Identification of an endonuclease and N6-adenine methyltransferase from Ureaplasma parvum SV3F4 strain.

Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5' extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.

Prenatal exposure to intra-amniotic infection with Ureaplasma species increases the prevalence of bronchopulmonary dysplasia.

OBJECTIVES: The present study investigated the relationship between bronchopulmonary dysplasia (BPD) and intra-amniotic infection with Ureaplasma species. METHODS: This was a single-center, retrospective cohort study. Patients with singleton pregnancies who underwent inpatient management at our department for preterm premature rupture of membranes (PPROM), preterm labor, cervical insufficiency, and asymptomatic cervical shortening at 22-33 gestational weeks were included. Amniocentesis was indicated for patients with PPROM or an elevated maternal C-reactive protein level (≥0.58 mg/dL). Patients with an amniotic fluid IL-6 concentration ≥3.0 ng/mL were diagnosed with intra-amniotic inflammation, while those with positive aerobic, anaerobic, M. hominis, and Ureaplasma spp. cultures were diagnosed with microbial invasion of the amniotic cavity (MIAC). Patients who tested positive for both intra-amniotic inflammation and MIAC were considered to have intra-amniotic infection. An umbilical vein blood IL-6 concentration >11.0 pg/mL indicated fetal inflammatory response syndrome (FIRS). The maternal inflammatory response (MIR) and fetal inflammatory response (FIR) were staged using the Amsterdam Placental Workshop Group Consensus Statement. RESULTS: Intra-amniotic infection with Ureaplasma spp. was diagnosed in 37 patients, intra-amniotic infection without Ureaplasma spp. in 28, intra-amniotic inflammation without MIAC in 58, and preterm birth without MIR/FIR and FIRS in 86 as controls. Following an adjustment for gestational age at birth, the risk of BPD was increased in patients with intra-amniotic infection with Ureaplasma spp. (adjusted odds ratio: 10.5; 95% confidence interval: 1.55-71.2), but not in those with intra-amniotic infection without Ureaplasma spp. or intra-amniotic inflammation without MIAC. CONCLUSION: BPD was only associated with intra-amniotic infection with Ureaplasma species.