Enhancement of a prime editing system via optimal recruitment of the pioneer transcription factor P65.

Prime editing is a versatile gene editing tool that enables precise sequence changes of all types in the genome, but its application is rather limited by the editing efficiency. Here, we first apply the Suntag system to recruit the transcription factor P65 and enhance the desired editing outcomes in the prime editing system. Next, MS2 hairpins are used to recruit MS2-fused P65 and confirmed that the recruitment of the P65 protein could effectively improve the prime editing efficiency in both the PE3 and PE5 systems. Moreover, this suggests the increased editing efficiency is most likely associated with the induction of chromatin accessibility change by P65. In conclusion, we apply different systems to recruit P65 and enhance the prime editing efficiency of various PE systems. Furthermore, our work provides a variety of methods to work as protein scaffolds for screening target factors and thus supports further optimization of prime editing systems.

Preparation of ethylenediamine-modified pectin/alginate/Fe3O4 microsphere and its efficient Pb2+ adsorption properties.

As a common macromolecular carbohydrate, pectin has a strong affinity for Pb2+. An ethylenediamine modified pectin (EP48) with 48 % of amidation was prepared and exhibited great removal efficiency towards Pb2+ in our previous study. However, the EP48 has drawbacks in adsorption including low mechanical strength and difficulty in separation. In this study, EP48 was compounded with sodium alginate (Alg) and Fe3O4 to synthesize EP48/Alg/Fe3O4 microsphere. The physicochemical properties and Pb2+ adsorption characteristics of microsphere were analyzed. It was found that the microsphere exhibited good thermal stability, mechanical strength, porous structure, as well as acid tolerance. The pseudo-second-order model well described the kinetics of adsorption process, indicating the chemical adsorption is dominant. The Langmuir model fitted the experimental data well, and the maximum adsorption capacity reached 175.19 mg/g. Adsorption-desorption experiments showed that the removal rate of the microsphere maintained over 98.9 % after 10 cycles. The X-ray photoelectron spectroscopy (XPS) analyses revealed that the potential adsorption mechanism included ion-exchange and chelation. The above results suggested its potential use for the removal of Pb2+ from wastewater.

Pseudomonas aeruginosa Polynucleotide Phosphorylase Controls Tolerance to Aminoglycoside Antibiotics by Regulating the MexXY Multidrug Efflux Pump.

Pseudomonas aeruginosa is an opportunistic pathogen that shows high intrinsic resistance to a variety of antibiotics. The MexX-MexY-OprM efflux pump plays an important role in bacterial resistance to aminoglycoside antibiotics. Polynucleotide phosphorylase (PNPase) is a highly conserved exonuclease that plays important roles in RNA processing and the bacterial response to environmental stresses. Previously, we demonstrated that PNPase controls the tolerance to fluoroquinolone antibiotics by influencing the production of pyocin in P. aeruginosa In this study, we found that mutation of the PNPase-encoding gene (pnp) in P. aeruginosa increases bacterial tolerance to aminoglycoside antibiotics. We further demonstrate that the upregulation of the mexXY genes is responsible for the increased tolerance of the pnp mutant. Furthermore, our experimental results revealed that PNPase controls the translation of the armZ mRNA through its 5' untranslated region (UTR). ArmZ had previously been shown to positively regulate the expression of mexXY Therefore, our results revealed a novel role of PNPase in the regulation of armZ and subsequently the MexXY efflux pump.

Endoribonuclease YbeY Is Essential for RNA Processing and Virulence in Pseudomonas aeruginosa.

Posttranscriptional regulation plays an essential role in the quick adaptation of pathogenic bacteria to host environments, and RNases play key roles in this process by modifying small RNAs and mRNAs. We find that the Pseudomonas aeruginosa endonuclease YbeY is required for rRNA processing and the bacterial virulence in a murine acute pneumonia model. Transcriptomic analyses reveal that knocking out the ybeY gene results in downregulation of oxidative stress response genes, including the catalase genes katA and katB Consistently, the ybeY mutant is more susceptible to H2O2 and neutrophil-mediated killing. Overexpression of katA restores the bacterial tolerance to H2O2 and neutrophil killing as well as virulence. We further find that the downregulation of the oxidative stress response genes is due to defective expression of the stationary-phase sigma factor RpoS. We demonstrate an autoregulatory mechanism of RpoS and find that ybeY mutation increases the level of a small RNA, ReaL, which directly represses the translation of rpoS through the 5' UTR of its mRNA and subsequently reduces the expression of the oxidative stress response genes. In vitro assays demonstrate direct degradation of ReaL by YbeY. Deletion of reaL or overexpression of rpoS in the ybeY mutant restores the bacterial tolerance to oxidative stress and the virulence. We also demonstrate that YbeZ binds to YbeY and is involved in the 16S rRNA processing and regulation of reaL and rpoS as well as the bacterial virulence. Overall, our results reveal pleiotropic roles of YbeY and the YbeY-mediated regulation of rpoS through ReaL.IMPORTANCE The increasing bacterial antibiotic resistance imposes a severe threat to human health. For the development of effective treatment and prevention strategies, it is critical to understand the mechanisms employed by bacteria to grow in the human body. Posttranscriptional regulation plays an important role in bacterial adaptation to environmental changes. RNases and small RNAs are key players in this regulation. In this study, we demonstrate critical roles of the RNase YbeY in the virulence of the pathogenic bacterium Pseudomonas aeruginosa We further identify the small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated pathway on the expression of bacterial virulence factors. Our results shed light on the complex regulatory network of P. aeruginosa and indicate that inference with the YbeY-mediated regulatory pathway might be a valid strategy for the development of a novel treatment strategy.

Pseudomonas aeruginosa Polynucleotide Phosphorylase Contributes to Ciprofloxacin Resistance by Regulating PrtR.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes various acute and chronic infections. It is intrinsically resistant to a variety of antibiotics. However, production of pyocins during SOS response sensitizes P. aeruginosa to quinolone antibiotics by inducing cell lysis. The polynucleotide phosphorylase (PNPase) is a conserved phosphate-dependent 3'-5' exonuclease that plays an important role in bacterial response to environmental stresses and pathogenesis by influencing mRNA and small RNA stabilities. Previously, we demonstrated that PNPase controls the type III and type VI secretion systems in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) increases the bacterial resistance to ciprofloxacin. Gene expression analyses revealed that the expression of pyocin biosynthesis genes is decreased in the pnp mutant. PrtR, a negative regulator of pyocin biosynthesis genes, is upregulated in the pnp mutant. We further demonstrated that PNPase represses the expression of PrtR on the post-transcriptional level. A fragment containing 43 nucleotides of the 5' untranslated region was found to be involved in the PNPase mediated regulation of PrtR. Overall, our results reveled a novel layer of regulation on the pyocin biosynthesis by the PNPase in P. aeruginosa.

Identification of a small RNA that directly controls the translation of the quorum sensing signal synthase gene rhlI in Pseudomonas aeruginosa.

The biofilm formation by Pseudomonas aeruginosa highly increases the bacterial resistance to antimicrobial agents and host immune clearance. The biofilm formation is positively regulated by two small RNAs, RsmY and RsmZ. Previously, we reported that mutation in the polynucleotide phosphorylase (PNPase) coding gene pnp increases the levels of RsmY/Z. However, in this study, we found that the biofilm formation is decreased in the pnp mutant, which is due to a defect in rhamnolipids production. The rhamnolipids production is regulated by the RhlI-RhlR quorum sensing system. We found that PNPase influences the translation of RhlI through its 5'-untranslated region (UTR) and identified that the sRNA P27 is responsible for the translational repression. In vitro translation experiments demonstrated that P27 directly represses the translation of the rhlI mRNA through its 5'UTR in an Hfq-dependent manner. Point mutations in the rhlI 5'UTR or P27, which abolish the pairing between the two RNAs restore the rhlI expression and rhamnolipids production as well as the biofilm formation in the pnp mutant. Overall, our results reveal a novel layer of regulation of the Rhl quorum sensing system by the sRNA P27.

Effect of simulation education and case management on glycemic control in type 2 diabetes.

BACKGROUND: The aim of the study was to investigate whether simulation education (SE) and case management had any effect on glycemic control in type 2 diabetes (T2DM) patients. METHODS: In this single center pilot trial, 100 T2DM patients who received medication and basic diabetes self-management education (DSME) were randomly divided into a control group (n = 50) and an experimental group (n = 50), who received SE and a case management program. Evaluation of biochemical indices was conducted at baseline and after 6 months. DSME consisted of 2-hour group trainings weekly for 2 consecutive weeks followed by 2 × 30 minute education sessions after 3 and 6 months. The SE program comprised additional 50-minute video sessions 3 times in the first week and twice in the second week. The experimental group was supervised by a nurse case manager, who followed up participants at least once a month, and who conducted group sessions once every 3 months, focusing on realistic aspects of physical activity and nutrition, with open discussions about setting goals and strategies to overcome barriers. RESULTS: After 6 months, HbA1c, fasting plasma glucose, and postprandial blood glucose level improvements were superior in the experimental group compared with the control group (P < 0.05). Self-care behavior adherence scores of healthy diet (P = 0.001), physical activity (P = 0.043), self-monitoring of blood glucose (P < 0.001), and reducing risks (P < 0.001) were significantly increased in the experimental group compared with the control group. CONCLUSIONS: Simulation education and case management added to routine DSME effectively improved glycemic control in T2DM patients.

Antimicrobial effect of black pepper petroleum ether extract for the morphology of Listeria monocytogenes and Salmonella typhimurium.

This study aimed to evaluate the effects of black pepper petroleum extract (BPPE) on pathogenic bacteria. The extraction from black pepper showed intense antimicrobial activity against the Gram-positive Listeria monocytogenes ATCC 19115 and the Gram-negative bacteria Salmonella typhimurium ATCC 14028. The minimum inhibitory concentrations of BPPE against L. monocytogenes and S. typhimurium were 0.625 and 1.25 mg/ml, respectively. Detection of Alkaline phosphatase outside the cell revealed that BPPE treatment destroyed the cell wall integrity. BPPE also altered the membrane integrity, thereby causing leaching of 260 and 280 nm UV-absorbing materials into the medium, particularly, nucleic acids and proteins. Propidium iodide infiltration experiments also indicated that BPPE treatment altered the permeability of bacterial cell membrane. Moreover, Na+/K+-ATPase activity was inhibited by BPPE. And the results of scanning electron microscopy showed that BPPE treatment damaged the morphology of the tested bacteria. These results indicated that BPPE could destroy cell wall integrity, alter the permeability of cell membrane, and inhibit the activity of intracellular enzyme, which could kill bacteria.

Pseudomonas aeruginosa Enolase Influences Bacterial Tolerance to Oxidative Stresses and Virulence.

Pseudomonas aeruginosa is a Gram negative opportunistic pathogenic bacterium, which causes acute and chronic infections. Upon entering the host, bacteria alter global gene expression to adapt to host environment and avoid clearance by the host. Enolase is a glycolytic enzyme involved in carbon metabolism. It is also a component of RNA degradosome, which is involved in RNA processing and gene regulation. Here, we report that enolase is required for the virulence of P. aeruginosa in a murine acute pneumonia model. Mutation of enolase coding gene (eno) increased bacterial susceptibility to neutrophil mediated killing, which is due to reduced tolerance to oxidative stress. Catalases and alkyl hydroperoxide reductases play a major role in protecting the cell from oxidative damages. In the eno mutant, the expression levels of catalases (KatA and KatB) were similar as those in the wild type strain in the presence of H2O2, however, the expression levels of alkyl hydroperoxide reductases (AhpB and AhpC) were significantly reduced. Overexpression of ahpB but not ahpC in the eno mutant fully restored the bacterial resistance to H2O2 as well as neutrophil mediated killing, and partially restored bacterial virulence in the murine acute pneumonia model. Therefore, we have identified a novel role of enolase in the virulence of P. aeruginosa.

HigB of Pseudomonas aeruginosa Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells.

Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA::Tn mutant revealed up regulation of type III secretion systems (T3SS) genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells.

DeaD contributes to Pseudomonas aeruginosa virulence in a mouse acute pneumonia model.

DExD/H box RNA helicases play essential roles in various biological processes in prokaryotes and eukaryotes. By screening Pseudomonas aeruginosa strains with mutations in various DExD/H box helicase genes, we identified that deaD was required for bacterial cytotoxicity and virulence in a mouse acute pneumonia model. Compared to a wild-type strain and its complementation strain, the deaD mutant induced less production of proinflammatory cytokines, neutrophil infiltration and lung damage during infection. We further found that the RNA helicase activity of DeaD was required for the expression of type III secretion system (T3SS) genes. Overexpression of ExsA, a master activator of the T3SS, restored the expression of T3SS genes as well as the virulence of the deaD mutant, suggesting that the attenuated virulence of the deaD mutant was mainly due to the defective T3SS. Overall, our results reveal a role of DeaD in the virulence of P. aeruginosa.

Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa.

Oligoribonuclease (Orn) is a 3' to 5' exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn's substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the Δorn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the Δorn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the Δorn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa.

PA3297 Counteracts Antimicrobial Effects of Azithromycin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes acute and chronic infections in human. Its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Among the alternatives is the unconventional usage of conventional antibiotics, of which the macrolide antibiotic azithromycin (AZM) provides a paradigmatic example. AZM therapy is associated with a small but consistent improvement in respiratory function of cystic fibrosis patients suffering from chronic P. aeruginosa infection. Besides immunomodulating activities, AZM represses bacterial genes involved in virulence, quorum sensing, biofilm formation, and motility, all of which are due to stalling of ribosome and depletion of cellular tRNA pool. However, how P. aeruginosa responds to and counteracts the effects of AZM remain elusive. Here, we found that deficiency of PA3297, a gene encoding a DEAH-box helicase, intensified AZM-mediated bacterial killing, suppression of pyocyanin production and swarming motility, and hypersusceptibility to hydrogen peroxide. We demonstrated that expression of PA3297 is induced by the interaction between AZM and ribosome. Importantly, mutation of PA3297 resulted in elevated levels of unprocessed 23S-5S rRNA in the presence of AZM, which might lead to increased susceptibility to AZM-mediated effects. Our results revealed one of the bacterial responses in counteracting the detrimental effects of AZM.

Polynucleotide Phosphorylase Regulates Multiple Virulence Factors and the Stabilities of Small RNAs RsmY/Z in Pseudomonas aeruginosa.

Post-transcriptional regulation enables bacteria to quickly response to environmental stresses. Polynucleotide phosphorylase (PNPase), which contains an N-terminal catalytic core and C-terminal RNA binding KH-S1 domains, is involved in RNA processing. Here we demonstrate that in Pseudomonas aeruginosa the KH-S1 domains of PNPase are required for the type III secretion system (T3SS) and bacterial virulence. Transcriptome analysis revealed a pleiotropic role of PNPase in gene regulation. Particularly, the RNA level of exsA was decreased in the ΔKH-S1 mutant, which was responsible for the reduced T3SS expression. Meanwhile, the pilus biosynthesis genes were down regulated and the type VI secretion system (T6SS) genes were up regulated in the ΔKH-S1 mutant, which were caused by increased levels of small RNAs, RsmY, and RsmZ. Further studies revealed that deletion of the KH-S1 domains did not affect the transcription of RsmY/Z, but increased their stabilities. An in vivo pull-down and in vitro electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction between RsmY/Z and the KH-S1 fragment. Overall, this study reveals the roles of PNPase in the regulation of virulence factors and stabilities of small RNAs in P. aeruginosa.

PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosa that are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtR results in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tac promoter repressed the endogenous prtR promoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

SuhB is a regulator of multiple virulence genes and essential for pathogenesis of Pseudomonas aeruginosa.

UNLABELLED: During initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa. IMPORTANCE: A variety of bacterial pathogens, such as Pseudomonas aeruginosa, cause acute and chronic infections in humans. During infections, pathogens produce different sets of virulence genes for colonization, tissue damage, and dissemination and for countering host immune responses. Complex regulatory networks control the delicate tuning of gene expression in response to host environments to enable the survival and growth of invading pathogens. Here we identified suhB as a critical gene for the regulation of virulence factors in P. aeruginosa. The expression of suhB was upregulated during acute infection in an animal model, and mutation of suhB rendered P. aeruginosa avirulent. Moreover, we demonstrate that SuhB is required for the activation of virulence factors associated with acute infections while suppressing virulence factors associated with chronic infections. Our report provides new insights into the multilayered regulatory network of virulence genes in P. aeruginosa.