Hybrid Antigens Expressing Surface Loops of ZnuD From Acinetobacter baumannii Is Capable of Inducing Protection Against Infection.

Acinetobacter baumannii is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from A. baumannii as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the Neisseria meningitidis TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the E. coli cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an A. baumannii sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.

Bacillus phage endolysin, lys46, bactericidal properties against Gram-negative bacteria.

BACKGROUND AND OBJECTIVES: The great potential of bacteriophage for removing pathogen bacteria via targeting the cell wall is highly concerned. With a priority for overcoming drug-resistance, we screened against endolysins targeting Gram-negative bacteria to introduce a new antibacterial agent. This study was aimed to identify endolysins from the lysogenic phage of the Siphoviridea family in Bacillus subtilis. MATERIALS AND METHODS: The Bacillus subtilis strain DDBCC46 was isolated from a preliminary antibacterial screening program. The endolysin (s) was extracted, concentrated with ammonium sulfate saturation, and their activity evaluated against the indicator bacteria. The phage particles were extracted from the bacteria using the minimum inhibition concentration of mitomycin C, followed by testing the phage inhibitory effect on the growth of indicator bacteria. The NCBI, Virus-Host DB, and EXPASY databases were used to obtain and confirm the sequences of the genes encoding PG hydrolases in Siphoviridea phages hosted in B. subtilis. RESULTS: An 816 bp gene encoding an endolysin enzyme, was approved in the B. subtilis DDBCC 46, with specific primers of Bacillus phage SPP1. The purified-endolysin indicated antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, Proteus (sp), and Escherichia coli. SDS-PAGE profiling followed by silica gel purification, led to introduce Lys4630 as a therapeutic product and food preservative. CONCLUSION: lys4630 showed antibacterial effects on the common Gram-negative pathogens in clinics and food industries; E. coli, P. aeruginosa and Salmonella (sp).

Identification and characterization of an endolysin - Like from Bacillus subtilis.

Drug-resistant Gram-positive pathogens have been a rising risk in hospitals and food industries from the last decades. Here in, the potential of endolysin production in Dasht Desert Bacterial Culture Collection (DDBCC), against indicator bacteria, was investigated. DDBCC was screened against autoclaved-indicator bacteria; Streptococcus faecalis, Streptococcus pyogenes, Bacillus sp, Bacillus subtilis and Staphylococcus aureus as the substrates for the endolysin enzymes. The endolysins were produced in BHI medium followed by ammonium sulfate purification. Peptidoglycan hydrolytic activity was tested by zymogram method. Lysogenic bacteria were induced by 0.1 µg/ml mitomycin C for bacteriophages extraction. The lysogenic bacteria inhibited S. pyogenes, S. faecalis, Bacillus sp. and B. subtilis. The strain DDBCC10 was selected for further experiments on its higher and specific activity against the cell wall of S. faecalis. The highest activity for the endolysin was obtained at 50-60% ammonium sulfate saturation as 8 U/ml. Lys10, a 22 kDa enzyme, digested the cell wall of S. faecalis in 15 min while the whole phage from DDBCC10 could form plaque on S. faecalis and S. pyogenes. In a Transmission Electron Microscopy assay (TEM), the phage was distinguished as a member of Siphoviridae. Here; Lys10 is introduced as a new biocontrol agent against S. faecalis for therapeutics, disinfection, and food preservatives purposes at a much lower expense than recombinant endolysins.

Adjuvant role of Pseudomonas flagellin for Acinetobacter baumannii biofilm associated protein.

AIM: To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii (A. baumannii) biofilm associated protein (Bap). METHODS: The N terminal flagellin gene was amplified. The pET28a (+) and polymerase chain reaction products were digested with HindIII and EcoR I. The ligation of N terminal flagellin into pET28a ( +) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21 (DE3) as a suitable expression host. pET28a ( +) vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purified by affinity chromatography with His-Tag residues followed by confirmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally (i.n) challenged with A. baumannii and Pseudomonas aeruginosa (P. aeruginosa). RESULTS: The flagellin enhanced the immunogenicity of Bap causing an increase in specific IgG titers in serum (P < 0.001). Internal organs, i.e., liver, lung and spleen of the Bap-Flagellin immunized group challenged with A. baumannii showed significantly lower bacterial load compared to the control group. The bacterial loads were studied in internal organs. A. baumannii infected immunized animals with Bap-Flagellin exhibited internal organs with minor bacterial load while P. aeruginosa PAO1 infected group showed heavy bacterial load of (4.3 ± 0.12) × 10(6), (1.1 ± 0.01) × 10(6) and (2.2 ± 0.22) × 10(6) per gram of lungs, liver and spleen respectively. Bacterial loads were detected per gram of lungs, liver and spleen of the mice group immunized with Bap were (1.2 ± 0.06) × 10(7), (11.1 ± 0.041) × 10(5) and (3.6 ± 0.42) × 10(6) respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was significantly (P < 0.05) protected against A. baumannii. CONCLUSION: These results demonstrate that P. aeruginosa Flagellin as an adjuvant for Bap A. baumannii could be a useful model to evaluate new vaccine against A. baumannii.