scholarly journals Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

2010 ◽  
Vol 192 (8) ◽  
pp. 2044-2052 ◽  
Author(s):  
Jyl S. Matson ◽  
Hyun Ju Yoo ◽  
Kristina Hakansson ◽  
Victor J. DiRita
Keyword(s):  
Antimicrobial Peptides ◽  
Vibrio Cholerae ◽  
Lipid A ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Peptide Antibiotic ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Antimicrobial Peptide Resistance ◽  
El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

scholarly journals Identification of Proteus mirabilisMutants with Increased Sensitivity to Antimicrobial Peptides

2001 ◽  
Vol 45 (7) ◽  
pp. 2030-2037 ◽  
Author(s):  
Andrea J. McCoy ◽  
Hongjian Liu ◽  
Timothy J. Falla ◽  
John S. Gunn
Keyword(s):  
Antimicrobial Peptides ◽  
Lipid A ◽  
Bacterial Species ◽  
Polymyxin B ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Swarming Motility ◽  
Spectrometry Analysis ◽  
O Antigen ◽  
Flight Mass Spectrometry

ABSTRACT Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example,Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilismutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization–time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.

scholarly journals Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

2015 ◽  
Vol 83 (3) ◽  
pp. 1199-1209 ◽  
Author(s):  
Kivanc Bilecen ◽  
Jiunn C. N. Fong ◽  
Andrew Cheng ◽  
Christopher J. Jones ◽  
David Zamorano-Sánchez ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Lipid A ◽  
Response Regulator ◽  
Regulatory Region ◽  
Regulatory System ◽  
Polymyxin B ◽  
Content Type ◽  
Two Component ◽  
Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

scholarly journals Modification of Lipooligosaccharide with Phosphoethanolamine by LptA in Neisseria meningitidis Enhances Meningococcal Adhesion to Human Endothelial and Epithelial Cells

2008 ◽  
Vol 76 (12) ◽  
pp. 5777-5789 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Russel W. Carlson ◽  
Artur Muszynski ◽  
Biswa Choudhury ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Inner Core ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Flight Mass Spectrometry ◽  
Epithelial Cell Lines

ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.

scholarly journals The Vibrio cholerae ToxR-Regulated Porin OmpU Confers Resistance to Antimicrobial Peptides

2004 ◽  
Vol 72 (6) ◽  
pp. 3577-3583 ◽  
Author(s):  
Jyoti Mathur ◽  
Matthew K. Waldor
Keyword(s):  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Surface Protein ◽  
Polymyxin B ◽  
Common Mechanism ◽  
Antimicrobial Protein ◽  
Wild Type ◽  
Human Gastrointestinal Tract ◽  
Serovar Typhimurium

ABSTRACT BPI (bactericidal/permeability-increasing) is a potent antimicrobial protein that was recently reported to be expressed as a surface protein on human gastrointestinal tract epithelial cells. In this study, we investigated the resistance of Vibrio cholerae, a small-bowel pathogen that causes cholera, to a BPI-derived peptide, P2. Unlike in Escherichia coli and Salmonella enterica serovar Typhimurium, resistance to P2 in V. cholerae was not dependent on the BipA GTPase. Instead, we found that ToxR, the master regulator of V. cholerae pathogenicity, controlled resistance to P2 by regulating the production of the outer membrane protein OmpU. Both toxR and ompU mutants were at least 100-fold more sensitive to P2 than were wild-type cells. OmpU also conferred resistance to polymyxin B sulfate, suggesting that this porin may impart resistance to cationic antibacterial proteins via a common mechanism. Studies of stationary-phase cells revealed that the ToxR-repressed porin OmpT may also contribute to P2 resistance. Finally, although the mechanism of porin-mediated resistance to antimicrobial peptides remains elusive, our data suggest that the BPI peptide sensitivity of OmpU-deficient V. cholerae is not attributable to a generally defective outer membrane.

scholarly journals A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
Prosenjit Samanta ◽  
Rahul Shubhra Mandal ◽  
Rudra Narayan Saha ◽  
Sreeja Shaw ◽  
Priyanka Ghosh ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Lipid A ◽  
Polymyxin B ◽  
Sensitive Strain ◽  
Two Component System ◽  
Content Type ◽  
Vibrio Cholerae O1 ◽  
Binding Cleft ◽  
Culturing Conditions ◽  
El Tor

ABSTRACT Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

Mass spectrometry analysis of intact Francisella bacteria identifies lipid A structure remodeling in response to acidic pH stress

Biochimie ◽  
2017 ◽  
Vol 141 ◽  
pp. 16-20 ◽  
Author(s):  
Camille B. Robert ◽  
Michael Thomson ◽  
Alain Vercellone ◽  
Francesca Gardner ◽  
Robert K. Ernst ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lipid A ◽  
Mass Spectrometry Analysis ◽  
Acidic Ph ◽  
Spectrometry Analysis ◽  
Ph Stress ◽  
Lipid A Structure

scholarly journals Oncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer

2021 ◽  
Vol 118 (21) ◽  
pp. e2016904118
Author(s):  
Derek K. Cheng ◽  
Tobiloba E. Oni ◽  
Jennifer S. Thalappillil ◽  
Youngkyu Park ◽  
Hsiu-Chi Ting ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Clinical Success ◽  
Treatment Options ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Ras Signaling ◽  
Wild Type ◽  
Feedback Mechanisms ◽  
Spectrometry Analysis ◽  
Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC.

scholarly journals Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

2010 ◽  
Vol 192 (18) ◽  
pp. 4651-4659 ◽  
Author(s):  
Wendy D. Smith ◽  
Jonathan A. Pointon ◽  
Emily Abbot ◽  
Hae Joo Kang ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pyogenes ◽  
Human Keratinocytes ◽  
Mass Spectrometry Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Spectrometry Analysis ◽  
Liquid Chromatography Tandem Mass ◽  
Protein Adhesion ◽  
Culture Supernatants

ABSTRACT Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.

scholarly journals Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Mei-Ling Han ◽  
Yan Zhu ◽  
Darren J. Creek ◽  
Yu-Wei Lin ◽  
Dovile Anderson ◽  
...  
Keyword(s):  
Lipid A ◽  
Therapeutic Option ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Wild Type ◽  
Metabolomics Data ◽  
Content Type ◽  
In The Wild ◽  
High Level ◽  
Acyl Coenzyme A

ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.

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