The specific binding domain between PGK

The specific binding domain between PGK in group B strepotocci (GBS) and actin had been reported . PGK as the actin-binding protein identified in TMW 1.1434, which displayed highly significant adhesion was investigated for the binding sites and compared to bacteria with less strong adhesion to actin. To amplify from 10 lactobacilli, genomes from overnight-cultured lactobacilli were extracted with E.Z.N.A. bacterial DNA kit (Omega, Biotek, Germany) and used as template for polymerase chain reaction. Fp 5′-GGCTAAATTAATYGTTTCAG-3′, Rp 5′-CAGAAATAGCWGCRATACC-3′ were designed for amplification of from and . Fb 5′-GGCTAAATTAACAGTTTCCG-3′, Rb 5′-CTTCAAGGTATTCCAGAGA-3′ were designed for amplification of from . Amplified was purified with E.Z.N.A. cycle pure kit (Omega, Biotek, Germany) and sent for sequencing (GATC Biotech, Germany). According to the PGK (from GBS)-actin domain KKESKNDEE reported by Boone and Tyrrell , partial amino acids of PGK from 10 strains were aligned with reported sequence by GENtle software (). It was found lysine (K) at amino MRS1523 synthesis position 127 in PGK from GBS and TMW 1.1434 was replaced by arginine (R) in PGK from and . To our knowledge, no paper on actin-binding protein in lactobacilli has been published. In the 96-well plate assay, TMW 1.1434 displayed highly significant adhesion to actin followed by TMW 1.1734 and TMW 1.1434 with significant adhesion. Actin-binding proteins from lactobacilli through repeated experiments were identified as: PK, PGI and PGK from TMW 1.1434; PK, GroEL and EF-Tu from TMW 1.1734; GroEL from TMW 1.485. Regardless of bacterial origins, PK, PGI, PGK, EF-Tu and GroEL being isolated as actin-binding protein is not surprising, in that these proteins from pathogenic bacteria as well as lactobacilli have been reported as host-targeting in many other studies. For example, chemically purified EF-Tu was found to bind to actin in murine cells . GroEL and EF-Tu from NCC533 La1 were successively found to bind to gastrointestinal mucosa , . Since the term “moonlighting proteins” introduced by Jeffery in year 1999 to represent multifunctional proteins , more and more moonlighting proteins have been discovered. PK, PGI, and PGK were previously defined as moonlighting proteins as a result of their additional ability to bind to plasminogen besides the basic function of metabolic catalysis . In the light of the multiple tasks of moonlighting proteins, these proteins are not exclusively binding to just one substance, but are able to anchor several targets as receptor. Cell surface protein PK from is capable of binding to mannan of yeast . PGK from oral streptococci could bind to plasminogen , and PGK from group B streptococci could bind to cellular actin, which is in accordance with our present study. The recombinant crystallized PGI from (formerly ) had cell-motility-stimulating activity on mouse colon cells and enhanced neurite outgrowth on neuronal progenitor cells , suggesting this bacterium could utilize PGI to elicit signals once it colonized host cells. We report that PK, PGI, and PGK from TMW 1.1434, and PK from TMW 1.1734 could bind to actin besides the essential function of glycolysis, confirming the multifunction of PK, PGI, and PGK. Moreover, PGK-actin binding domain analysis revealed that lysine at amino acid position 127 in PGK from TMW 1.1434, which displayed highly significant adhesion to actin is replaced by arginine in PGK from and strains, which displayed less strong adhesion. This is in accordance with Boone's study, reporting that substitution of the lysine residues with other amino acid residues resulted in significantly reduced bacterial binding to actin . Therefore, lysine in the consensus sequence of the binding domain is assumed to be important for actin-binding, which is to be verified by point mutation in future work. Author contribution
Conflict of interests
Acknowledgements The authors would like to thank China Scholarship Council (CSC) for financial support and Dr. Hua Wei for genuine academic suggestions.