Characterizing the interplay between Acinetobacter baumannii, A549 cells, and anti-Omp34 antibodies: implications for adherence, internalization, and cytotoxicity.

Acinetobacter baumannii thrives within eukaryotic cells, influencing persistence, treatment approaches, and progression of disease. We probed epithelial cell invasion by A. baumannii and the influence of antibodies raised to outer membrane protein 34 (Omp34) on epithelial interactions. We expressed and purified recombinant Omp34 and induced anti-Omp34 antibodies in Bagg albino or BALB/c mice. Omp34 was evaluated for acute toxicity in mice through histological analysis of six organs. The host cell line, A549, was exposed to both A. baumannii 19606 and a clinical isolate. The study also investigated serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells, with and without anti-Omp34 sera, utilizing cell culture techniques and light microscopy. A549 cell viability was evaluated by A. baumannii challenge and exposure to anti-Omp34 sera. Actin disruption experiments using cytochalasin D probed microfilament and microtubule roles in A. baumannii invasion. Omp34 prompted antibody production without toxicity in mice. The serum showed bactericidal effects on both strains. Additionally, both A. baumannii strains were found to form biofilms. Omp34 serum was observed to decrease biofilm formation, bacterial adherence, internalization, and proliferation in A549 cells. Furthermore, the use of anti-Omp34 serum enhanced the post-infection survival of the host cell. Pre-exposure of A549 cells to cytochalasin D reduced bacterial internalization, highlighting the role of actin polymerization in the invasion process. Microscopic analysis revealed various interactions, such as adherence, membrane alterations, vacuolization, apoptosis, and cellular damage. Anti-Omp34 serum-exposed A549 cells were protected and showed reduced damage. The findings reveal that A. baumannii can significantly multiply intracellularly within host cells. This suggests the bacterium's ability to establish an environment conducive to its replication by preventing fusion with degradative lysosomes and inhibiting acidification. This finding contributes to the understanding of A. baumannii's intracellular persistence and highlights the role of Omp34 in influencing apoptosis, autophagy, and bacterial adherence, which may impact the development of effective treatments against A. baumannii infections.

In silico design of a novel hybrid epitope-based antigen harboring highly exposed immunogenic peptides of BamA, OmpA, and Omp34 against Acinetobacter baumannii.

Acinetobacter baumannii, is among the highest priority bacteria according to the WHO categorization which necessitate the exploration of alternative strategies such as vaccination. OmpA, BamA, and Omp34 are assigned as appropriate antigens to serve in vaccine development against this pathogen. Experimentally validated exposed epitopes of OmpA and Omp34 along with selected exposed epitopes predicted by an integrative in silico approach were represented by the barrel domain of BamA as a scaffold. Among the 8 external loops of BamA, 5 loops were replaced with selected loops of OmpA and Omp34. The designed antigen was analyzed regarding the physicochemical properties, antigenicity, epitope retrieval, topology, structure, and safety. BamA is a two-domain OMP with a 16-stranded barrel in which L4, L6, and L7 were the longest loops of BamA in order. The designed antigen consisted of 478 amino acids with antigen probability of 0.7793. The novel antigen was a 16-stranded barrel. No identical 8-meric peptides were found in the human proteome against the designed antigen sequence. The designed construct was safe regarding the allergenicity, toxicity, and human proteome reactivity. The designed antigen could develop higher protection against A. baumannii in comparison to either OmpA, BamA, or Omp34 alone.

A two-protein cocktail elicits a protective immune response against Acinetobacter baumannii in a murine infection model.

PURPOSE: Due to its high drug resistance, Acinetobacter baumannii is a priority for new therapeutic measures like vaccines. In this study, the protectivity of a combination cocktail of Omp34 and BauA as a vaccine against A. baumannii was studied in a murine sepsis model. METHODS: The antibody titers were raised to Omp34 and BauA in BALB/c mice and assessed by indirect ELISA. The immunized mice were challenged with A. baumannii ATCC 19606. The bacterial loads in the liver, spleen, and lungs were also determined. RESULTS: A significant increase in survival of the immunized mice was noted. In active immunity, the survival rates in mice receiving Omp34 and BauA alone or in combination were 100%. A significant decrease in the bacterial load was observed in the spleens, livers, and lungs of vaccinated mice. Anti-BauA and anti-Omp34 sera crossly detected Omp34 and BauA respectively. The decrease in bacterial load in body organs of mice vaccinated with a combination of the two proteins was significantly higher than those of the single proteins in both actively and passively immunized mice. In passive immunity, the survival rate of mice receiving specific sera raised to the combination of these proteins was 85.7%. CONCLUSION: Higher protection by a combination of Omp34 and BauA than Omp34 or BauA could be attributed to targeting simultaneously both surface antigens indicating the synergistic effect of Omp34 and BauA as suitable vaccine candidates in the prevention or treatment of A. baumannii infections.

Immunogenicity of loop 3 of Omp34 from A. Baumannii in loopless C-lobe of TbpB of N. meningitidis.

Acinetobacter baumannii is a common causative agent of nosocomial infections, with a mortality rate of 43% in infected patients. Due to the emergence of multidrug-resistant (MDR) strains, vaccine development has become necessary. Since the 34 kDa outer membrane protein Omp34 has been identified as a potential vaccine target, we implemented a hybrid antigen approach to target its extracellular loops. Using bioinformatic and structural analyses, we selected Loop 3 from Omp34 and displayed it on the loopless C-lobe (LCL) of TbpB of Neisseria meningitidis. The hybrid antigen and the LCL were produced and used to immunize mice for passive and active immunization and challenge experiments in which the reactivity of the sera was assessed by ELISAs, the bacterial load in the tissues measured and the survival of immunized mice compared. LCL was ineffective in immunization against A. baumannii thus the resulting immunity was due to the presence of Omp34 loop 3. It resulted in increased survival and a reduced bacterial load in the tissues compared to the control groups. The findings indicate that the immunogenicity of Omp34 loops can induce protection against A. baumannii infection, and it could probably be used as a vaccine candidate to control the pathogenesis of A. baumannii.

BauA and Omp34 surface loops trigger protective antibodies against Acinetobacter baumannii in a murine sepsis model.

The complexity of treating Acinetobacter baumannii infections with the newly developed resistant strains has led researchers to confront this pathogen by developing vaccines. In this study, we used two important virulence factors of A. baumannii to elicit immunity against the A. baumannii. The immunogenic loops were from Baumannii acinetobactin utilization A (BauA) and 34kD outer membrane protein (Omp34). C-lobe derivative of the TbpB surface lipoprotein was used to display the superficial epitopes of the TbpA receptor protein of Neisseria meningitidis. The resulting loopless C-lobe (LCL) with implanted nucleotide sequences of the immunogenic loops from BauA and Omp34 was used as a hybrid antigen. The hybrid antigens were expressed in the E. coli and were used to immunize mice. The mice were challenged with a clinical isolate of A. baumannii (ABI022). Immunization with the hybrid antigens of the BauA loop 7 (BauAL7P3), Omp34 loop 3 Omp34L3P1, and the combination of both loops (BauAL7P3Omp34L3P1) brought about 42.86%, 42.86%, and 71.43% protection against A. baumannii infection. Histopathological findings in the immunized mice showed bronchioles clear from inflammatory cells and normal texture of the spleen and liver. The findings support the use of a multivalent vaccine to induce broadly reactive antibody responses against heterologous A. baumannii strains.

Anti-Omp34 antibodies protect against Acinetobacter baumannii in a murine sepsis model.

Acinetobacter baumannii, an opportunistic extracellular pathogen is one of the major causes of nosocomial infections. Omp34, also known as Omp33-36, is a bacterial porin protein involved in the virulence and fitness of this pathogen by adhesion to the host cell. This antigen nominated as an appropriate candidate for immunization against A. baumannii. In this study, the expression of the recombinant Omp34 (rOmp34) was carried out in E. coli BL21 (DE3). The immunogenicity of the rOmp34 in A. baumannii was studied in a murine sepsis model. Antibody response in mice injected with the recombinant protein was assessed using indirect ELISA. Bactericidal activity of rOmp34-immunized mice sera (1:10 dilution) against A. baumannii ATCC 19606 after 0, 1, 2, 4, and 8 h of incubation at 37 °C was assessed. In addition to survival rate, load of bacteria in liver and spleen of the infected mice were evaluated. A high titer of specific antibody equivalent to optical density of 1.54 ± 0.06 against rOmp34 was elicited in the immunized mice sera. Viability of the A. baumannii incubated 8 h with immunized mice sera was 64%. Homogenized liver and spleen samples of the control mice challenged with A. baumannii were loaded with 8 × 103 and 9 × 103 CFU per gram tissue respectively 48 h post-challenge as against complete clearance of A. baumannii in the immunized group. The protective immunity was achieved by challenging the mice groups with 5 × LD50 of live A. baumannii. Omp34 can be nominated as an immunogen that can bring about protection against Acinetobacter baumannii.

Specific egg yolk antibodies (IgY) confer protection against Acinetobacter baumannii in a murine pneumonia model.

AIM: Acinetobacter baumannii, an increasingly serious health threat, is considered as one of the six most dangerous microbes of high mortality rate. However, treatment of its infections is difficult because of the lack of efficient antibiotic or commercial vaccines. Passive immunization through administration of specific antibodies such as IgY, could be an attractive practical solution. METHODS AND RESULTS: In the current study, antigenicity of two recombinant outer membrane proteins (OmpA and Omp34) as well as inactivated whole cell of A. baumannii was assessed by ELISA. Moreover, prophylactic effects of specific IgY antibodies (avian antibody) raised against these antigens were evaluated in a murine pneumonia model. The specific IgY antibodies had various prophylactic effects in the pneumonia model. OmpA was the most potent antigen in terms of triggering antibody and conferring protection. While a synergic effect was observed in ELISA for antibodies raised against a combination of OmpA and Omp34 (which are known as Omp33-36 and Omp34 kDa), an antagonistic effect was unexpectedly seen in challenges. The reason for this phenomenon remains to be precisely addressed. CONCLUSION: All the specific IgY antibodies could protect mice against pneumonia caused by A. baumannii. SIGNIFICANCE AND IMPACT OF THE STUDY: The specific IgY antibodies could be employed as a pharmaceutical against pneumonia caused by A. baumannii.

An integrative in silico approach to the structure of Omp33-36 in Acinetobacter baumannii.

Omp33-36 in A. baumannii, a bacterium causing serious nosocomial infections, is a virulence factor associated with the pathogen metabolic fitness as well as its adherence and invasion to human epithelial cells. This protein is also involved in interaction of the bacteria with host cells by binding to fibronectin. Moreover, Omp33-36 renders cytotoxicity to A. baumannii in addition to inducing apoptosis and modulation of autophagy. In the present study, an integrated strategy is launched to pierce into the 3D structure of Omp33-36 protein. The signal peptide within the sequence was determined, then, topology as well as secondary and tertiary structures of the protein were predicted. The mature protein assigned as a 14-stranded barrel in which residues 1-19 is removed as signal peptide. The obtained 3D models were evaluated in terms of quality; and then, served as queries to find similar protein structures. The hits were analyzed regarding topology among which 14-stranded were considered. The most qualified model was refined and then its sequence aligned to its counterpart similar structure protein (CymA from Klebsiella oxytoca). The determined structure of Omp33-36 could justify its porin function and carbapenem-resistance associated with the loss of this protein.