| Shigella spp. is a group of Gram-negative, non-spore forming, facultative pathogens. The bacteria cause a disease called shigellosis in humans, an infection of the large bowel characterized by abdominal cramps, diarrhea, and fever. There are four different species of Shigella based on the differences in O antigen and some biochemical reactions. Among those, Shigella flexneri is responsible for the majority of cases of endemic dysentery prevalent in developing countries where sanitation is poor. Most affected by this organism are children under 5 years old. In the recent 3 years, about 20 million people were infected every year and its one-year cumulative incidence rate was at the third place merely inferior to the pulmonary tuberculosis and hepatitis B. Owing to the low infectious dose (10 to 100 bacteria) and the emergence of multiple resistance strains, the traditional treatment by antimicrobial agents encounters big problems. Because the mechanism of pathogenicity and immunoprotection are unknown, no ideal and effective vaccine has been developed till now.Protein HtpG of S. flexneri is the homolog of the eukaryotic Hsp90 family of proteins. It has been showed that the expression of HtpG is induced by heat shock and acid shock in bacteria; it participates in folding of the newly synthesized protein under mild heat shock conditions; and its expression is repressed by nitrogen starvation. Furthermore, Mason C.A. et al. found an interesting phenomenon that the expression of HtpG of E. coli is dependent on the growth environment: its expression is induced by heat shock condition in a complex medium (such as LB broth), whereas its expression is unaffected by the external environmental temperature in the glucose minimal medium. This phenomenon suggested that the expression of HtpG might be under the control of other signal molehacules besides environmental temperature; its functions were more than those found in heat shock condition.To explore the function of htpG gene of S. flexneri, we firstly successfully knocked out this gene using an improved method based on theλ-Red recombination system established by Datsenko and Wanner. Then, a recovery mutant was also obtained by introducing a low-copy plasmid containing one copy of htpG gene into the deletion mutant. Subsequently, some experiments were carried out as following: 1) the growth curves of wild-type strain, deletion mutant and recovery mutant were measured respectively. 2) some basic biochemical events were also investigated. 3) the Sereny tests were performed to evaluate the virulence of these strains. All of the results of above studies showed no significant differences between three strains, suggesting that the function of this protein might be unrelated to the metabolism and invasion ability of S. flexneri. However, the titers of some inflammatory factors evoked by wild-type strain, deletion mutant and recovery mutant in intraperitoneal injected mice were quite different. These results indicated that the HtpG protein of S. flexneri 2a strain 2457T might be involved in the immunopathogenesis.After those analyses, comparative proteomic studies of whole cell proteins of these three strains were performed by means of two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF MS. The results showed that the expressions of 16 proteins were induced when htpG gene were absent, while the expressions of 13 proteins were repressed. These results showed that HtpG, a heat shock protein, also took part in some metabolic pathways, expecially oxidative defense mechanism and purine synthesis pathway. In the htpG gene deletion mutant, the expression of KatG was increased, while the expression of SodA was decreased; the expressions of proteins involved in the de novo synthesis of purine nucleotide, such as PurH, PurB and PurC, were also decreased. These findings extend our knowledge of the function of HtpG protein and are useful for the research of pathogenesis of S. flexneri.
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