Gene polA as a Suitable Reference for Studying Antibacterial Effect of Hydroxyapatite.
Hydroxyapatite (HAP: Ca10(PO4)6(OH)2) is a widely used biocompatible material; however, information on the reaction mechanism between HAP and microorganisms is insufficient. This study aimed to identify a stable reference gene for studying the antibacterial activity of HAP. The half maximal inhibitory concentration (C50) and gene expression of a human symbiotic Escherichia coli strain TOP10, was investigated at various concentrations of HAP. Our uniformly sized HAP nanoparticles (HANPs) had a high surface area and an estimated IC50 of 75 mg/mL. The expressions of genes, including those of DNA polymerase I (poIA), DNA polymerase II (poIB), cytochrome d complex (cyd), glucan biosynthesis protein G (mdoG), D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 16S ribosomal RNA (16S rRNA), were analyzed by performing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and using reported and newly designed primers. The changes in the copy numbers compared to non-HANPs-treated conditions for polB, cyd, mdoG, GAPDH, and 16S rRNA were 50%-381%, 0.7%-66.3%, 1.1%-7.8%, 1.6%-86.3%, and 0.3%-8.1%, respectively. The expressions of polA remained stable under all conditions (62.8%-88.4%). Therefore, we identified polA as a suitable reference gene. Moreover, the expressions of cyd and mdoG were inhibited, indicating that the antibacterial activity of HANPs is related to cell membrane or cell wall proteins. Our findings provide a thorough understanding of HAP-microorganism interactions for potential biomedical applications.
Yu-Tzu
YT
Huang
Yusuke
Y
Yamauchi
Ankit
A
Rawat
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004256:DNA Polymerase I / Q000235:genetics*; D017886:Durapatite / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000592:standards; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000592:standards; D053758:Nanoparticles; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards
D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D012336:RNA, Ribosomal, 16S; C074431:polB protein, E coli; D017886:Durapatite; D004256:DNA Polymerase I