Cloning, Expression and Functional Analysis of the Streptococcus pyogenes M1 Pilins using Lactococcus lactis

Abstract

Streptococcus pyogenes is a highly destructive, globally spread and re-emergent human pathogen. This bacterium primarily colonizes and causes infection through either the skin or throat. To date, no vaccine which prevents it has been fully commercialized. Pili (the hair-like cell appendages) in S. pyogenes are essential for colonization. Around nine types of the pili have been identified. Each is encoded from a discrete genomic region, namely the fibronectin and collagen-binding T-antigen (FCT) pathogenic island. In this report, the FCT2 type of pilus from the most invasive S. pyogenes M1 strain, SF370, was studied. It consists of a homo-polymeric shaft of Spy0128, a tip of Spy0125 and a base of Spy0130. The aim was to clone those pilin subunits into L. lactis and use them as pre-clinical models to study pilin expressions and biological functions. Our results indicated, first, Spy0130 functions as a pilus-base adaptor. Both Spy0125 and Spy0128 can directly anchor to bacteria cell walls. However, Spy0130 appears to be necessary to maintain high levels of pilus anchorage. Second, using human skin and throat cell-lines, both the cell-surface Spy0125 and Spy0130 can elicit weak adherence to the skin. The pili can elicit strong adherence to both the skin and throat. Third, the pili appeared to elicit more adherences to the skin than the throat. This is somewhat inconsistent with previous studies which indicated that many S. pyogenes FCT2- containing strains are throat specialists. Fourth, Spy0125 can promote bacteria adherence to a plastic surface. This indicates Spy0125 may participate in S. pyogenes transmission and biofilm formation. Fifth, purified Spy0125 antibodies can neutralize the adherence of S. pyogenes but not L. lactis piliated mutants. It appears that the antibodies can specifically inhibit the primary adherence of pili, which is required for S. pyogenes to initiate colonization. In conclusion, each pilin was shown to possess multiple roles in association with its cell surface localizations. Furthermore, Spy0125 is a potential vaccine candidate and future in vivo work will be required to tailor and to improve its therapeutic potential.