Lactococcus lactis secreting phage lysins as a potential antimicrobial against multi-drug resistant Staphylococcus aureus

Abstract

Background: Staphylococcus aureus is an opportunistic Gram-positive bacterium that can form biofilm and become resistant to many types of antibiotics. The treatment of multi-drug resistant Staphylococcus aureus (MDRSA) infection is difficult since it possesses multiple antibiotic-resistant mechanisms. Endolysin and virion-associated peptidoglycan hydrolases (VAPGH) enzymes from bacteriophage have been identified as potential alternative antimicrobial agents. This study aimed to assess the ability of Lactococcus lactis NZ9000 secreting endolysin and VAPGH from S. aureus bacteriophage 88 to inhibit the growth of S. aureus PS 88, a MDRSA.

Method: Endolysin and VAPGH genes were cloned and expressed in L. lactis NZ9000 after fusion with the SPK1 signal peptide for secretion. The recombinant proteins were expressed and purified, then analyzed for antimicrobial activity using plate assay and turbidity reduction assay. In addition, the spent media of the recombinant lactococcal culture was analyzed for its ability to inhibit the growth of the S. aureus PS 88.

Results: Extracellular recombinant endolysin (Endo88) and VAPGH (VAH88) was successfully expressed and secreted from L. lactis which was able to inhibit S. aureus PS 88, as shown by halozone formation on plate assays as well as inhibition of growth in the turbidity reduction assay. Moreover, it was observed that the spent media from L. lactis NZ9000 expressing Endo88 and VAH88 reduced the viability of PS 88 by up to 3.5-log reduction with Endo88 being more efficacious than VAH88. In addition, Endo88 was able to lyse all MRSA strains tested and Staphylococcus epidermidis but not the other bacteria while VAH88 could only lyse S. aureus PS 88.

Conclusion: Recombinant L. lactisNZ9000 expressing phage 88 endolysin may be potentially developed into a new antimicrobial agent for the treatment of MDRSA infection.

Keywords: Bacteriophage; Endolysin; Lactococcus lactis; Multi-drug resistant Staphylococcus aureus; Virion associated peptidoglycan hydrolase.

Figure 1. Multiple sequence alignment analysis and prediction of domains of Endo88 and VAH88.

Multiple sequence alignment was done by MAFFT. Fully conserved regions are denoted with the symbol (*), conservative substitution regions are denoted with the symbol (:) and semi-conservative substitution regions are denoted signed with symbol. Multiple alignment of Endo88 with LysGH15 (ADG26756.1), MVL (BAF33253.1), LysK (YP_009041293), Twort (AAX92311.1) and LysH5 (ACJ64589.1). Blue, orange and green represent the CHAP, Amidase, and SH3 domains respectively. The conserved region of the catalytic site and peptidoglycan binding site are indicated by black and red boxes respectively (CHAP: C32, H95, E111, N113; SH3: N390, Y392, G393 & T394). (ii) Multiple sequence alignment of VAH88 with HydH5 (BAF79638.1) and YP_002332533.1. Blue and orange represent CHAP and LytD, respectively. The conserved region of the catalytic site are indicated by black boxes (CHAP: C38 & H100; LytD: E509, F528, Y590, W596).

Figure 2. Predicted 3D structure of Endo88 and VAH88 generated by MODELLER and viewed in PyMol.

The catalytic site and binding site are shown in grey boxes. (A) Superimposition of Endo88 with respective templates. Green: Endo88; Yellow: 6ist; Purple: 4ols; Cyan: 2mk5. (B) Superimposition of VAH88 with respective templates. Green: VAH88; purple: 6ist; cyan: 6fxp.

Figure 3. Western blot analysis showing intracellularly and extracellularly expressed VAH88 (A and B) and Endo88 (C and D) by recombinant L. lactis.

Both lysins were successfully expressed and secreted and the slightly smaller size of the extracellular protein compared to its intracellular counterpart showed that the SPK1 signal peptide was also correctly cleaved. Lane 1: Uninduced, 2: Induced. M: PageRulerTM Plus Prestained Protein Ladder (Thermo Fisher Scientific, USA). The arrow indicates position of the expressed VAH88 and Endo88 recombinant protein.

Figure 4. SDS-PAGE analysis showing intracellularly and extracellularly purified VAH88 (A and B) and Endo88 (C and D) by recombinant L. lactis.

The extracellular protein showed a homogenous single band as there are very few extracellular lactococcal endogenous proteins while residual endogenous proteins were observed in the intracellular fraction. Lane M: PageRulerTM Plus Prestained Protein Ladder (Thermo Fisher Scientific, Waltham, MA, USA). Lane 1: Flow through, Lane 2: Wash 1, Lane 3: Wash 2, Lane 4: Wash 3, Lane 5: Elute 1 and Lane 6: Elute 2. The arrow indicates position of the recombinant purified protein of VAH88 and Endo88.

Figure 5. Lytic activity of intracellular and extracellular VAH88 and Endo88 expressed by recombinant L. lactis NZ9000.

Plate assay showed that only the extracellular fraction was active (A). Well 1: Phosphate Saline Buffer (PBS), Well 2: Chloroform, Well 3: 1 µg, Well 4: 5 µg, Well 5: 10 µg, Well 6: 25 µg, Well 7: 50 µg Well 8: 100 µg. The zone of inhibition for the extracellular fractions indicated that Endo88 was more inhibitory compared to VAH88 (B). Values are the mean of three independent experiments with standard deviation indicated by error bars. An asterisk (*) indicates significance with p < 0.05.

Figure 6. Growth of PS 88 in the presence and absence of recombinant phage lysins.

Endo88 inhibited growth of PS88 at a higher rate than VAH88 while the control was not inhibited as expected. Values are the mean of three independent experiments with standard deviation indicated by error bars.

Figure 7. Survival of PS 88 in spent media of L. lactis secreting VAH88 and Endo88, respectively.

Endo88 was able to reduce growth of PS88 by 3.5 log reductions in 12 h. Values are the mean of three biological replicates and three technical replicates each, with standard deviation indicated by error bars. An asterisk (*) indicates significance with p < 0.05.

Figure 8. Lytic activity of extracellular protein produced by recombinant clone in plate assay calculated according to the zone size.

Endo88 was able to lyse all MRSA strains and S. epidermidis but not the other bacteria tested. Values are the mean of three independent experiments with standard deviation indicated by error bars. An asterisk (*) indicates significance with p < 0.05.