ABSTRACT
<p><b>OBJECTIVE</b>Cytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.</p><p><b>METHODS</b>Akt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.</p><p><b>RESULTS</b>CagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.</p><p><b>CONCLUSIONS</b>CagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.</p>
Subject(s)
Humans , Antigens, Bacterial , Genetics , Physiology , Bacterial Proteins , Genetics , Physiology , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Gastric Mucosa , Cell Biology , Microbiology , Helicobacter pylori , Metabolism , Virulence , Physiology , Interleukin-8 , Bodily Secretions , Intracellular Signaling Peptides and Proteins , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transcription, Genetic , VirulenceABSTRACT
Objective To analyze the predominant genotypes of outer membrane porin I (PI)and to determine the correlation between G120 as well as A121 mutations in PI proteins and drug resistance in Neisseria gonorrhoeae isolates in the local area.Methods A double PCR to simultaneously detect both pIA and pIB genes,was established in this study.The target amplification products were T-A cloned and then sequenced to determine the mutations at G120,A121 and the specificity of double PCR.By using acidity slip method and double agar dilution method,the β-lactamase production and resistance to six antibiotics of pIA~+ and pIB~+ gonoeoeeal isolates were detected.Results Double PCR could be used to accurately genotyping pI genes in all the tested gonococcal isolates with the sensitivity of 1 ng DNA template.In the 116 N.gonorrhoeae isolates,30.2%(35/116) were pIA~+ strains and 69.8%(81/136) were pIB~+ strains.All the pIA~+ strains presented G120D/A121G double mutations (88.6%) or A121G single mutation (11.4%).98.8% of the pIB +strains presented G120K/A121D (65.0%),G120K/A121G or G120N/A121D ( 13.8% ) double mutations,and G120D/N/K single mutation (21.3% ).34.5% (40/116) of the isolates produced β-lactamase,and the enzyme-produced rate (20%) in pIA~+ strains was significantly lower than that in pIB~+ strains (40.7%) with P＜0.05.No spectinomycin-resistant swains were identified but three ceftriaxoneresistant strains were presented.However,the resistance ratios to penicilin,terramycin,ciprofloxacin and azithromycin of all the isolates were as high as 75.0%-90.5%.100% and 71.4% of the pIA~+strains without β-lactamase production and with G120 and/or A121 mutations were sensitive topenicillin and terramycin,respectively.On the contrast,100% of the pIB~+ strains without β-1actamase production and with G120 and/or A121 mutations were resistant to both the two antibiotics.Conclusion The established double PCR method could be used for fast and accurate genotyping of N.gonorrhoeae pI genes.The N.gonorrhoeae strains prevalent in the local areas mainly possessed pIB gene.Both spectinomycin and ceftriaxone could still be chosen to treat gonorrhea.The resistance enhancement caused by G120 and/or A121 mutations to penicillin and tetramycin was only presented in pIB~+ gonococci.