Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.
Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.
Zhang
Xue-han
XH
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014 China.
He
Kong-wang
KW
Duan
Zhi-tao
ZT
Zhou
Jun-ming
JM
Yu
Zheng-yu
ZY
Ni
Yan-xiu
YX
Lu
Cheng-ping
CP
D000818:Animals; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D055786:Gene Knockout Techniques; D007168:IMP Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013290:Streptococcal Infections / Q000382:microbiology; D017011:Streptococcus suis / Q000201:enzymology* / Q000472:pathogenicity*; D016019:Survival Analysis; D013552:Swine
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D007168:IMP Dehydrogenase