scholarly journals Pseudomonas aeruginosa High-Level Resistance to Polymyxins and Other Antimicrobial Peptides RequirescprA, a Gene That Is Disrupted in the PAO1 Strain

2015 ◽  
Vol 59 (9) ◽  
pp. 5377-5387 ◽  
Author(s):  
Alina D. Gutu ◽  
Nicole S. Rodgers ◽  
Jihye Park ◽  
Samuel M. Moskowitz
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Lipid A ◽  
Full Length ◽  
Open Reading Frame ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Reading Frame ◽  
High Level ◽  
Pao1 Strain

ABSTRACTThearnlocus, found in many Gram-negative bacterial pathogens, mediates resistance to polymyxins and other cationic antimicrobial peptides through 4-amino-l-arabinose modification of the lipid A moiety of lipopolysaccharide. InPseudomonas aeruginosa, several two-component regulatory systems (TCSs) control thearnlocus, which is necessary but not sufficient for these resistance phenotypes. A previous transposon mutagenesis screen to identify additional polymyxin resistance genes that these systems regulate implicated an open reading frame designated PA1559 in the genome of theP. aeruginosaPAO1 strain. Resequencing of this chromosomal region and bioinformatics analysis for a variety ofP. aeruginosastrains revealed that in the sequenced PAO1 strain, a guanine deletion at the end of PA1559 results in a frameshift and truncation of a full-length open reading frame that also encompasses PA1560 in non-PAO1 strains, such asP. aeruginosaPAK. Deletion analysis in the PAK strain showed that this full-length open reading frame, designatedcprA, is necessary for polymyxin resistance conferred by activating mutations in the PhoPQ, PmrAB, and CprRS TCSs. ThecprAgene was also required for PmrAB-mediated resistance to other cationic antimicrobial peptides in the PAK strain. Repair of the mutatedcprAallele in the PAO1 strain restored polymyxin resistance conferred by an activating TCS mutation. The deletion ofcprAdid not affect thearn-mediated lipid A modification, indicating that the CprA protein is necessary for a different aspect of polymyxin resistance. This protein has a domain structure with a strong similarity to the extended short-chain dehydrogenase/reductase family that comprises isomerases, lyases, and oxidoreductases. These results suggest a new avenue through which to pursue targeted inhibition of polymyxin resistance.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

scholarly journals PmrAB, a Two-Component Regulatory System of Pseudomonas aeruginosa That Modulates Resistance to Cationic Antimicrobial Peptides and Addition of Aminoarabinose to Lipid A

2004 ◽  
Vol 186 (2) ◽  
pp. 575-579 ◽  
Author(s):  
Samuel M. Moskowitz ◽  
Robert K. Ernst ◽  
Samuel I. Miller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Peptides ◽  
Lipid A ◽  
Regulatory System ◽  
Signal Transduction System ◽  
Cationic Antimicrobial Peptides ◽  
Two Component ◽  
Serovar Typhimurium ◽  
Two Component Signal Transduction ◽  
Resistant Mutants

ABSTRACT Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.

scholarly journals PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

2011 ◽  
Vol 56 (2) ◽  
pp. 1019-1030 ◽  
Author(s):  
Samuel M. Moskowitz ◽  
Mark K. Brannon ◽  
Nandini Dasgupta ◽  
Miyuki Pier ◽  
Nicole Sgambati ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Clinical Isolates ◽  
Gain Of Function ◽  
Content Type ◽  
Regulatory Systems ◽  
Repeated Passage ◽  
High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance,P. aeruginosaisolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-functionpmrBalleles that conferred polymyxin resistance to strains with a wild-type orpmrABdeletion background. Double mutantpmrBalleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutantpmrBalleles induced transcription from the promoter of thearnBoperon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate thatpmrBgain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains ofP. aeruginosa.

scholarly journals Phase-Variable Expression oflptAModulates the Resistance of Neisseria gonorrhoeae to Cationic Antimicrobial Peptides

2014 ◽  
Vol 58 (7) ◽  
pp. 4230-4233 ◽  
Author(s):  
Justin L. Kandler ◽  
Sandeep J. Joseph ◽  
Jacqueline T. Balthazar ◽  
Vijaya Dhulipala ◽  
Timothy D. Read ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antimicrobial Peptides ◽  
High Frequency ◽  
Lipid A ◽  
Bacterial Resistance ◽  
Phase Variable ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Variable Expression

ABSTRACTPhosphoethanolamine (PEA) decoration of lipid A produced byNeisseria gonorrhoeaehas been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) andin vivofitness during experimental infection. We now report that thelptAgene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat withinlptAcan influence gonococcal resistance to CAMPs.

scholarly journals The Vibrio cholerae VprA-VprB Two-Component System Controls Virulence through Endotoxin Modification

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Carmen M. Herrera ◽  
Alexander A. Crofts ◽  
Jeremy C. Henderson ◽  
S. Cassandra Pingali ◽  
Bryan W. Davies ◽  
...  
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Vibrio Cholerae ◽  
Lipid A ◽  
Host Immune System ◽  
Two Component System ◽  
Cationic Antimicrobial Peptides ◽  
Component System ◽  
Content Type ◽  
Two Component

ABSTRACTThe bacterial cell surface is the first structure the host immune system targets to prevent infection. Cationic antimicrobial peptides of the innate immune system bind to the membrane of Gram-negative pathogens via conserved, surface-exposed lipopolysaccharide (LPS) molecules. We recently reported that modern strains of the global intestinal pathogenVibrio choleraemodify the anionic lipid A domain of LPS with a novel moiety, amino acids. Remarkably, glycine or diglycine addition to lipid A alters the surface charge of the bacteria to help evade the cationic antimicrobial peptide polymyxin. However, the regulatory mechanisms of lipid A modification inV. choleraeare unknown. Here, we identify a novel two-component system that regulates lipid A glycine modification by responding to important biological cues associated with pathogenesis, including bile, mildly acidic pH, and cationic antimicrobial peptides. The histidine kinase Vc1319 (VprB) and the response regulator Vc1320 (VprA) respond to these signals and are required for the expression of thealmEFGoperon that encodes the genes essential for glycine modification of lipid A. Importantly, both the newly identified two-component system and the lipid A modification machinery are required for colonization of the mammalian host. This study demonstrates howV. choleraeuses a previously unknown regulatory network, independent of well-studiedV. choleraevirulence factors and regulators, to respond to the host environment and cause infection.IMPORTANCEVibrio cholerae, the etiological agent of cholera disease, infects millions of people every year.V. choleraeEl Tor and classical biotypes have been responsible for all cholera pandemics. The El Tor biotype responsible for the current seventh pandemic has displaced the classical biotype worldwide and is highly resistant to cationic antimicrobial peptides, like polymyxin B. This resistance arises from the attachment of one or two glycine residues to the lipid A domain of lipopolysaccharide, a major surface component of Gram-negative bacteria. Here, we identify the VprAB two-component system that regulates the charge of the bacterial surface by directly controlling the expression of genes required for glycine addition to lipid A. The VprAB-dependent lipid A modification confers polymyxin B resistance and contributes significantly to pathogenesis. This finding is relevant for understanding howVibrio choleraehas evolved mechanisms to facilitate the evasion of the host immune system and increase bacterial fitness.

scholarly journals PhoQ Mutations Promote Lipid A Modification and Polymyxin Resistance of Pseudomonas aeruginosa Found in Colistin-Treated Cystic Fibrosis Patients

2011 ◽  
Vol 55 (12) ◽  
pp. 5761-5769 ◽  
Author(s):  
Amanda K. Miller ◽  
Mark K. Brannon ◽  
Laurel Stevens ◽  
Helle Krogh Johansen ◽  
Sara E. Selgrade ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Loss Of Function ◽  
Content Type ◽  
Clinical Strains ◽  
Regulatory Systems ◽  
Polymyxin E ◽  
High Level

ABSTRACTPseudomonas aeruginosacan develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance,P. aeruginosastrains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-functionphoQalleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations inphoPwere identified in some cystic fibrosis strains with resistance-conferringphoQmutations, suggesting that additional loci can be involved in polymyxin resistance inP. aeruginosa. Disruption of chromosomalphoQin the presence of an intactphoPallele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of thepmrH(arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate thatphoQloss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains ofP. aeruginosa.

scholarly journals Perturbations of Phosphatidate Cytidylyltransferase (CdsA) Mediate Daptomycin Resistance in Streptococcus mitis/oralis by a Novel Mechanism

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Nagendra N. Mishra ◽  
Truc T. Tran ◽  
Ravin Seepersaud ◽  
Cristina Garcia-de-la-Maria ◽  
Kym Faull ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Cross Resistance ◽  
Human Neutrophils ◽  
Small Subset ◽  
Streptococcus Mitis ◽  
Anionic Phospholipid ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
High Level

ABSTRACT Streptococcus mitis/oralis is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant S. mitis/oralis strains due to its in vitro bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, S. mitis/oralis has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both in vivo and in vitro. Using isogenic DAP-susceptible and DAP-resistant S. mitis/oralis strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in cdsA (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant S. mitis/oralis strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in S. mitis/oralis and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

scholarly journals Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Jie Lin ◽  
Chunquan Xu ◽  
Renchi Fang ◽  
Jianming Cao ◽  
Xiucai Zhang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Population Analysis ◽  
Colistin Resistance ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Expression Levels ◽  
Maldi Tof ◽  
Broth Microdilution Method ◽  
Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

scholarly journals Spatiotemporal Distribution of Pseudomonas aeruginosa Alkyl Quinolones under Metabolic and Competitive Stress

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Tianyuan Cao ◽  
Jonathan V. Sweedler ◽  
Paul W. Bohn ◽  
Joshua D. Shrout
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Species ◽  
Metabolic Stress ◽  
Opportunistic Pathogen ◽  
Cell Interaction ◽  
Chemical Imaging ◽  
Carbon Limitation ◽  
Bacterial Strains ◽  
Content Type ◽  
High Level

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen important to diseases such as cystic fibrosis. P. aeruginosa has multiple quorum-sensing (QS) systems, one of which utilizes the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). Here, we use hyperspectral Raman imaging to elucidate the spatiotemporal PQS distributions that determine how P. aeruginosa regulates surface colonization and its response to both metabolic stress and competition from other bacterial strains. These chemical imaging experiments illustrate the strong link between environmental challenges, such as metabolic stress caused by nutritional limitations or the presence of another bacterial species, and PQS signaling. Metabolic stress elicits a complex response in which limited nutrients induce the bacteria to produce PQS earlier, but the bacteria may also pause PQS production entirely if the nutrient concentration is too low. Separately, coculturing P. aeruginosa in the proximity of another bacterial species, or its culture supernatant, results in earlier production of PQS. However, these differences in PQS appearance are not observed for all alkyl quinolones (AQs) measured; the spatiotemporal response of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) is highly uniform for most conditions. These insights on the spatiotemporal distributions of quinolones provide additional perspective on the behavior of P. aeruginosa in response to different environmental cues. IMPORTANCE Alkyl quinolones (AQs), including Pseudomonas quinolone signal (PQS), made by the opportunistic pathogen Pseudomonas aeruginosa have been associated with both population density and stress. The regulation of AQ production is known to be complex, and the stimuli that modulate AQ responses are not fully clear. Here, we have used hyperspectral Raman chemical imaging to examine the temporal and spatial profiles of AQs exhibited by P. aeruginosa under several potentially stressful conditions. We found that metabolic stress, effected by carbon limitation, or competition stress, effected by proximity to other species, resulted in accelerated PQS production. This competition effect did not require cell-to-cell interaction, as evidenced by the fact that the addition of supernatants from either Escherichia coli or Staphylococcus aureus led to early appearance of PQS. Lastly, the fact that these modulations were observed for PQS but not for all AQs suggests a high level of complexity in AQ regulation that remains to be discerned.

scholarly journals A rhlI 5′ UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Maureen K. Thomason ◽  
Maya Voichek ◽  
Daniel Dar ◽  
Victoria Addis ◽  
David Fitzgerald ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Regulatory Elements ◽  
Content Type ◽  
Reading Frame ◽  
Bacterial Physiology ◽  
Small Regulatory Rna ◽  
Key Enzyme ◽  
Opportunistic Human Pathogen

ABSTRACT N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5′ untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS. IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

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