DOI: 10.14457/tu.the.2015.160
title: GENETIC DIVERSITY OF STREPTOCOCCUS SUIS THAI ISOLATES AND CHARACTERIZATION OF ARGININE DEIMINASE

description: Streptococcus suis (S. suis) is an important zoonotic bacterium causing life threatening diseases in piglets and humans. S. suis isolates from various Western countries as well as those from China and Vietnam have been previously characterized. At the commencement of this thesis, the genetic characteristics and relationship between S. suis strains isolated from both humans and pigs in Thailand were unknown. In this study, a total of 245 S. suis isolates were collected from both human cases (epidemic and sporadic) and pigs (diseased and healthy) in Thailand. S. suis were identified by conventional biochemical tests and PCR targeting 16S rDNA and gdh genes. Thirty-six isolates were identified as S. suis serotype 2 (SS2) based on serotyping and the cps2-PCR. Among the remaining 209 non-SS2 isolates, the serotype was untypable for the majority of them (67.7%). For the known serotypes, serotype 22 was the most common among the 20 presented serotypes (9.6%). All of S. suis isolates were tested for the presence of six virulence-associated genes i. e., arginine deiminase (arcA), 38-kDa protein and protective antigen (bay046), extracellular protein factor (epf), hyaluronidase (hyl), muramidase-released protein (mrp) and suilysin (sly). The genetic diversities of these isolates were characterized by random amplified polymorphic DNA (RAPD) PCR. For the SS2 isolates, multilocus sequence typing (MLST) analysis was also performed as an additional genotyping method. Four virulence-associated gene patterns (VAGP1 to 4) were obtained and the majority of isolates (32/36) carried all tested genes (VAGP1). Each of the three OPB primers used provided four patterns designated RAPD-A to RAPD-D. Furthermore, MLST analysis could also distinguish the 36 isolates into four sequence types (STs) i. e., ST1 (n = 32), ST104 (n = 2), ST233 (n = 1) and a newly identified ST, ST336 (n = 1). Dendrogram constructions based on RAPD patterns indicated that SS2 isolates from Thailand could be divided into four groups and the characteristics of the individual groups were in complete agreement with the virulence gene profiles and STs. The majority (32/36) of SS2 isolates recovered from diseased pigs, slaughterhouse pigs and human patients could be classified into a single group (VAGP1, RAPD-A and ST1). This genetic information strongly suggests the transmission of SS2 isolates from pigs to humans in Thailand. Non-SS2 isolates carried 13 VAGPs (excluding VAGP3). The 69 non-SS2 isolates had both arcA and bay046 genes (VAGP11). The phylogenic trees of non-SS2 S. suis isolates constructed from RAPD PCR banding patterns indicated that they were highly diverse. However, the genetic relationship between non-SS2 isolates carrying VAGP1−4 was found to relate to the SS2 isolates carrying the same VAGP indicating that a minority of non-SS2 isolates carried the potential to transmit to human. This study is the first report for genetic characteristics of strains from Thailand and to elucidate the genetic relationship among S. suis isolates from human and pig origins.ArcA of S. suis is one of the putative virulence factors. It could play an important role to facilitate intracellular survival of S. suis under acidic conditions. However, the biological properties and function of SS2 ArcA have not yet been fully elucidated. This study revealed physical characteristics and enzyme kinetic properties of SS2 ArcA. The SS2 arcA was cloned into Escherichia coli (E. coli) and expressed as a soluble protein under T7 RNA polymerase/promoter system. Recombinant SS2 ArcA (rSS2 ArcA) was successfully purified using Ni-NTA affinity chromatography with EDTA gradient elution, yielding a 47 kDa protein. Multiple sequence alignment of SS2-arcA deduced-amino acid sequence revealed that it contained a highly conserved catalytic triad (Cys399-His273-Glu218) and had a high sequence similarity with that of S. pyogenes (89.98%). Enzymatic activity assay demonstrated that the rSS2 ArcA was functionally active protein. The optimum temperature and pH of rSS2 ArcA and crude native ArcA were 42 ºC and 7.2, respectively. The rSS2 ArcA and crude native SS2 ArcA were stable at 4 ºC and 25 ºC for 3 h but stability of crude native ArcA could be exceed to 30 ºC for 3 h. The pH stability and dependency tests suggested that the rSS2 ArcA and crude native SS2 ArcA were functionally active in acidic conditions. Enzyme kinetic studies demonstrated that the specific activity of rSS2 ArcA was 16.00 U/mg and L-arginine substrate binding affinity (Km) value was 0.058 mM. Whilst, specific activity and Km values of crude native SS2 ArcA were 0.23 U/mg and 0.157 mM, respectively. Compared to their orthologous proteins, the rSS2-ArcA exhibited a weak binding affinity with a common ArcA inhibitor, L-canavanine and L-NIO; therefore, neither rSS2 nor crude native ArcAs were completely inactivated by these inhibitors. These results suggested that the inactivation mechanism of SS2 ArcA might be distinct from other ArcA. Furthermore, the partial inactivation of ArcA significantly impaired the viability and growth of SS2 at pH 4.0, 6.0 and 7.5 indicating the significant role of SS2 ArcA for the survival of SS2 under acidic conditions.