Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis.
OBJECTIVE
In vivo induced genes are thought to play an important role during infection of host. AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET), but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneri.


METHODS
PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively.


RESULTS
Deletion mutant of S. flexneri 2a alkA was successfully constructed by gamma Red recombination system. The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay.


CONCLUSIONS
AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.
Shi
Zhao-Xing
ZX
Beijing Institute of Biotechnology, Beijing 100071, China.
Wang
Heng-Liang
HL
Hu
Kun
K
Feng
Er-Ling
EL
Yao
Xiao
X
Su
Guo-Fu
GF
Huang
Pei-Tang
PT
Huang
Liu-Yu
LY
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003227:Conjugation, Genetic; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004195:Disease Models, Animal; D004405:Dysentery, Bacillary / Q000382:microbiology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012763:Shigella flexneri / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity*; D013045:Species Specificity; D014774:Virulence / Q000235:genetics*
D017931:DNA Primers; C023091:rubredoxin-NAD+ reductase; D009247:NADH, NADPH Oxidoreductases