Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

BACKGROUND: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility. METHODS: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His)6-tagged uvsY. RESULTS: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY. CONCLUSIONS: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.

A phase II, multicenter, nonblinded, randomized controlled trial for evaluating protective effects of ABPC/SBT plus, azithromycin versus erythromycin, in pregnant women with pPROM occurring at <28 weeks of gestation on the development of BPD in neonates: Study protocol.

This is a protocol for PPROM-AZM Study, phase II, nonblinded, randomized controlled trial. Bronchopulmonary dysplasia (BPD) at a postmenstrual age of 36 weeks (BPD36) is often observed in infants with preterm premature rupture of the membranes (pPROM). A regimen of ampicillin (ABPC) intravenous infusion for 2 days and subsequent amoxicillin (AMPC) oral administration for 5 days plus erythromycin (EM) intravenous infusion for 2 days followed by EM oral administration for 5 days is standard treatment for pPROM. However, the effect on the prevention of moderate/severe BPD36 using the standard treatment has not been confirmed. Recently, it is reported that ampicillin/sulbactam (ABPC/SBT) plus azithromycin (AZM) was effective for the prevention of moderate/severe BPD36 in pPROM patients with amniotic infection of Ureaplasma species. Therefore, our aim is to evaluate the occurrence rate of the composite outcome of "incidence rate of either moderate/severe BPD36 or intrauterine fetal death, and infantile death at or less than 36 weeks 0 days" comparing subjects to receive ABPC/SBT for 14 days plus AZM for 14 days (intervention group) and those to receive ABPC/SBT for 14 days plus EM for 14 days (control group), in a total of 100 subjects (women with pPROM occurring at 22-27 weeks of gestation) in Japan. The recruit of subjects was started on April 2022, and collection in on-going. We also investigate the association between the detection of Ureaplasma species and occurrence of BPD36. In addition, information on any adverse events for the mother and fetus and serious adverse events for infants are collected during the observation period. We allocate patients at a rate of 1:1 considering two stratification factors: onset of pPROM (22-23 or 24-27 weeks) and presence/absence of a hospital policy for early neonatal administration of caffeine. Trial registration: The trial number in the Japan Registry of Clinical Trials is jRCTs031210631.

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.

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.

Expression of a recombinant protein by an acetic acid bacterial host.

We evaluated the suitability of Komagataeibacter europaeus, a vinegar production organism adept at synthetic media growth, as a host for heterologous gene expression. Cryptic plasmids (pGE1 and pGE2 derivatives) from K. europaeus strain KGMA0119 were employed as vectors for heterologous gene expression. The focus was placed on the groES promoter as a potential inducible switch. The groES promoter was fused with the EGFP gene and introduced into a pGE1 derivative to assess its suitability. Ethanol, acetic acid, and heat stresses were examined under various conditions for induction. EGFP transcription surged 600-fold when late logarithmic phase K. europaeus cells, cultured at 30 °C, endured heat stress at 40 °C, coupled with 20% acetic acid and 30% ethanol stress after an additional 6-hour cultivation. This robust induction system was then applied to express two proteins, Tth pol from the thermophilic bacterium Thermus thermophilus strain M1 and UPV230, a restriction enzyme from the acid-tolerant microorganism Ureaplasma parvum, known to cause vaginal infections and miscarriages. Both Tth pol and UPV230 were successfully expressed in K. europaeus cells and purified. The recovery of Tth pol from K. europaeus cells (480 µg protein per liter culture) was approximately half that from E. coli (960 µg protein per liter culture). In contrast, UPV230 recovery from K. europaeus cells (640 µg protein per liter culture) was nearly 10 times higher than that from Escherichia coli (66 µg protein per liter). The data highlights the potential of acetic acid bacteria as a host for producing acidophilic proteins. The shift in recognition from a 6-base sequence to a 4-base sequence of UPV230 was observed, accompanied by a change in structure as the pH transitioned from acidic pH to near-neutral pH.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.