an in vivo assay with microinjection of rAbOmpA into the nucleus of fertilized Xenopus laevis eggs, rAbOmpA degraded chromosomal DNA with the characteristic DNA ladders and induced degeneration of the embryos. These results suggest that AbOmpA translocates into the nuclei of host cells and degrades chromosomal DNA by DNAse I-like enzymatic activity, which is a new pathogenic strategy of A. baumannii.

Anti-tumor activity of Acinetobacter baumannii outer membrane protein A on dendritic cell-based immunotherapy against murine melanoma

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-gamma+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens.

CarO, an Acinetobacter baumannii outer membrane protein involved in carbapenem resistance, is essential for l-ornithine uptake

We previously associated the emergence of carbapenem resistance in Acinetobacter baumannii with the loss of an outer membrane (OM) protein designated CarO. CarO was found essential for l-ornithine uptake: CarO-deficient strains were specifically impaired to grow only on l-ornithine, and failed to incorporate l-[ 14C] ornithine from the medium. l-arginine, and histidine and lysine to a lower extent, could effectively compete for l-[ 14C] ornithine uptake. l-ornithine also reduced A. baumannii sensitivity to imipenem, suggesting that both compounds compete for uptake. The overall results indicate that CarO participates in the selective uptake of l-ornithine, carbapenems, and other basic amino acids in A. baumannii.

Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity

Acinetobacter baumannii is an emerging opportunistic pathogen responsible for healthcare-associated infections. The outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting. The nuclear translocation of AbOmpA and the subsequent pathology on host cells were further investigated. AbOmpA directly binds to eukaryotic cells. AbOmpA translocates to the nucleus by a novel monopartite nuclear localization signal (NLS). The introduction of rAbOmpA into the cells or a transient expression of AbOmpA-EGFP causes the nuclear localization of these proteins, while the fusion proteins of AbOmpADeltaNLS-EGFP and AbOmpA with substitutions in residues lysine to alanine in the NLS sequences represent an exclusively cytoplasmic distribution. The nuclear translocation of AbOmpA induces cell death in vitro. Furthermore, the microinjection of rAbOmpA into the nucleus of Xenopus laevis embryos fails to develop normal embryogenesis, thus leading to embryonic death. We propose a novel pathogenic mechanism of A. baumannii regarding the nuclear targeting of the bacterial structural protein AbOmpA.