Uncovering the GacS-mediated role in evolutionary progression through trajectory reconstruction in Pseudomonas aeruginosa.

The genetic diversities of subpopulations drive the evolution of pathogens and affect their ability to infect hosts and cause diseases. However, most studies to date have focused on the identification and characterization of adaptive mutations in single colonies, which do not accurately reflect the phenotypes of an entire population. Here, to identify the composition of variant subpopulations within a pathogen population, we developed a streamlined approach that combines high-throughput sequencing of the entire population cells with genotyping of single colonies. Using this method, we reconstructed a detailed quorum-sensing (QS) evolutionary trajectory in Pseudomonas aeruginosa. Our results revealed a new adaptive mutation in the gacS gene, which codes for a histidine kinase sensor of a two-component system (TCS), during QS evolution. This mutation reduced QS activity, allowing the variant to sweep throughout the whole population, while still being vulnerable to invasion by the emerging QS master regulator LasR-null mutants. By tracking the evolutionary trajectory, we found that mutations in gacS facilitated QS-rewiring in the LasR-null mutant. This rapid QS revertant caused by inactive GacS was found to be associated with the promotion of ribosome biogenesis and accompanied by a trade-off of reduced bacterial virulence on host cells. In conclusion, our findings highlight the crucial role of the global regulator GacS in modulating the progression of QS evolution and the virulence of the pathogen population.

New Evolutionary Insights into RpoA: A Novel Quorum Sensing Reprograming Factor in Pseudomonas aeruginosa.

Quorum-sensing (QS) coordinates the expression of virulence factors in Pseudomonas aeruginosa, an opportunistic pathogen known for causing severe infections in immunocompromised patients. QS has a master regulator, the lasR gene, but in clinical settings, P. aeruginosa isolates have been found that are QS-active but LasR-null. In this study, we developed an experimental evolutionary approach to identify additional QS-reprogramming determinants. We began the study with a LasR-null mutant with an extra copy of mexT, a transcriptional regulator gene that is known to be able to reprogram QS in laboratory LasR-null strains. In this strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. Using this novel method, which we have named "targeted gene duplication followed by mutant screening", we identified QS-active revertants with mutations in genes other than mexT. One QS-active revertant had a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the downregulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Our results indicate that a RpoA-dependent regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

Novel antibiotic susceptibility of an RNA polymerase α-subunit mutant in Pseudomonas aeruginosa.

BACKGROUND: RNA polymerase (RNAP) is highly conserved and essential for prokaryotic housekeeping activities, making it an important target for the development of new antibiotics. The rpoB gene, encoding a ß-subunit of bacterial RNAP, has a well-known association with rifampicin resistance. However, the roles of other RNAP component genes such as rpoA, encoding an α-subunit of RNAP, in antibiotic resistance remain unexplored. OBJECTIVES: To characterize the antibiotic resistance-related role of RpoA. METHODS: We measured the expression of the MexEF-OprN efflux pump in an RpoA mutant using a transcriptional reporter. The MICs of various antibiotics for this RpoA mutant were determined. RESULTS: We uncover a novel role of antibiotic susceptibility for an RpoA mutant in Pseudomonas aeruginosa. We found that a single amino acid substitution in RpoA resulted in reduced activity of the MexEF-OprN efflux pump, which is responsible for the exportation of various antibiotics, including ciprofloxacin, chloramphenicol, ofloxacin and norfloxacin. This attenuated efflux pump activity, caused by the RpoA mutation, conferred the bacteria further susceptibility to antibiotics regulated by MexEF-OprN. Our work further revealed that certain clinical P. aeruginosa isolates also contained the same RpoA mutation, providing clinical relevance to our findings. Our results elucidate why this new antibiotic-susceptible function of RpoA mutants would have remained undetected in conventional screens for mutants involving antibiotic resistance. CONCLUSIONS: The discovery of antibiotic susceptibility in an RpoA mutant implicates a new therapeutic approach for treating clinical isolates of P. aeruginosa with RpoA mutations, using specific antibiotics regulated by MexEF-OprN. More generally, our work suggests that RpoA could serve as a promising candidate target for anti-pathogen therapeutic purposes.

Improved probe detection sensitivity by flexible tandem biotin labeling.

We develop a biotin-based tandem labeling approach to improve detection sensitivity of DNA probe. Through DNA polymerase-mediated overhand filling, the 3'end of DNA probe was tandemly labeled with biotin molecules. The intensity of biotin signals could be flexibly manipulated by controlling the introduced length of poly(A) in the 5' overhang.

Modifying Escherichia coli to mimic Shigella for medical microbiology laboratory teaching: a new strategy to improve biosafety in class.

Introduction: Laboratory teaching of medical microbiology involves highly pathogenic microorganisms, thus posing potential biosafety risks to the students and the teacher. To address these risks, non/low-pathogenic microorganisms were modified to mimic highly pathogenic ones or highly pathogenic microorganisms were attenuated directly using the CRISPR/Cas9 technology. This study describes the modification of Escherichia coli DH5α to mimic Shigella and its evaluation as a safe alternative for medical laboratory teaching. Methods: To generate E. coli DH5α△FliC△tnaA2a, the tnaA and FliC genes in E. coli DH5α were knocked out using CRISPR/Cas9 technology; a plasmid bearing the O-antigen determinant of S. flexneri 2a was then constructed and transformed. Acid tolerance assays and guinea pig eye tests were used to assess the viability and pathogenicity, respectively. Questionnaires were used to analyze teaching effectiveness and the opinions of teachers and students. Results: The survey revealed that most teachers and students were inclined towards real-time laboratory classes than virtual classes or observation of plastic specimens. However, many students did not abide by the safety regulations, and most encountered potential biosafety hazards in the laboratory. E. coli DH5α△FliC△tnaA2a was biochemically and antigenically analogous to S. flexneri 2a and had lower resistance to acid than E. coli. There was no toxicity observed in guinea pigs. Most of teachers and students were unable to distinguish E. coli DH5α△FliC△tnaA2a from pure S. flexneri 2a in class. Students who used E. coli DH5α△FliC△tnaA2a in their practice had similar performance in simulated examinations compared to students who used real S. flexneri 2a, but significantly higher than the virtual experimental group. Discussion: This approach can be applied to other high-risk pathogenic microorganisms to reduce the potential biosafety risks in medical laboratory-based teaching and provide a new strategy for the development of experimental materials.

Spontaneous quorum-sensing hierarchy reprogramming in Pseudomonas aeruginosa laboratory strain PAO1.

Pseudomonas aeruginosa strain PAO1 has been commonly used in the laboratory, with frequent genome variations reported. Quorum sensing (QS), a cell-cell communication system, plays important role in controlling a variety of virulence factors. However, the evolution and adaptability of QS in those laboratory strains are still poorly understood. Here we used the QS reporter and whole-genome sequencing (WGS) to systematically investigate the QS phenotypes and corresponding genetic basis in collected laboratory PAO1 strains. We found that the PAO1-z strain has an inactive LasR protein, while bearing an active Rhl QS system and exhibiting QS-controlled protease-positive activity. Our study revealed that an 18-bp insertion in mexT gene gave rise to the active QS system in the PAO1-z strain. This MexT inactivation restored the QS activity caused by the inactive LasR, showing elevated production of pyocyanin, cyanide and elastase. Our results implied the evolutionary trajectory for the PAO1-z strain, with the evulutionary order from the first Las QS inactivation to the final Rhl QS activation. Our findings point out that QS homeostasis occurs in the laboratory P. aeruginosa strain, offering a potential platform for the QS study in clinical isolates.

Efficacy and safety of alprostadil cream for the treatment of female sexual arousal disorder: a double-blind, placebo-controlled study in chinese population.

INTRODUCTION: To date, no approved medication is available for the treatment of female sexual arousal disorder (FSAD). AIM: The purpose of this study was to evaluate the clinical efficacy and safety of a novel alprostadil topical cream for the treatment of FSAD. METHODS: This was a multicenter, randomized, double blind, placebo-controlled, parallel design dose-ranging study. Four hundred female patients with FSAD (22-62 years of age), after a 4-week nontreatment baseline period, were provided with 10 blinded doses of 500, 700, or 900 mcg alprostadil or a placebo cream to be applied to the clitoris and the G-spot in the vagina prior to vaginal intercourse. MAIN OUTCOME MEASURES: The primary efficacy end point was the arousal success rate (equal number of the Yes responses to Question 3 of the Female Sexual Encounter Profile [FSEP] or number of the sexual encounters). Secondary endpoints included the Female Sexual Function Index (FSFI), Global Assessment Questionnaire, other FSEP question responses, and post-treatment changes in Female Sexual Distress Scale. RESULTS: A total of 374 FSAD patients completed the study. Primary efficacy analysis of the intent-to-treat (ITT) population showed a significant increase in arousal success rates with dose. Arousal success rates at the end of the total evaluation period were 33.1%, 46.3% (P = 0.0161), 43.5% (P = 0.0400), and 53.9% (P = 0.0002) in the placebo, 500, 700, and 900 mcg alprostadil groups, respectively. The changes of the FSFI score, relative to baseline were 14.7%, 20.7% (P = 0.067), 21.7% (P = 0.035), and 22.9% (P = 0.002) for the placebo, 500, 700, and 900 mcg treatment groups, respectively. The other secondary efficacy end point values showed a consistent trend in support of the primary efficacy results. CONCLUSION: These results demonstrated that the application of topical alprostadil prior to vaginal intercourse significantly improved the sexual arousal rate of the subjects with FSAD.

Effect of zinc on biochemical parameters and changes in related gene expression assessed by cDNA microarrays in pituitary of growing rats.

OBJECTIVE: The present study simultaneously investigated the effects of different zinc (Zn) levels on the growth performance and relative biochemical parameters in growing rats and analyzed the molecular mechanism of zinc influencing food intake. METHODS: Three diets with different Zn levels--Zn adequate (ZA; 35.94 mg/kg, control), Zn deficient (ZD; 3.15 mg/kg), and Zn overdose (ZO; 347.50 mg/kg)--were fed to rats for 6 wk. Dietary Zn was supplemented with ZnSO4. The relation between zinc and food intake was studied by pituitary cDNA microarrays. RESULTS: Compared with ZA group, rats fed the ZD diet showed decreases in body weight (P < 0.01), food intake (P < 0.05), tissue zinc concentrations (P < 0.01), and specific activities of alkaline phosphatase (P < 0.01) and copper/Zn superoxide dismutase (P < 0.05), whereas the ZO diet had positive effects on body weight (P < 0.05), zinc concentrations (P < 0.01), and alkaline phosphatase activity (P < 0.05). The villi of the jejunum became shorter (P < 0.01), shriveled, and flattened. This change in morphology decreased absorption surface area, and there was a substantial decrease (P < 0.01) in villi number per unit area in ZD rats. Metallothionein concentration was increased in livers of rats fed ZD (P < 0.01) and ZO (P < 0.05) diets. Moreover, ZD and ZO influenced normal growth and development of organs. The results from pituitary cDNA arrays indicated that different Zn levels affect gene expression of appetite-related peptides, including neuropeptide-Y, melanin-concentrating hormone, ghrelin, calcitonin gene-related product, and serotonin. CONCLUSION: The present results showed that zinc deficiency has a negative effect on the growth performance and biochemical parameters of rats. The ZO diet increased body weight (P < 0.05) but had no effect (P > 0.05) on food intake, copper/Zn superoxide dismutase activity, and intestinal morphology. The ZD diet decreased rat food intake by regulating appetite-related gene expression in the pituitary gland.

[The therapeutic efficacy for ED patients treated with low dosage of PGE1].

OBJECTIVES: To evaluate the efficacy of intrameatal application of low dosage alprostadil (PGE1) cream (300 mcg) for the treatment of erectile dysfunction (ED). METHODS: A total of 43 ED patients were selected in the study based on the inclusion criteria. All of the patients signed informed consent forms and entered a 4-week open-label clinical study. A dosage of 300 mg PGE1 in 75 mg cream was applied intrameatally. RESULTS: The results showed that the primary efficacy (IIEF Q3 + Q4) reached 70.73% after application of the cream. The successful intercourse rate was 86.41%. Based on the GAQ (global assessment Question); 73.17% of the patients were satisfied with their sexual life. At the same time, all of the secondary criteria supported the primary efficacy results. Two patients withdrew during the study period. Six patients (14.63%) had urethral pain or penile redness, which were mostly mild and transient. CONCLUSIONS: With intrameatal low dosage (300 mcg PGE1) of the PGE1 cream can achieve an equivalent efficacy as that with the full dosage.