Mechanism Of PhoP-PhoQ Two-component Signal Transduction System In Regulating The Virulence Of Shigella Flexneri

Shigella is a Gram-negative bacterium responsible for the majority of bacillary dysentery, which can spread to mucous membrane of colon by the fecal-oral route. In China shigellosis is becoming the third infectious disease which only next to virus hepatitis and tuberculosis. The major serological type in China is flexneri2a. Because of the abuse of antibiotic and horizontal transmission of antibiotic resistance genes, Shigella is becoming multidrug resistance in clinic, especially resistance to ampicillin, sulfanilamide and tetracycline, even to carbostyril and cephalosporin, which are usually used in clinic now. New therapeutic targets and drugs are thus urgently needed to decrease the incidence of shigellosis worldwide. Inhibit bacteria virulence can bring theory evidence to treat shigellosis. The main pathopoiesis steps of Shigella are invade host cell, intercellular spread and release anatoxin. Its virulence are relate to its adhesion, invasiveness, enterotoxin, regulate protein and many other pathopoiesis factors. Among these factors, PhoP-PhoQ is important to regulate Shigella virulence, but its mechanism of regulating Shigella virulence is unknown which need in-depth study.The aim of this study is to explore the mechanism of regulating virulence by two-component signal transduction systems （TCSTS） PhoP-PhoQ in Shigella flexneri. First, the effects of four PhoQ inhibitors which were found by former experiments were detected:the HeLa cell invasion assay and mouse sereny test were used to detect the effects of four PhoQ inhibitors on the virulence of Shigella flexneri. Real-time PCR was used to compare the transcription level of virulence genes of Shigella flexneri which inoculated with or without PhoQ inhibitors. Base on this, the technique of homologous recombination was used to construct the phoP-phoQ gene deletion mutant of Shigella flexneri301. The HeLa cell invasion test and guinea pig sereny test were used to detect the virulence of Sf301and Sf301ΔphoP-phoQ. Then Real-time PCR was used to compare the transcription level of virulence genes between Sf301 and ΔphoP-phoQ stain, investigate the mechanism of virulence which regulated by PhoP-PhoQ in Shigella.Chapter1Bioinformatic analysis of PhoP-PhoQ two-component signal transduction systemsBecause the PhoP-PhoQ TCSTS play an important role in regulating the virulence of Shigella, we analyzed this TCSTS in Shigella based on bioinformatic analysis. The homo logy of phoQ gene in Shigella flexneri2a str.301is more than99%compare to other six Shigella strains.In other Gram negative bacteria, the homology of phoQ is more than76%compare to Escherichia coli or Salmonella enterica subsp. enterica serovar Typhi. PhoQ in Gram negative bacteria is a sensory protein, contains486aa. It encodes transmembrane histidine protein kinases. The homology of amino acid sequence of PhoQ is more than99%in Shigella, more than85%in other Gram negative bacteria. The homology of phoP gene in Shigella flexneri2a str.301is more than98%compare to other six Shigella strains. In other Gram negative bacteria, the homology of phoP is more than80%compare to Escherichia coli or Salmonella enterica subsp. enterica serovar Typhi. In Gram negative bacteria PhoP is a response regulator protein, contains223aa, encodes intracellular response regulator protein. The homology of amino acid sequence of PhoP is more than98%, more than80%in other Gram negative bacteria, lower than32%in Gram positive bacteria.Chapter2The effects of the PhoQ histidine kinase inhibitors on the virulence of Shigella flexneriPhoQ can regulate the virulence of Shigella flexneri, four PhoQ inhibitors which targeting in PhoQ protein were found by former experiments. In this chapter, HeLa cell invasion test and mouse sereny test were used to detect the effects of PhoQ inhibitors on the virulence of Shigella flexneri. First we tested the effects of four PhoQ inhibitors on the growth of Shigella: Sf9380inoculated with PhoQ inhibitors （final concentration100μmol/L）, and count CFU of Sf9380every1h, continue12h. The result suggested that four PhoQ inhibitors have no effects on the growth of Sf9380.The HeLa cell invasion assay was used as a cellular model to evaluate the inhibitory effects of the four potential PhoQ inhibitors:The bacteria were treated with each of the four potential PhoQ inhibitors （100μmo/L） grown for4h,6h,8h. Then the bacteria were inoculated with HeLa cells for60min prior to the addition of gentamicin to kill extracellular bacteria. After the incubation, the Hela cells in each well were lysed in1ml of PBS containing0.1%Triton X-100. The results of the assays were expressed as the number of bacteria recovered from gentamicin-treated cells divided by the number of inoculated bacteria added to the cell. The colonies of lysates containing bacterial cells on LB plates were counted. Result indicated that the PhoQ inhibitors which co-culture with bacterial for4h have no effects on Shigella flexneri invation. PhoQ inhibitors which co-culture with bacterial for6h have no obvious effects on Shigella flexneri invation. PhoQ inhibitors which co-culture with bacterial for8h have obvious effects on Shigella flexneri invation. Based on these results, we used Sf9380which co-culture with bacterial for8h for this research, invitation time is60min. Results shown that the PhoQ inhibitor1（12.5μmo/L）,2（25μmo/L）,3（100μmo/L） could weaken the HeLa cell invasiveness of Sf9380（p<0.05） while inhibitor4has no effects on Shigella flexneri invation. To future test the PhoQ inhibitors effect on bacteria invation, we used Salmonella which has high homogeneity compare to Shigella flexneri. Result shown that inhibitor1（12.5μmo/L）,2（25μmo/L）,3（100μmo/L） could reduced invasion of HeLa cells by Salmonella, the invasion rate are0.89×10-2,1.564×10-2,0.036×10-2, respectively. But inhibitor4could not reduced invasion of HeLa cells by Salmonella, the invasion rate is5.7×10-2. The invasion rate of Salmonella is6×10-2.The mouse keratoconjunctivitis model was used to evaluate the effects of these PhoQ inhibitors on the virulence of Shigella flexneri infection. Keratoconjunctivitis in the mice infected with bacteria was observed at24h,48h,72h and96h after inoculation. Result shown that inhibitor1（12.5μmo/L）,2（25μmo/L） and inhibitor4（100μmol/L） could greatly reduce the keratoconjunctivitis in24h and48h infected by Sf9380and accelerate the recovery of the inflammation, after72h the inflammation were weaken obviously （P<0.05）. Inhibitor3could reduce the inflammation in24h but could not release the inflammation in48h, and also could not accelerate the recovery of the inflammation （P>0.05）.Then we used Real-time PCR to test the PhoQ inhibitors effects on transcription level of Sf9380virulence genes. The results shown that the transpiration level of ipaA, ipaB, ipaC, ipaD, ipaH, mxiA,virB and virF in Sf9380which treated with inhibitor1（12.5μmol/L）,2（25μmol/L） or3（100μmol/L） were down regulated. But the transpiration level of ipaA, ipaB, ipaC, ipaD, ipaH, mxiA, virB and virF in Sf9380which treated with inhibitor4（100μmol/L） were not down regulated. The results suggested that PhoQ inhibitors we obtained could affect the virulence of Shigella and PhoP-PhoQ TCSTS could regulate the virulence of Shigella.Chapter3The effects of the phoP-phoQ on the virulence of Shigella flexneriPhoP-PhoQ TCSTS is a major regulator of virulence in Gram-negative bacteria such as Salmonella and E.coli. It can mediate transcription and expression of some virulence genes. In the research of PhoQ inhibitors, we found that the PhoQ inhibitors could inhibit the invasion of Shigella flexneri infection. At the same time, we also found that the invasion rate of Sf301wide type were low. So the method of continuous passage in sensitive animals was used to recovery the virulence of Sf301wide type. With continuous passage in corneal of guinea pig, the virulence of Sf301were enhance obvious. After48h purulent secretion were found and after72h the corneal of guinea pig were cloudiness and become off-white besides purulence, score were increase from "++" to "+++". Base on this, the biology characteristics of Sf301and Sf301Full virulence stain （Sf301Fv） were detected. Compare to Sf301, the growth curve of Sf301Fv was changed:for Sf301Fv,3-4h was early stage of logarithmic growth phase,5-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9h-12h was plateau phase; for Sf301,1-2h was early stage of logarithmic growth phase;3-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9-12h was plateau phase. By using HeLa cell invasion test, it was found that invasion ability of the Sf301Fv was enhanced compared to Sf301, which from5×10-6to2.6×10-3. Real-time PCR was used to compare the transcription level of virulence genes in early stage, intermediate stage and late stage of logarithmic growth phase. The results shown that the transcription level of cpxA, icsA（virG）, ipaA, ipaB, ipaC, mxiA, phoR, sly A, virB, virF, wzzE and ycfC were up-regulation and envZ, ipaD, ompC, ompF and rep were down regulation. It was suggested that by continuous passage in sensitive animals, Sf301could recovery its virulence by induce some virulence genes. The Sf301Fv we obtained in this chapter could be used to study the effects of phoP-phoQ on regulating virulence in Shigella flexneri.To further verify the effects of PhoP-PhoQ TCSTS on regulating the Shigella flexneri virulence, the vecter pSB890was used to construct a phoP-phoQ knockout stain of Shigella flexneri. Because the transcription level of some virulence genes in Sf301Fv were changed, so sf301ΔphoP-phoQ and Sf301Fv ΔphoP-phoQ were constructed. Two homologous arms were designed base on the genome sequence of Sf301which were published by NCBI （NC₀04337）. Because sf301and sf301Fv are resistance to Tet which pSB890contains, so we should inserted an antibiotic resistant gene into vecter pSB890. After screen the antibiotic, we constructed plasmid pSB890ΔphoP-phoQ which contains two homologous arms of phoP-phoQ. Then an ampicillinum resistant gene（amp） was inserted into pSB890ΔphoP-phoQ and plasmid pSBS90ΔphoP-phoQ-amp was obtained. With conjugation and twice homologous recombinations, two phoP-phoQ knockout strains of Shigella flexneri were obtained. Base on the Sf301ΔphoP-phoQ and Sf301Fv ΔphoP-phoQ we obtained, the growth curve of Sf301,Sf301ΔphoP-phoQ,Sf301Fv and Sf301Fv ΔphoP-phoQ were detected. The growth curve of Sf301Fv and Sf301Fv ΔphoP-phoQ were similar:3-4h was early stage of logarithmic growth phase,5-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9h-12h was plateau phase. The growth curve of Sf301and Sf301ΔphoP-phoQ were similar:1-2h was early stage of logarithmic growth phase;3-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9-12h was plateau phase. Then biochemistry characteristic of these stains were tesed. Results shown that the ARA （Arabinose） of Sf301ΔphoP-phoQ was negative while Sf301was positive which was similar to the change between Sf301Fv and Sf301Fv ΔphoP-phoQ. The results of drug-resistance test were shown that, for Sf301Fv AphoP-phoQ, the sensibility of NAL and STR were increased compare to Sf301Fv, while sensibility of CAZ and CFT were decreased which were similar to the change between Sf301and Sf301ΔphoP-phoQ.By using HeLa cell invasion test, the virulence of Sf301,Sf301ΔphoP-phoQ, Sf301Fv and Sf301Fv ΔphoP-phoQ were detected. The results of these assays were expressed as the number of bacteria recovered from gentamicin-treated cells divided by the number of inoculated bacteria added to the cell. It was found that invasion ability of the Sf301Fv ΔphoP-phoQ was decreased compared to Sf301Fv, from2.6×10-3to4.9×10-5, which was similar to the change between Sf301and Sf301ΔphoP-phoQ. Guinea pig sereny test was used to detected infectivity of Sf301,Sf301 AphoP-phoQ, Sf301Fv and Sf301Fv AphoP-phoQ. Keratoconjunctivitis in the guinea pig infected with bacteria was observed at24h,48h and72h after inoculation. The results of guinea pig sereny test shown that the infectivity of mutant was reduced compared to Sf301Fv, and could not cause inflammation in guinea pig. The score were decrease from "+++" to "-" which was similar to the change between Sf301and Sf301AphoP-phoQ. Real-time PCR was used to compare the transcription level of virulence genes between Sf301and Sf301AphoP-phoQ or Sf301Fv and Sf301Fv AphoP-phoQ in early stage, intermediate stage and late stage of logarithmic growth phase. The results shown that the transcription level of icsA（virG）, ipaA, ipaB, ipaC, ipaD, mxiA, ompC and virB were down regulation and cpxA, ipaH, ompF, ompR,phoB, slyA, sod,B wzzE and ycfC were have no change. These results suggested that phoP-phoQ could regulate some virulence genes to affect the virulence of Shigella.