Elucidating the mechanism by which synthetic helper peptides sensitize Pseudomonas aeruginosa to multiple antibiotics

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reduce ATP production and inhibit the activity of efflux pumps), impairs the respiration chain, promote the production of reactive oxygen species (ROS) in bacterial cells and induce intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa

Scutellarin Ameliorates Renal Injury via Increasing CCN1 Expression and Suppressing NLRP3 Inflammasome Activation in Hyperuricemic Mice

Considerable evidences have indicated that elevated uric acid (UA) was involved in renal tubular injury leading to hyperuricemic nephropathy (HN). Scutellarin is a biologically active flavonoid derived from the Chinese traditional herb Erigeron breviscapus Hand-Mazz, which has been widely used in the treatment of cardiovascular and cerebrovascular diseases. In the present study, we analyzed the effect of scutellarin on HN, by using C57BL/6 mice and human renal tubular epithelial cell line HK-2 which was subjected to adenine/potassium oxonate and UA to mimic a HN injury. The HN mice showed a significant decrease in renal function with the increased SCr and blood urea nitrogen (BUN) (p < 0.05). Hematoxylin-eosin staining results showed a histological injury in HN mice kidney tissues with severe tubular damage. Scutellarin dose dependently alleviated the renal injury of the HN model (p < 0.05), and a dose of 20 mg/kg/day remarkably reduced the Scr level (26.10 +/- 3.23 mu mol/ml vs. 48.39 +/- 7.51 mu mol/ml, p < 0.05) and BUN (151.12 +/- 30.24 mmol/L vs. 210.43 +/- 45.67 mmol/L, p < 0.05) compared with the HN model group. Similarly, scutellarin decreased NGAL, Kim-1, cystatin C, and IL-18 protein expression levels in HN mouse (p < 0.05). Overexpressed CCN1 could not induce NLRP3 inflammasome activation, with no change of mRNA and protein expression levels of NLRP3, ASC, and pro-caspase-1 compared with the control HK-2. However, HK-2 showed a significant NLRP3 inflammasome activation and apoptosis. Importantly, knockdown of CCN1 not only aggravated NLRP3 inflammasome activation and apoptosis but also abrogated the protective effect of scutellarin in UA-induced HK-2 injury. Thus, scutellarin might alleviate HN progression via a mechanism involved in CCN1 regulation on NLRP3 inflammasome activation

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.</p

Critical Role of Cysteine-Rich Protein 61 in Mediating the Activation of Renal Fibroblasts

ObjectiveTo explore the expression of cysteine-rich protein 61 (Cyr61) in ischemic renal fibrosis and the role of Cyr61 in mediating the activation of renal fibroblasts.Methods(1) The rat model of renal fibrosis was established after ischemia-reperfusion acute renal injury (IR-AKI). We detected the renal function by biochemical test, evaluated the fibrosis by Masson staining, and detected the expression of Cyr61 by western blotting. (2) Bioinformatics technique was adopted to analyze the expression of Cyr61 in activated renal fibroblasts. (3) Normal rat kidney fibroblast cells (NRK-49F cells) with over-expression of Cyr61 (Cyr61+) and low-expression of it (Cyr61--) were established by plasmid transfection. Then part of the cells were activated by TGF-β1 and NRK-49F cells were divided into control group, activated group, Cyr61+/Cyr61-- group and Cyr61+/Cyr61-- activated group. The expression of Cyr61 and fibrosis related factors (Col1α1, Col3α1, MMP9, and MMP13) were ascertained by PCR and western blotting. Cell proliferation was discovered by CCK8 method, cell cycle was analyzed by flow cytometry, and the transcription of cell senescence related factors (P53, P21, Rb, and P16) were ascertained by PCR method.Results(1) In the process of fibrosis after IR-AKI, the area of collagen fiber was most obviously at AKI 1W, while the Cyr61 protein was at the lowest level at AKI 1W. (2) Gene chip analysis showed that the expression of Cyr61 was decreased in renal fibroblasts after IR. (3) Compared with control group, Cyr61+ group expressed less Col1α1 or Col3α1, as well as more MMP9 and MMP13. At the same time, the proliferation of Cyr61+ group decreased and cells in G1 phases increased with more transcription of P53, P21, and Rb (all P &lt; 0.05). Compared with activated group, the results of Cyr61+ activated group were similar to the above. The above effects of low expression group were just the opposite. In addition, there was no difference in the transcription of P16 among these groups (P &gt; 0.05).ConclusionCyr61 may not only inhibit the fibrotic phenotype of fibroblasts, but may also inhibit proliferation by promoting fibroblasts arrest in G1 phase through the P53/P21/Rb interrelated cell senescence pathway, subsequently affecting the process of ischemic renal fibrosis

The cathelicidin-derived close-to-nature peptide D-11 sensitizes Klebsiella pneumoniae to a range of antibiotics in vitro, ex vivo and in vivo

The outer membrane of Gram-negative bacteria constitutes a permeability barrier that prevents certain antibiotics to reach their targets, thus conferring a high tolerance to a wide range of antibiotics. Combined therapies of antibiotics and outer membrane-perturbing drugs have been proposed as an alternative treatment to extend the use of antibiotics against Gram-positive bacteria to Gram-negative bacteria. Among the outer-membrane active compounds, the outer-membrane permeabilizing peptides, play a prominent role. They form a group of small cationic and amphipathic molecules with the ability to insert specifically into bacterial membranes inducing their permeabilization and/or disruption. Here we assess the combined effect of several compounds belonging to main antibiotic families and the cathelicidin close-to-nature outer membrane peptide D-11 against four clinically relevant Gram-negative bacteria. The results show that the peptide D-11 displays strong synergistic activity with several antibiotics belonging to different families, in particular against Klebsiella pneumoniae, even better than some other outer membrane-active peptides that are currently in a clinical trial such as SPR741. Notably, we observed this activity in vitro, ex vivo in a newly designed bacteremia model and in vivo in a mice abscess infection model. Overall, our results suggest that D-11 is a good candidate to repurpose the activity of traditional antibiotics against Klebsiella pneumoniae

Spatiotemporal Distribution of Soil Moisture and Salinity in the Taklimakan Desert Highway Shelterbelt

Salinization and secondary salinization often appear after irrigation with saline water. The Taklimakan Desert Highway Shelterbelt has been irrigated with saline ground water for more than ten years; however, soil salinity in the shelterbelt has not been evaluated. The objective of this study was to analyze the spatial and temporal distribution of soil moisture and salinity in the shelterbelt system. Using a non-uniform grid method, soil samples were collected every two days during one ten-day irrigation cycle in July 2014 and one day in spring, summer, and autumn. The results indicated that soil moisture declined linearly with time during the irrigation cycle. Soil moisture was greatest in the southern and eastern sections of the study area. In contrast to soil moisture, soil electrical conductivity increased from 2 to 6 days after irrigation, and then gradually decreased from 6 to 8 days after irrigation. Soil moisture was the greatest in spring and the least in summer. In contrast, soil salinity increased from spring to autumn. Long time drip-irrigation with saline groundwater increased soil salinity slightly. The soil salt content was closely associated with soil texture. The current soil salt content did not affect plant growth, however, the soil in the shelterbelt should be continuously monitored to prevent salinization in the future

Spatial Distribution and Structural Characteristics for Haloxylon ammodendron Plantation on the Southwestern Edge of the Gurbantünggüt Desert

Haloxylon ammodendron (C.A.Mey.) Bge. is crucially important for stabilizing sand dunes in the desert area of the Junggar Basin and has thus been widely planted in the oasis–desert ecotone for windbreak and sand fixation purposes since the 1980s. The spatial distribution and structural characteristics of Haloxylon ammodendron plantations of three different ages—planted in 1983 (36a), 1997 (22a), and 2004 (15a)—on the southwestern edge of the Gurbantünggüt Desert were studied. The results showed that the spatial distribution patterns for the different stages of growth showed a trend of cluster that was random during the transformation from seedlings to juvenile and mature trees. Forest density for the 15a, 22a, and 36a plantations was, respectively, 1110, 1189, and 1933 plants ha−1; the base stem diameter for the main forest layer was 5.85, 8.77, and 6.17 cm, respectively, and the tree height was concentrated in the range of 1.5–3.0 m, 2.0–3.5 m, and 1.5–2.5 m. In the regeneration layers, the proportion of seedlings was the largest in all three stand ages, followed by juvenile trees, and mature trees only appeared in the 22a plantation. The proportion of deadwood in the 36a forest was the highest, and there were no mature trees in the regeneration layer. These results indicate that the three Haloxylon ammodendron plantation stages were in the period of rising at 15a, stable and degenerate with increasing age at 22a, and at 36a the regeneration ability was very weak and presented degradation due to species competition for soil moisture, because of too many seedlings and mature plants. In this case, measures such as thinning could be taken to prevent rapid degradation and to accelerate regeneration when the stand age exceeds 20 years. Considering the sand fixation effect, the pressure of competition for water resources, and forest capacity for renewal and sustainability, the most suitable forest density in the Haloxylon ammodendron plantation would be 8.5–9 m2 per plant

Moderate irrigation intervals facilitate establishment of two desert shrubs in the Taklimakan Desert Highway Shelterbelt in China.

Water influences various physiological and ecological processes of plants in different ecosystems, especially in desert ecosystems. The purpose of this study is to investigate the response of physiological and morphological acclimation of two shrubs Haloxylon ammodendron and Calligonum mongolicunl to variations in irrigation intervals.The irrigation frequency was set as 1-, 2-, 4-, 8- and 12-week intervals respectively from March to October during 2012-2014 to investigate the response of physiological and morphological acclimation of two desert shrubs Haloxylon ammodendron and Calligonum mongolicunl to variations in the irrigation system. The irrigation interval significantly affected the individual-scale carbon acquisition and biomass allocation pattern of both species. Under good water conditions (1- and 2-week intervals), carbon assimilation was significantly higher than other treatments; while, under water shortage conditions (8- and 12-week intervals), there was much defoliation; and under moderate irrigation intervals (4 weeks), the assimilative organs grew gently with almost no defoliation occurring.Both studied species maintained similar ecophysiologically adaptive strategies, while C. mongolicunl was more sensitive to drought stress because of its shallow root system and preferential belowground allocation of resources. A moderate irrigation interval of 4 weeks was a suitable pattern for both plants since it not only saved water but also met the water demands of the plants

Diurnal pattern of leaf water potential of <i>H</i>. <i>ammodendron</i> (a, b) and <i>C</i>. <i>mongolicunl</i> (c, d) before and after irrigation for the five irrigation intervals.

<p>The data points for the 2-week interval fell between those of the 1 and 4 weeks; those of the 8-week interval fell between those of 4 and 12 weeks. Error bars represent the standard deviation of the mean.</p

YbeY controls the type III and type VI secretion systems and biofilm formation through RetS in Pseudomonas aeruginosa

YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa, we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosaIMPORTANCEPseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa