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75100.9564Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in Photorhabdus laumondii. In Gram-negative bacteria, resistance-nodulation-division (RND)-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. Photorhabdus laumondii is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrent properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in P. laumondii TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of ∆acrA compared to wild type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbene, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact-dependent growth inhibition systems were upregulated in ∆acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in ∆acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the ∆acrA mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting acrAB causes transcriptional and metabolic dysregulation in P. laumondii, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators.IMPORTANCERecent discoveries have highlighted Photorhabdus laumondii as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in P. laumondii TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of resistance-nodulation-division (RND) pump-mediated signaling pathways in bacteria, thereby aiding in combating multidrug-resistant infections.202540920493
80510.9557LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus. Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.202337166326
886420.9555Resistance, mechanism, and fitness cost of specific bacteriophages for Pseudomonas aeruginosa. The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa (P. aeruginosa) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU. It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy.IMPORTANCEThe bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy, involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.202438299825
819430.9552Role of the phenazine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis. The triggering of antibiotic production by various environmental stress molecules can be interpreted as bacteria's response to obtain increased fitness to putative danger, whereas the opposite situation - inhibition of antibiotic production - is more complicated to understand. Phenazines enable Pseudomonas species to eliminate competitors for rhizosphere colonization and are typical virulence factors used for model studies. In the present work, we have investigated the negative effect of subinhibitory concentrations of NaCl, fusaric acid and two antibiotics on quorum-sensing-controlled phenazine production by Pseudomonas chlororaphis. The selected stress factors inhibit phenazine synthesis despite sufficient cell density. Subsequently, we have identified connections between known genes of the phenazine-inducing cascade, including PsrA (Pseudomonas sigma regulator), RpoS (alternative sigma factor), Pip (phenazine inducing protein) and PhzI/PhzR (quorum-sensing system). Under all tested conditions, overexpression of Pip or PhzR restored phenazine production while overexpression of PsrA or RpoS did not. This forced restoration of phenazine production in strains overexpressing regulatory genes pip and phzR significantly impairs growth and stress resistance; this is particularly severe with pip overexpression. We suggest a novel physiological explanation for the inhibition of phenazine virulence factors in pseudomonas species responding to toxic compounds. We propose that switching off phenazine-1-carboxamide (PCN) synthesis by attenuating pip expression would favour processes required for survival. In our model, this 'decision' point for promoting PCN production or stress resistance is located downstream of rpoS and just above pip. However, a test with the stress factor rifampicin shows no significant inhibition of Pip production, suggesting that stress factors may also target other and so far unknown protagonists of the PCN signalling cascade.201121030433
1140.9548Diffusible signal factor primes plant immunity against Xanthomonas campestris pv. campestris (Xcc) via JA signaling in Arabidopsis and Brassica oleracea. BACKGROUND: Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas-plant interactions remain unclear. METHODS: Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc). Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance. RESULTS: We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana. Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes (AOC2, AOS, LOX2, OPR3 and JAR1), transportor gene (JAT1), regulator genes (JAZ1 and MYC2) and responsive genes (VSP2, PDF1.2 and Thi2.1) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1. CONCLUSION: These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea.202337404719
843450.9547A potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with LED209 targeting QseC receptor to inhibit the virulence genes of gram negative bacteria. The pandemic of multidrug-resistant Gram negative bacteria (GNB) is a worldwide healthcare concern, and very few antibiotics are being explored to match the clinical challenge. Recently, amino-terminated poly(amidoamine) (PAMAM) dendrimers have shown potential to function as broad antimicrobial agents. However, PAMAM displays a generation dependent cytotoxicity to mammalian cells and low selectivity on bacterial cells, which limits PAMAM to be developed as an antibacterial agent for systemic administration. We conjugated G3 PAMAM with LED209, a specific inhibitor of quorum sensor QseC of GNB, to generate a multifunctional agent PAMAM-LED209. Intriguingly, PAMAM-LED209 showed higher selectivity on GNB and lower cytotoxicity to mammalian cells, yet remained strong antibacterial activity. PAMAM-LED209 also inhibited virulence gene expression of GNB, and did not induce antibiotic-resistance. The present work firstly demonstrated that PAMAM-LED209 conjugate had a highly selective anti-GNB activity and low cytotoxicity, which offered a feasible strategy for combating multidrug-resistant GNB infections. FROM THE CLINICAL EDITOR: This research team demonstrated that a novel PAMAM-LED209 conjugate had highly selective activity against Gram-negative bacteria, coupled with low cytotoxicity, offering a potential strategy for combating multidrug-resistant infections.201525461286
883760.9547Phage resistance formation and fitness costs of hypervirulent Klebsiella pneumoniae mediated by K2 capsule-specific phage and the corresponding mechanisms. INTRODUCTION: Phage is promising for the treatment of hypervirulent Klebsiella pneumoniae (hvKP) infections. Although phage resistance seems inevitable, we found that there still was optimization space in phage therapy for hvKP infection. METHODS: The clinical isolate K. pneumoniae FK1979 was used to recover the lysis phage ΦFK1979 from hospital sewage. Phage-resistant bacteria were obtained on LB agar and used to isolate phages from sewage. The plaque assay, transmission electron microscopy (TEM), multiplicity of infection test, one-step growth curve assay, and genome analysis were performed to characterize the phages. Colony morphology, precipitation test and scanning electron microscope were used to characterize the bacteria. The absorption test, spot test and efficiency of plating (EOP) assay were used to identify the sensitivity of bacteria to phages. Whole genome sequencing (WGS) was used to identify gene mutations of phage-resistant bacteria. The gene expression levels were detected by RT-qPCR. Genes knockout and complementation of the mutant genes were performed. The change of capsules was detected by capsule quantification and TEM. The growth kinetics, serum resistance, biofilm formation, adhesion and invasion to A549 and RAW 264.7 cells, as well as G. mellonella and mice infection models, were used to evaluate the fitness and virulence of bacteria. RESULTS AND DISCUSSION: Here, we demonstrated that K2 capsule type sequence type 86 hvKP FK1979, one of the main pandemic lineages of hvKP with thick capsule, rapidly developed resistance to a K2-specific lysis phage ΦFK1979 which was well-studied in this work to possess polysaccharide depolymerase. The phage-resistant mutants showed a marked decrease in capsule expression. WGS revealed single nucleotide polymorphism (SNP) in genes encoding RfaH, galU, sugar glycosyltransferase, and polysaccharide deacetylase family protein in the mutants. RfaH and galU were further identified as being required for capsule production and phage sensitivity. Expressions of genes involved in the biosynthesis or regulation of capsule and/or lipopolysaccharide significantly decreased in the mutants. Despite the rapid and frequent development of phage resistance being a disadvantage, the attenuation of virulence and fitness in vitro and in vivo indicated that phage-resistant mutants of hvKP were more susceptible to the immunity system. Interestingly, the newly isolated phages targeting mutants changed significantly in their plaque and virus particle morphology. Their genomes were much larger than and significantly different from that of ΦFK1979. They possessed much more functional proteins and strikingly broader host spectrums than ΦFK1979. Our study suggests that K2-specific phage has the potential to function as an antivirulence agent, or a part of phage cocktails combined with phages targeting phage-resistant bacteria, against hvKP-relevant infections.202337538841
62370.9545The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin. The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.202031964794
818880.9544Biofilm in implant infections: its production and regulation. A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intraocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intra-ocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.200516353112
902290.9544Drug repositioning: doxazosin attenuates the virulence factors and biofilm formation in Gram-negative bacteria. The resistance development is an increasing global health risk that needs innovative solutions. Repurposing drugs to serve as anti-virulence agents is suggested as an advantageous strategy to diminish bacterial resistance development. Bacterial virulence is controlled by quorum sensing (QS) system that orchestrates the expression of biofilm formation, motility, and virulence factors production as enzymes and virulent pigments. Interfering with QS could lead to bacterial virulence mitigation without affecting bacterial growth that does not result in bacterial resistance development. This study investigated the probable anti-virulence and anti-QS activities of α-adrenoreceptor blocker doxazosin against Proteus mirabilis and Pseudomonas aeruginosa. Besides in silico study, in vitro and in vivo investigations were conducted to assess the doxazosin anti-virulence actions. Doxazosin significantly diminished the biofilm formation and release of QS-controlled Chromobacterium violaceum pigment and virulence factors in P. aeruginosa and P. mirabilis, and downregulated the QS encoding genes in P. aeruginosa. Virtually, doxazosin interfered with QS proteins, and in vivo protected mice against P. mirabilis and P. aeruginosa. The role of the membranal sensors as QseC and PmrA was recognized in enhancing the Gram-negative virulence. Doxazosin downregulated the membranal sensors PmR and QseC encoding genes and could in silico interfere with them. In conclusion, this study preliminary documents the probable anti-QS and anti-virulence activities of doxazosin, which indicate its possible application as an alternative or in addition to antibiotics. However, extended toxicological and pharmacological investigations are essential to approve the feasible clinical application of doxazosin as novel efficient anti-virulence agent. KEY POINTS: • Anti-hypertensive doxazosin acquires anti-quorum sensing activities • Doxazosin diminishes the virulence of Proteus mirabilis and Pseudomonas aeruginosa • Doxazosin could dimmish the bacterial espionage.202337079062
6220100.9543Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa. Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.201424770387
8799110.9541The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence. Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.202236008485
9019120.9540Deleting qseC downregulates virulence and promotes cross-protection in Pasteurella multocida. QseC, a histidine sensor kinase of the QseBC two-component system, acts as a global regulator of bacterial stress resistance, biofilm formation, and virulence. The function of QseC in some bacteria is well understood, but not in Pasteurella multocida. We found that deleting qseC in P. multocida serotype A:L3 significantly down-regulated bacterial virulence. The mutant had significantly reduced capsule production but increased resistance to oxidative stress and osmotic pressure. Deleting qseC led to a significant increase in qseB expression. Transcriptome sequencing analysis showed that 1245 genes were regulated by qseC, primarily those genes involved in capsule and LPS biosynthesis and export, biofilm formation, and iron uptake/utilization, as well as several immuno-protection related genes including ompA, ptfA, plpB, vacJ, and sodA. In addition to presenting strong immune protection against P. multocida serotypes A:L1 and A:L3 infection, live ΔqseC also exhibited protection against P. multocida serotype B:L2 and serotype F:L3 infection in a mouse model. The results indicate that QseC regulates capsular production and virulence in P. multocida. Furthermore, the qseC mutant can be used as an attenuated vaccine against P. multocida strains of multiple serotypes.202134801081
5170130.9540Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
9027140.9539Scorpion Venom Antimicrobial Peptides Induce Siderophore Biosynthesis and Oxidative Stress Responses in Escherichia coli. The increasing development of microbial resistance to classical antimicrobial agents has led to the search for novel antimicrobials. Antimicrobial peptides (AMPs) derived from scorpion and snake venoms offer an attractive source for the development of novel therapeutics. Smp24 (24 amino acids [aa]) and Smp43 (43 aa) are broad-spectrum AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio mauruspalmatus and subsequently characterized. Using a DNA microarray approach, we examined the transcriptomic responses of Escherichia coli to subinhibitory concentrations of Smp24 and Smp43 peptides following 5 h of incubation. Seventy-two genes were downregulated by Smp24, and 79 genes were downregulated by Smp43. Of these genes, 14 genes were downregulated in common and were associated with bacterial respiration. Fifty-two genes were specifically upregulated by Smp24. These genes were predominantly related to cation transport, particularly iron transport. Three diverse genes were independently upregulated by Smp43. Strains with knockouts of differentially regulated genes were screened to assess the effect on susceptibility to Smp peptides. Ten mutants in the knockout library had increased levels of resistance to Smp24. These genes were predominantly associated with cation transport and binding. Two mutants increased resistance to Smp43. There was no cross-resistance in mutants resistant to Smp24 or Smp43. Five mutants showed increased susceptibility to Smp24, and seven mutants showed increased susceptibility to Smp43. Of these mutants, formate dehydrogenase knockout (fdnG) resulted in increased susceptibility to both peptides. While the electrostatic association between pore-forming AMPs and bacterial membranes followed by integration of the peptide into the membrane is the initial starting point, it is clear that there are numerous subsequent additional intracellular mechanisms that contribute to their overall antimicrobial effect.IMPORTANCE The development of life-threatening resistance of pathogenic bacteria to the antibiotics typically in use in hospitals and the community today has led to an urgent need to discover novel antimicrobial agents with different mechanisms of action. As an ancient host defense mechanism of the innate immune system, antimicrobial peptides (AMPs) are attractive candidates to fill that role. Scorpion venoms have proven to be a rich source of AMPs. Smp24 and Smp43 are new AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, and these peptides can kill a wide range of bacterial pathogens. By better understanding how these AMPs affect bacterial cells, we can modify their structure to make better drugs in the future.202133980680
9014150.9539Role of acid responsive genes in the susceptibility of Escherichia coli to ciclopirox. Antibiotic resistance poses a huge threat to the effective treatment of bacterial infections. To circumvent the limitations in developing new antibiotics, researchers are attempting to repurpose pre-developed drugs that are known to be safe. Ciclopirox, an off-patent antifungal agent, inhibits the growth of Gram-negative bacteria, and genes involved in galactose metabolism and lipopolysaccharide (LPS) biosynthesis are plausible antibacterial targets for ciclopirox, since their expression levels partially increase susceptibility at restrictive concentrations. In the present study, to identify new target genes involved in the susceptibility of Escherichia coli to ciclopirox, genome-wide mRNA profiling was performed following ciclopirox addition at sublethal concentrations, and glutamate-dependent acid resistance (GDAR) genes were differentially regulated. Additional susceptibility testing, growth analyses and viability assays of GDAR regulatory genes revealed that down-regulation of evgS or hns strongly enhanced susceptibility to ciclopirox. Further microscopy and phenotypic analyses revealed that down-regulation of these genes increased cell size and decreased motility. Our findings could help to maximise the efficacy of ciclopirox against hard-to-treat Gram-negative pathogens.201829654752
18160.9538Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells. BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.202438336608
9997170.9537RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.201021209831
9062180.9536Biological cost of pyocin production during the SOS response in Pseudomonas aeruginosa. LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance to clinically important antibiotics and impairs the survival of bacteria exposed to one of several genotoxic agents. Two distinct mechanisms mediated these effects. Cell lysis genes that are induced following PrtR autocleavage reduced resistance to bactericidal levels of ciprofloxacin, and production of extracellular R2 pyocin was lethal to cells that initially survived UV light treatment. Although typically resistant to R2 pyocin, P. aeruginosa becomes transiently sensitive to R2 pyocin following UV light treatment, likely because of the strong downregulation of lipopolysaccharide synthesis genes that are required for resistance to R2 pyocin. Our results demonstrate that pyocin production during the P. aeruginosa SOS response carries both expected and unexpected costs.201425022851
806190.9536A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069