scholarly journals The RNA Chaperone Hfq Participates in Persistence to Multiple Antibiotics in the Fish Pathogen Yersinia ruckeri

2021 ◽  
Vol 9 (7) ◽  
pp. 1404
Author(s):  
Iván L. Calderón ◽  
María José Barros ◽  
Fernanda Montt ◽  
Fernando Gil ◽  
Juan A. Fuentes ◽  
...  
Keyword(s):  
Growth Curves ◽  
Polymyxin B ◽  
Yersinia Ruckeri ◽  
Knockout Mutant ◽  
Persister Cells ◽  
Rna Chaperone ◽  
Time Kill ◽  
Sublethal Concentrations ◽  
Multiple Antibiotics

Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon.

scholarly journals Role of RppA in the Regulation of Polymyxin B Susceptibility, Swarming, and Virulence Factor Expression in Proteus mirabilis

2008 ◽  
Vol 76 (5) ◽  
pp. 2051-2062 ◽  
Author(s):  
Won-Bo Wang ◽  
I-Chun Chen ◽  
Sin-Sien Jiang ◽  
Hui-Ru Chen ◽  
Chia-Yu Hsu ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Proteus Mirabilis ◽  
Response Regulator ◽  
Polymyxin B ◽  
Wild Type ◽  
High Concentration ◽  
Knockout Mutant ◽  
Two Component System ◽  
Tract Infections

ABSTRACT Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (>160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg2+. Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

Bacteriophages immunomodulate the response of monocytes

2021 ◽  
pp. 153537022199515
Author(s):  
Lídia Perea ◽  
Lorena Rodríguez-Rubio ◽  
Juan C Nieto ◽  
Carlos Zamora ◽  
Elisabet Cantó ◽  
...  
Keyword(s):  
Cytokine Production ◽  
Statistical Significance ◽  
Control Sample ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Direct Role ◽  
Gram Positive Bacteria ◽  
Hla Dr ◽  
Tnf Α

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

scholarly journals The Periplasmic α-Carbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation

2005 ◽  
Vol 187 (2) ◽  
pp. 729-738 ◽  
Author(s):  
Elizabeth A. Marcus ◽  
Amiel P. Moshfegh ◽  
George Sachs ◽  
David R. Scott
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Potential ◽  
Carbonic Anhydrase ◽  
Membrane Integrity ◽  
Carbonic Anhydrase Activity ◽  
Knockout Mutant ◽  
Inner Membrane ◽  
Acid Acclimation

ABSTRACT The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3 −, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3 − by the periplasmic α-CA.

Double deletion of cpxR and tolC significantly increases the susceptibility of Salmonella enterica serovar Typhimurium to colistin

2021 ◽  
Author(s):  
Meng-Ke Zhang ◽  
Meng-Yao Zhang ◽  
Shuo-Bo Liu ◽  
Ying-Ying Yang ◽  
Ya-Jun Zhai ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Molecular Mechanisms ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Growth Curves ◽  
Tca Cycle ◽  
Lower Growth ◽  
Double Deletion ◽  
Broth Microdilution Method ◽  
Serovar Typhimurium ◽  
Time Kill

Abstract Background The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. Objectives To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin. Methods A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time–kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis. Results We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time–kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration. Conclusions Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin.

scholarly journals Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Anna Olsson ◽  
Pikkei Wistrand-Yuen ◽  
Elisabet I. Nielsen ◽  
Lena E. Friberg ◽  
Linus Sandegren ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Time Lapse ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Positive Interactions ◽  
Synergistic Activity ◽  
Content Type ◽  
Time Lapse Microscopy ◽  
Time Kill ◽  
Antibiotic Combination

ABSTRACT Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (≤106 CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal β-lactams, ciprofloxacin, and amikacin. Genes encoding β-lactamases (e.g., blaPAO and blaOXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Autophagy Suppresses Invasiveness of Endometrial Cells through Reduction of Fascin-1

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Xiaomei Luo ◽  
Wei Cheng ◽  
Shizhang Wang ◽  
Zhihong Chen ◽  
Jieqiong Tan
Keyword(s):  
Metabolic Diseases ◽  
Growth Curves ◽  
Cellular Growth ◽  
Control Group ◽  
Scratch Assay ◽  
Endometrial Cells ◽  
Autophagy Inhibitor ◽  
Exogenous Expression ◽  
Potential Therapeutics

Objective. Autophagy has been reported to be involved in the development of various disorders such as neurodegenerative and metabolic diseases and tumors. Autophagy activators and inhibitors are also potential therapeutics for these diseases. However, the mechanism of autophagic involvement in different diseases is not the same, and the role of autophagy in endometriosis (EM) has not yet been elucidated. This research investigated the mechanism by which autophagy acts in EM, with the aim of establishing a theoretical basis for its prevention and treatment through the targeted interference with autophagy. Methods. We used an RNA interference fragment targeting ATG5, the autophagy activator rapamycin, and the autophagy inhibitor 3-MA or overexpression of filopodia-related protein fascin-1, in conjunction with clonogenic assays, growth curves, and scratch assay to investigate the influence of autophagy on cellular growth, proliferation, and invasiveness. We collected specimens from 20 clinical cases of EM and investigated the protein expression of the autophagic marker LC3-II, the autophagic substrate p62, and fascin-1. Results. Rapamycin was able to inhibit the proliferation and colony formation of the endometriotic cell line CRL-7566, whereas the autophagy inhibitor 3-MA as well as the interference with the autophagy-related gene ATG5 had the opposite effect. More importantly, the autophagy activator rapamycin was able to inhibit the growth of filopodia in the endometriotic cells, and the overexpression of the fascin-1 restored the rapamycin-induced decrease of invasiveness. We found that the expression of the autophagy marker LC3-II was significantly reduced among the clinical EM specimens compared to the control group, while the expressions of fascin-1 and autophagic substrate p62 were increased. Conclusion. Our results indicate that the inhibition of autophagy and exogenous expression of fascin-1 may promote the invasiveness of endometrial cells. As a corollary, autophagy represents a potential target for the treatment of EM.

RNA chaperone hfq mediates persistence to multiple antibiotics in Aeromonas veronii

2019 ◽  
Vol 132 ◽  
pp. 124-128 ◽  
Author(s):  
Lin Zhang ◽  
Wenjing Yu ◽  
Yanqiong Tang ◽  
Hong Li ◽  
Xiang Ma ◽  
...  
Keyword(s):  
Rna Chaperone ◽  
Aeromonas Veronii ◽  
Multiple Antibiotics
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