Identification of Potential Gene Determinants Associated with Mouse Infectivity-Related Immune Modulation in Helicobacter pylori

availability strongly influence bacterial adaptation and colonization (43).Essential growth factors-including dextrose, sodium chloride, and glucose-support survival in the gastric environment (44)(45)(46)(47).As H. pylori divides more slowly than E. coli or the resident gastric microbiota (48), exogenous nutrient supplementation such as Albumin-Dextrose-Saline (ADS) is required, similar to Mycobacterium spp.(49,50).Therefore, ADS was used as a serum substitute to enhance the transformation efficiency of H. pylori.In this study, we compared the gene expression profiles of H. pylori strains Hp52, Hp26695, and HpJ99-which exhibit distinct levels of infectivity in mice-using microarray analysis, and constructed a gene library comprising candidates presumed to contribute to these differences in infectivity.To investigate the immunological roles of these genes during H. pylori -host interactions, we generated individual gene-K/O mutants and analyzed DC immune responses to these mutants in mice.Through this approach, we aimed to identify genes associated with the infectivity-related immune modulation in H. pylori. MATERIALS AND METHODS Culture of H. pyloriAll H. pylori strains were provided by Fastidious Specialized Pathogen Resources Bank of Gyeongsang National University Hospital (Korea).Hp52 was isolated from a gastric cancer patient and possesses infectivity in mice.In contrast, the standard strains used in the experiment, Hp26695 and HpJ99, do not possess infectivity in mice.Half-thawed H. pylori stocks were inoculated onto Brucella agar plates (MP Biomedicals, Korea) supplemented with 10% horse serum (Gibco, USA) and incubated at 37C under a microaerophilic atmosphere containing 10% CO for 48 h.Following primary isolation, the bacteria were subcultured in the growth medium for at least three consecutive passages at 24 h intervals.The cultured medium was reinoculated into 3 mL of fresh medium and incubated for 16 h using the thin-layer liquid culture method (39). Microarray analysis using H. pylori total RNATotal RNA was extracted from cultured H. pylori using the TRIzol TM total RNA extraction kit (ThermoFisher, USA), and microarray analysis was performed (Genomictree Inc., Korea).Total RNA was quantified and amplified using Agilent's Low RNA Input Linear Amplification Kit PLUS (Agilent, USA), and subsequently labeled by the addition of 0.5 L of Cyanine 3-CTP (Cy3) according to the manufacturer's instructions.Hybridization was carried out for 17 h at 65C using Agilent's Gene Expression Hybridization Kit (Agilent, USA).The Cy3-hybridized samples were scanned using the SureScan Microarray Scanner (Agilent, USA), and fluorescence signals were extracted using Agilent's Feature Extraction Software.In this study, Hp52 was used as the reference strain.For each gene, probe recognition was achieved by aligning to a 120 bp region beginning at the N-terminus of the mRNA.Whole-genome clustering of H. pylori genes was performed using Agilent's GeneSpring Software (Agilent, USA).Differential gene expression between Hp52, Hp26695, and HpJ99 was assessed, and genes exhibiting statistically significant changes were identified using an ANOVA t-test. Verification of microarray resultsAll genes that showed significantly different expression in Hp26695 and HpJ99 compared to the Hp52 in the microarray analysis were verified using quantitative real-time RT-PCR (qRT-PCR) .The nucleotide sequences of selected target genes, along with the housekeeping gene (16S rRNA), were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/).Primer sequences were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and PrimerSelect (DNASTAR, USA).Total RNA was extracted from cultured H. pylori using the TRIzol Reagent (Invitrogen, USA).Complementary DNA (cDNA) was synthesized using the GoScript Reverse Transcription System (Promega, USA) with the following thermal cycling conditions: 5 min at 25C, 1 h at 42C, and 15 min at 70C.qRT-PCR was performed