scholarly journals Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa

2013 ◽  
Vol 57 (4) ◽  
pp. 1617-1624 ◽  
Author(s):  
Julia Gödeke ◽  
Christian Pustelny ◽  
Susanne Häussler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Opportunistic Pathogen ◽  
Phase Cell ◽  
Chronic Infections ◽  
Content Type ◽  
Mediated Effects ◽  
Protein Expression Profiles ◽  
The Impact

ABSTRACTAcute and chronic infections caused by the opportunistic pathogenPseudomonas aeruginosapose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronicP. aeruginosainfections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility inP. aeruginosaseem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of theP. aeruginosapeptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon inrhlRalso explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on theP. aeruginosaproteome and possibly also on the protein expression profiles of other bacterial pathogens.

scholarly journals Heterogenous Susceptibility to R-Pyocins in Populations of Pseudomonas aeruginosa Sourced from Cystic Fibrosis Lungs

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Madeline Mei ◽  
Jacob Thomas ◽  
Stephen P. Diggle
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Genotypic Diversity ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Heterogeneous Populations ◽  
Content Type ◽  
Lung Infections ◽  
The Impact ◽  
Lps Binding

ABSTRACT Bacteriocins are proteinaceous antimicrobials produced by bacteria that are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their antipseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa organisms from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection; however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1 to R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources, (ii) a large number of strains lack R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of intrastrain diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage. IMPORTANCE R-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection; however, little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics and to evolve phenotypic and genotypic diversity over time; thus, it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

scholarly journals Heterogenous response to R-pyocin killing in populations of Pseudomonas aeruginosa sourced from cystic fibrosis lungs

2020 ◽  
Author(s):  
Madeline Mei ◽  
Jacob Thomas ◽  
Stephen P. Diggle
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Genotypic Diversity ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Bacterial Populations ◽  
Heterogeneous Populations ◽  
Lung Infections ◽  
The Impact ◽  
Lps Binding

AbstractBacteriocins are proteinaceous antimicrobials produced by bacteria which are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa. Due to their anti-pseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection, however little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1-R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources and (ii) isolates collected from the same patient have the same R-type. We then assessed the impact of diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.ImportanceR-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection, however little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics, as well as evolve phenotypic and genotypic diversity over time; thus it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is likely heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Ziyu Sun ◽  
Jing Shi ◽  
Chang Liu ◽  
Yongxin Jin ◽  
Kewei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Mrna Level ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat 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 ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults 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 atacpromoter repressed the endogenousprtRpromoter 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.

scholarly journals Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

2022 ◽  
Author(s):  
Wai Leong ◽  
Wee Han Poh ◽  
Jonathan Williams ◽  
Carla Lutz ◽  
M. Mozammel Hoque ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Late Stage ◽  
Early Stage ◽  
Opportunistic Pathogen ◽  
Phenotypic Traits ◽  
Nucleotide Polymorphisms ◽  
Chronic Infections ◽  
The Impact

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

scholarly journals The Two-Component Sensor KinB Acts as a Phosphatase To Regulate Pseudomonas aeruginosa Virulence

2012 ◽  
Vol 194 (23) ◽  
pp. 6537-6547 ◽  
Author(s):  
Nikhilesh S. Chand ◽  
Anne E. Clatworthy ◽  
Deborah T. Hung
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Opportunistic Pathogen ◽  
Regulatory Control ◽  
Primary Role ◽  
Chronic Infections ◽  
Content Type ◽  
Two Component ◽  
Sense And Respond ◽  
Cognate Response Regulator

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that is capable of causing both acute and chronic infections.P. aeruginosavirulence is subject to sophisticated regulatory control by two-component systems that enable it to sense and respond to environmental stimuli. We recently reported that the two-component sensor KinB regulates virulence in acuteP. aeruginosainfection. Furthermore, it regulates acute-virulence-associated phenotypes such as pyocyanin production, elastase production, and motility in a manner independent of its kinase activity. Here we show that KinB regulates virulence through the global sigma factor AlgU, which plays a key role in repressingP. aeruginosaacute-virulence factors, and through its cognate response regulator AlgB. However, we show that rather than phosphorylating AlgB, KinB's primary role in the regulation of virulence is to act as a phosphatase to dephosphorylate AlgB and alleviate phosphorylated AlgB's repression of acute virulence.

scholarly journals Insights into the Assembly of the Alginate Biosynthesis Machinery in Pseudomonas aeruginosa

2013 ◽  
Vol 79 (10) ◽  
pp. 3264-3272 ◽  
Author(s):  
Zahid U. Rehman ◽  
Yajie Wang ◽  
M. Fata Moradali ◽  
Iain D. Hay ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stability Analysis ◽  
Experimental Evidence ◽  
Outer Membrane ◽  
Opportunistic Pathogen ◽  
Multiprotein Complex ◽  
Inner Membrane ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion ofalgKin an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.

scholarly journals A Large-Scale Whole-Genome Comparison Shows that Experimental Evolution in Response to Antibiotics Predicts Changes in Naturally Evolved Clinical Pseudomonas aeruginosa

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Samuel J. T. Wardell ◽  
Attika Rehman ◽  
Lois W. Martin ◽  
Craig Winstanley ◽  
Wayne M. Patrick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Experimental Evolution ◽  
Large Scale ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Genome Comparison ◽  
Chronic Infections ◽  
Content Type ◽  
Large Deletions ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations, we experimentally evolved the antibiotic-sensitive strain P. aeruginosa PAO1 to become resistant to three widely used antipseudomonal antibiotics, namely, ciprofloxacin, meropenem, and tobramycin. Mutants could tolerate up to 2,048-fold higher concentrations of antibiotics than strain PAO1. Genome sequences were determined for 13 mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic, at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics, mutations arose in genes known to be associated with resistance but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study, we analyzed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479 kb arose in experimentally evolved meropenem-resistant mutants, and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically relevant resistance-associated mutations.

scholarly journals Signal Sensing and Transduction Are Conserved between the Periplasmic Sensory Domains of BifA and SagS

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Jozef Dingemans ◽  
Rebecca E. Al-Feghali ◽  
Holger Sondermann ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Structural Similarity ◽  
Opportunistic Pathogen ◽  
Sequence Conservation ◽  
Sensor Kinase ◽  
Antibiotic Tolerance ◽  
Bacterial Cells ◽  
Chronic Infections ◽  
Content Type

ABSTRACT The hybrid sensor kinase SagS of Pseudomonas aeruginosa plays a key role in the transition from the planktonic to the biofilm mode of growth. Recently, we have shown that distinct sets of residues in its periplasmic HmsP sensory domain are involved in the regulation of biofilm formation or antibiotic tolerance. Interestingly, the HmsP domain of the phosphodiesterase BifA shows great predicted structural similarity to that of SagS, despite moderate sequence conservation and only a number of residues involved in SagS signaling being conserved between both proteins. Based on this observation, we hypothesized that BifA and SagS may use similar mechanisms to sense and transduce signals perceived at their periplasmic HmsP domains and, therefore, may be interchangeable. To test this hypothesis, we constructed SagS hybrids in which the HmsP domain of SagS was replaced by that of BifA (and vice versa) or by the DISMED2 sensory domain of NicD. The SagS-BifA hybrid restored attachment and biofilm formation by the ΔbifA mutant. Likewise, while the NicD-SagS hybrid was nonfunctional, the BifA-SagS hybrid partially restored pathways leading to biofilm formation and antibiotic tolerance in a ΔsagS mutant background. Furthermore, alanine substitution of key residues previously associated with the biofilm formation and antibiotic tolerance pathways of SagS impaired signal transduction by the BifA-SagS hybrid in a similar way to SagS. In conclusion, our data indicate that the nature of the sensory domain is important for proper functionality of the cytoplasmic effector domains and that signal sensing and transduction are likely conserved in SagS and BifA. IMPORTANCE Biofilms have been associated with more than 60% of all recalcitrant and chronic infections and can render bacterial cells up to a thousand times more resistant to antibiotics than planktonic cells. Although it is known that the transition from the planktonic to the biofilm mode of growth involves two-component regulatory systems, increased c-di-GMP levels, and quorum sensing systems among others, the exact signaling events that lead to biofilm formation remain unknown. In the opportunistic pathogen Pseudomonas aeruginosa, the hybrid sensor kinase SagS regulates biofilm formation and antibiotic tolerance through two independent pathways via distinct residues in its periplasmic sensory domain. Interestingly, the sensory domains of SagS and BifA show great predicted structural similarity despite moderate sequence conservation. Here we show that the sensory domains of BifA and SagS are functionally interchangeable and that they use a similar mechanism of signal sensing and transduction, which broadens our understanding of how bacteria perceive and transduce signals when transitioning to the biofilm mode of growth.

scholarly journals Intraspecies Signaling between Common Variants of Pseudomonas aeruginosa Increases Production of Quorum-Sensing-Controlled Virulence Factors

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Dallas L. Mould ◽  
Nico J. Botelho ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Loss Of Function ◽  
Wild Type ◽  
Chronic Infections ◽  
Content Type ◽  
Complex Interactions ◽  
Natural Settings ◽  
Citrate Release

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing (QS). The lasR gene, which encodes a central QS regulator, is frequently mutated in clinical isolates from chronic infections, and loss of LasR function (LasR−) generally impairs the activity of downstream QS regulators RhlR and PqsR. We found that in cocultures containing LasR+ and LasR− strains, LasR− strains hyperproduce the RhlR/RhlI-regulated antagonistic factors pyocyanin and rhamnolipids in diverse models and media and in different strain backgrounds. Diffusible QS autoinducers produced by the wild type were not required for this effect. Using transcriptomics, genetics, and biochemical approaches, we uncovered a reciprocal interaction between wild-type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin produced by the lasR mutant induced citrate release and cross-feeding from the wild type. Citrate, a metabolite often secreted in low iron environments, stimulated RhlR signaling and RhlI levels in LasR−but not in LasR+ strains. These studies reveal the potential for complex interactions between recently diverged, genetically distinct isolates within populations from single chronic infections. IMPORTANCE Coculture interactions between lasR loss-of-function and LasR+ Pseudomonas aeruginosa strains may explain the worse outcomes associated with the presence of LasR− strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote unpredictably high virulence factor production.

scholarly journals Regulation of AmpC-Driven β-Lactam Resistance in Pseudomonas aeruginosa: Different Pathways, Different Signaling

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Gabriel Torrens ◽  
Sara Belén Hernández ◽  
Juan Alfonso Ayala ◽  
Bartolome Moya ◽  
Carlos Juan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Molecular Mechanisms ◽  
Resistance Mechanism ◽  
Quantitative Information ◽  
Chronic Infections ◽  
Knockout Mutant ◽  
Major Step ◽  
Content Type ◽  
Soluble Cell

ABSTRACT The hyperproduction of the chromosomal AmpC β-lactamase is the main mechanism driving β-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-N-acetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of β-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent β-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling. IMPORTANCE The extensive use of β-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC β-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance.

Sign in / Sign up

Export Citation Format

Share Document