Autolysin (lytA) recombinant protein: a potential target for developing vaccines against pneumococcal infections

Abstract

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 lytA gene by polymerase chain reaction assay. The lytA amplicon and pET28a vector were separately double digested using Nde-1 and Xho1 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 lytA 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.

Keywords: Antibody; N-acetylmuramoyl-l-alanine amidase; Streptococcus pneumoniae; Surface protein; Western blotting.

Fig. 1. Gel electrophoresis of extracted plasmid and lytA amplicon. Lane pet, pPET28a; lane M, DNA ladder 100–10,000 bp; lane lytA, lytA amplicon; C, negative control.

Fig. 2. Polyacrylamide gel electrophoresis of samples collected from protein production and purification. M, protein marker; T0, E. coli BL21 before induction; T4, E. coli BL21 after 4 hours induction with IPTG (isopropyl-β-D-thiogalactopyranoside); B, unbounded proteins in flow-through fraction from binding step; DWB, flow-through fraction following washing column with denature wash buffer; W1–W5, flow-through fractions following washing column; NWB, flow-through fraction after washing with native wash buffer; E1–E2, sample from eluted proteins. E. coli, Escherichia coli.

Fig. 3. Western blot analysis of lytA recombinant protein.