Purpose N-acetylmuramoyl-l-alanine amidase known as lytA, is an immunogenic protein that plays an important role in the pathogenesis of Streptococcus pneumoniae . It is highly conserved among S. pneumoniae strains and is absent among other Streptococcus species. In the present study, the level of antibodies against the lytA recombinant protein was evaluated in healthy individuals' sera. Materials and Methods DNA was extracted from S. pneumoniae ATCC 49619 to amplify lyt A gene by polymerase chain reaction assay. The lytA amplicon and p ET28a vector were separately double digested using Nde -1 and Xho 1 restriction enzymes and then ligated together with ligase enzyme. The recombinant plasmid was expressed in Escherichia coli BL21 strain and the lytA recombinant protein purified using nickel-nitrilotriacetic acid affinity chromatography. Western blot was carried to detect lytA recombinant protein. Sixty healthy individual's sera (at three age groups: group 1, <2; group 2, 2–40; and group 3, 60–90 years old) were collected and the titers of anti-lytA antibodies were determined. Results The lyt A gene was highly expressed in E. coli BL21 host. The recombinant lytA protein was purified and confirmed by western blotting. Tukey test analysis showed that there were no significant differences among the age groups considering the anti-lytA titer of 10. However, at the anti-lytA titer of 60, significant differences were observed between group 1 vs. group 2 (p<0.001); group 1 vs. group 3 (p=0.003), and group 2 vs. group 3 (p=0.024). Conclusion The lytA protein seems to be a highly immunogenic antigen and a potential target for developing vaccines against pneumococcal infections.
Purpose Iron is a necessary element for the growth of bacteria; however, there are limited iron sources known for these microorganisms yet. Intracellular iron is stored as ferritin from, which releases iron in a gradual and controlled manner. The present study aimed to characterize ferritin-binding proteins (FBPs) of Streptococcus pneumoniae. Material and Methods S. pneumoniae species were cultured in BHI broth containing ferritin (1094 ng/mL) for 4h at 37°C. Ferritin level was measured using ELISA assay. Bacterial proteome was electrophoresed on SDS-PAGE and then transferred on PVDF nitrocellulose membrane. Afterward, the PVDF membranes were incubated with a ferritin solution. Identification of ferritin binding proteins was performed using anti-ferritin monoclonal antibody conjugated with HRP enzyme. Molecular docking was used to assess the interaction between pneumococcal proteases and FBPs applying phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor. Results No FBPs were identified in S. pneumoniae proteome. Moreover, ferritin levels have significantly (p<0.05) decreased following the growth of S. pneumoniae in ferritin-rich BHI medium. Also, molecular docking showed that RadA protease, ClpP hydrolase, and HtrA protease can potentially interact with PMSF protease inhibitors. On the other hand, the addition of the PMSF to the culture of S. pneumoniae prevented the reduction of ferritin, which indicates a potential role of RadA, ClpP, and HtrA proteases in ferritin degradation. Conclusion Our results suggest that S. pneumoniae produces no FBPs and also cannot directly use ferritin as an iron source. However, ferritin may be degraded through a protease-mediated mode.
Background and Objectives: Considering rising antibiotic resistance in various strains of Streptococcus pneumoniae, there is a need to find new immunogenic candidates for developing pneumococcal vaccines. Immunoglobulin A1 (IgA1) protease is one of the virulence factors playing an important role in the pathogenesis of S. pneumoniae infections. In the present study, we aimed to evaluate the titer of antibody against pneumococcal recombinant IgA1 protease in the serum of healthy humans. Materials and Methods: A part of the IgA1 protease gene (705 bp) from S. pneumonia ATCC 49619 was amplified by PCR and then digested using restriction enzymes and ligated by the pET28a expression vector. The recombinant protein was expressed in E. coli BL21 strain. Affinity chromatography was used to purify the protein. The titer of antibody against the recombinant protease was determined in healthy individuals in three age groups of <2, 2-40, and > 40 years using indirect Enzyme-Linked Immunosorbent Assay (ELISA). Results: The expression and purification of the IgA1 recombinant protease were successful. The concentration of the purified protein was determined as 1.013 mg/ml using the NanoDrop method. The titer of anti-recombinant IgA1 protease antibody (20, 40, 80 and 160) showed a significant correlation with age (p-value<0.05). According to our results, the antibody titer was desirable, especially in individuals over two years old. Conclusion: In the present study, desirable antibody titers against the pneumococcal recombinant IgA1 protease were seen in the three groups’ serum of healthy individuals. However, a significant correlation was not totally observed among groups.
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