A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae

AbstractIneffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

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Identification of Proteus mirabilisMutants with Increased Sensitivity to Antimicrobial Peptides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.7.2030-2037.2001 ◽

2001 ◽

Vol 45 (7) ◽

pp. 2030-2037 ◽

Cited By ~ 79

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.

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

Biochimie ◽

10.1016/j.biochi.2017.08.008 ◽

2017 ◽

Vol 141 ◽

pp. 16-20 ◽

Cited By ~ 8

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

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

Journal of Bacteriology ◽

10.1128/jb.00023-10 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2044-2052 ◽

Cited By ~ 31

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.

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Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes

Bulletin of Entomological Research ◽

10.1017/s0007485316000638 ◽

2016 ◽

Vol 107 (1) ◽

pp. 66-76 ◽

Cited By ~ 5

Author(s):

M.I. Boguś ◽

W. Wieloch ◽

M. Ligęza-Żuber

Keyword(s):

Entomopathogenic Fungus ◽

Galleria Mellonella ◽

Elongation Factor ◽

Peptide Sequence ◽

Mass Spectrometry Analysis ◽

Sequence Coverage ◽

Spectrometry Analysis ◽

Conidiobolus Coronatus ◽

Liquid Chromatography Tandem Mass

AbstractCoronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC–MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva−1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

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Molecular Characterization and Genomic Analysis of Novel Phage vB_ ShiP-A7 Infecting Multidrug-Resistant Shiglla Fexneri and Escherichia Coli

10.21203/rs.3.rs-96088/v1 ◽

2020 ◽

Author(s):

Jing Xu ◽

Yu Gu ◽

Xinyan Yu ◽

Ruiyang Zhang ◽

Xuesen Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Case Reports ◽

Burst Size ◽

Shigella Flexneri ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Open Reading Frames ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Spherical Head

Abstract BackgroundPhage therapy has regained more attention due to the rise of multidrug-resistant (MDR) bacteria. Several case reports demonstrated clinical application of phage in resolving infections caused by MDR bacteria in recent years. ResultsWe isolated a new phage, vB_ShiP-A7, and then investigated its characteristics. Phage vB_ShiP-A7 is a member of Podoviridae that has an icosahedral spherical head and a short tail. vB_ShiP-A7 has large burst size and short replication time. vB_ShiP-A7’s genome is linear double stranded DNA composed of 40058 bp, encoding forty-three putative open reading frames. Comparative genome analysis demonstrated vB_ShiP-A7’s genome sequence is closely related to fifteen different phages (coverage 74-88%, identity 86-93%). Mass Spectrometry analysis revealed that twelve known proteins and six hypothetical proteins exist in particles of vB_ShiP-A7. Genome and proteome analyses confirmed the absence of lysogen-related proteins and toxic proteins in this phage. In addition, phage vB_ShiP-A7 can significantly reduce the growth of clinical MDR stains of Shigella ﬂexneri and Escherichia coli in liquid culture. Furthermore, vB_ShiP-A7 can disrupt biofilms formed by Shigella ﬂexneri or Escherichia coli in vitro. ConclusionPhage vB_ShiP-A7 is a stable novel phage, which has a strong application potential to inhibit MDR stains of Shigella ﬂexneri and Escherichia coli. Comparing the genomes between vB_ShiP-A7 and other closely-related phages will help us better understand the evolutionary mechanism of phages.

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Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01586-18 ◽

2019 ◽

Vol 63 (3) ◽

Cited By ~ 18

Author(s):

Stefanie Gerson ◽

Jonathan W. Betts ◽

Kai Lucaßen ◽

Carolina Silva Nodari ◽

Julia Wille ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Lipid A ◽

Galleria Mellonella ◽

Resistance Mechanism ◽

Resistant Isolate ◽

Infection Model ◽

Colistin Resistance ◽

Strain Specificity ◽

Content Type

ABSTRACT Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

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2-Hydroxylation ofAcinetobacter baumanniiLipid A Contributes to Virulence

Infection and Immunity ◽

10.1128/iai.00066-19 ◽

2019 ◽

Vol 87 (4) ◽

Cited By ~ 6

Author(s):

Toby L. Bartholomew ◽

Timothy J. Kidd ◽

Joana Sá Pessoa ◽

Raquel Conde Álvarez ◽

José A. Bengoechea

Keyword(s):

Protein Kinase ◽

Lipid A ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Polymyxin B ◽

Interleukin 10 ◽

Multidrug Resistant ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

Wide Range

ABSTRACTAcinetobacter baumanniicauses a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasingly frequent isolation of multidrug-resistant strains. There is a major gap in knowledge on the infection biology ofA. baumannii, and only a few virulence factors have been characterized, including lipopolysaccharide. The lipid A expressed byA. baumanniiis hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of lipid A have been already characterized. Here, we report the characterization ofA. baumanniiLpxO, which encodes the enzyme responsible for the 2-hydroxylation of lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyzes the 2-hydroxylation of the laurate transferred byA. baumanniiLpxL. LpxO-dependent lipid A 2-hydroxylation protectsA. baumanniifrom polymyxin B, colistin, and human β-defensin 3. LpxO contributes to the survival ofA. baumanniiin human whole blood and is required for pathogen survival in the waxmothGalleria mellonella. LpxO also protectsAcinetobacterfromG. mellonellaantimicrobial peptides and limits their expression. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of lipid A limits the activation of the mitogen-activated protein kinase Jun N-terminal protein kinase to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) via the activation of the transcriptional factor CREB. IL-10 in turn limits the production of inflammatory cytokines followingA. baumanniiinfection. Altogether, our studies suggest that LpxO is a candidate for the development of anti-A. baumanniidrugs.

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

Infection and Immunity ◽

10.1128/iai.00676-08 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5777-5789 ◽

Cited By ~ 27

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.

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Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.778968 ◽

2021 ◽

Vol 12 ◽

Author(s):

Matteo Cervoni ◽

Alessandra Lo Sciuto ◽

Chiara Bianchini ◽

Carmine Mancone ◽

Francesco Imperi

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Cell Envelope ◽

Resistance Mechanism ◽

Multidrug Resistant ◽

Transferase Activity ◽

Colistin Resistance ◽

Phosphoethanolamine Transferases ◽

Positively Charged ◽

Inducible Gene

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

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Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding

10.1101/2021.08.27.457985 ◽

2021 ◽

Author(s):

R. Christopher D. Furniss ◽

Nikol Kaderabkova ◽

Declan Barker ◽

Patricia Bernal ◽

Evgenia Maslova ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Galleria Mellonella ◽

Specific Resistance ◽

Cell Envelope ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Protein Homeostasis ◽

Colistin Resistance ◽

Resistance Proteins ◽

Chemical Inhibition

Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.

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