2-Hydroxylation ofAcinetobacter baumanniiLipid A Contributes to Virulence

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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Identification and Characterization of Two Klebsiella pneumoniaelpxL Lipid A Late Acyltransferases and Their Role in Virulence

Infection and Immunity ◽

10.1128/iai.00068-17 ◽

2017 ◽

Vol 85 (9) ◽

Cited By ~ 15

Author(s):

Grant Mills ◽

Amy Dumigan ◽

Timothy Kidd ◽

Laura Hobley ◽

José A. Bengoechea

Keyword(s):

Lipid A ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Two Component System ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Candidate Target ◽

Tract Infections

ABSTRACT Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniae lpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs.

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Induction of Interleukin 10 byBorrelia burgdorferiIs Regulated by the Action of CD14-Dependent p38 Mitogen-Activated Protein Kinase and cAMP-Mediated Chromatin Remodeling

Infection and Immunity ◽

10.1128/iai.00781-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 4

Author(s):

Bikash Sahay ◽

Kathleen Bashant ◽

Nicole L. J. Nelson ◽

Rebeca L. Patsey ◽

Shiva Kumar Gadila ◽

...

Keyword(s):

Protein Kinase ◽

Lyme Borreliosis ◽

Human Peripheral Blood ◽

Interleukin 10 ◽

Mapk Signaling ◽

Lyme Arthritis ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

C3h Mice ◽

Mitogen Activated Protein

ABSTRACTHost genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacteriumBorrelia burgdorferi. C57BL/6 (B6) mice develop mild Lyme arthritis, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 mitogen-activated protein kinase (MAPK) signaling, B6 macrophages stimulated withB. burgdorferiproduce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including tumor necrosis factor (TNF). cAMP relaxes chromatin, while p38 increases binding of the transcription factors signal transducer and activator of transcription 3 (STAT3) and specific protein 1 (SP1) to the IL-10 promoter, leading to increased IL-10 production in B6 bone marrow-derived monocytes (BMDMs). Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill equipped to mitigate the damaging consequences ofB. burgdorferi-induced TNF. Intriguingly, an altered balance between anti-inflammatory and proinflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes, providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme borreliosis. Discovery of novel treatments or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain post-Lyme complications, in whom dysregulated immune responses may play a role.

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Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02656-17 ◽

2018 ◽

Vol 62 (6) ◽

Cited By ~ 15

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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Deciphering the Contribution of Human Meningothelial Cells to the Inflammatory and Antimicrobial Response at the Meninges

Infection and Immunity ◽

10.1128/iai.00477-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 4299-4310 ◽

Cited By ~ 9

Author(s):

Pierre-Joseph Royer ◽

Andrew J. Rogers ◽

Karl G. Wooldridge ◽

Patrick Tighe ◽

Jafar Mahdavi ◽

...

Keyword(s):

Protein Kinase ◽

Intracellular Signaling ◽

Cell Activation ◽

Mitogen Activated Protein Kinase ◽

Meningococcal Infection ◽

Minor Role ◽

Content Type ◽

Wide Range ◽

Mitogen Activated Protein ◽

High Level

ABSTRACTWe have investigated the response of primary human meningothelial cells toNeisseria meningitidis. Through a transcriptome analysis, we provide a comprehensive examination of the response of meningothelial cells to bacterial infection. A wide range of chemokines are elicited which act to attract and activate the main players of innate and adaptive immunity. We showed that meningothelial cells expressed a high level of Toll-like receptor 4 (TLR4), and, using a gene silencing strategy, we demonstrated the contribution of this pathogen recognition receptor in meningothelial cell activation. Secretion of interleukin-6 (IL-6), CXCL10, and CCL5 was almost exclusively TLR4 dependent and relied on MyD88 and TRIF adaptor cooperation. In contrast, IL-8 induction was independent of the presence of TLR4, MyD88, and TRIF. Transcription factors NF-κB p65, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal protein kinase (JNK1), IRF3, and IRF7 were activated after contact with bacteria. Interestingly, the protein kinase IRAK4 was found to play a minor role in the meningothelial cell response toNeisseriainfection. Our work highlights the role of meningothelial cells in the development of an immune response and inflammation in the central nervous system (CNS) in response to meningococcal infection. It also sheds light on the complexity of intracellular signaling after TLR triggering.

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Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02040-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 3

Author(s):

Fatma Ben Jeddou ◽

Léna Falconnet ◽

Alexandre Luscher ◽

Thissa Siriwardena ◽

Jean-Louis Reymond ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Polymyxin B ◽

Multidrug Resistant ◽

Sensor Kinase ◽

Loss Of Function ◽

Adaptive Responses ◽

Kinase Gene ◽

Polyamine Biosynthesis ◽

Content Type

ABSTRACT Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

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Comparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa

mSystems ◽

10.1128/msystems.00149-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 13

Author(s):

Mei-Ling Han ◽

Yan Zhu ◽

Darren J. Creek ◽

Yu-Wei Lin ◽

Alina D. Gutu ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Lipid A ◽

Therapeutic Option ◽

Polymyxin B ◽

Multidrug Resistant ◽

Central Carbon Metabolism ◽

Dynamic Interactions ◽

Content Type ◽

Comparative Metabolomics

ABSTRACT Polymyxins are a last-line therapy against multidrug-resistant Pseudomonas aeruginosa; however, resistance to polymyxins has been increasingly reported. Therefore, understanding the mechanisms of polymyxin activity and resistance is crucial for preserving their clinical usefulness. This study employed comparative metabolomics and transcriptomics to investigate the responses of polymyxin-susceptible P. aeruginosa PAK (polymyxin B MIC, 1 mg/liter) and its polymyxin-resistant pmrB mutant PAKpmrB6 (MIC, 16 mg/liter) to polymyxin B (4, 8, and 128 mg/liter) at 1, 4, and 24 h, respectively. Our results revealed that polymyxin B at 4 mg/liter induced different metabolic and transcriptomic responses between polymyxin-susceptible and -resistant P. aeruginosa. In strain PAK, polymyxin B significantly activated PmrAB and the mediated arn operon, leading to increased 4-amino-4-deoxy-L-arabinose (L-Ara4N) synthesis and the addition to lipid A. In contrast, polymyxin B did not increase lipid A modification in strain PAKpmrB6. Moreover, the syntheses of lipopolysaccharide and peptidoglycan were significantly decreased in strain PAK but increased in strain PAKpmrB6 due to polymyxin B treatment. In addition, 4 mg/liter polymyxin B significantly perturbed phospholipid and fatty acid levels and induced oxidative stress in strain PAK, but not in PAKpmrB6. Notably, the increased trehalose-6-phosphate levels indicate that polymyxin B potentially caused osmotic imbalance in both strains. Furthermore, 8 and 128 mg/liter polymyxin B significantly elevated lipoamino acid levels and decreased phospholipid levels but without dramatic changes in lipid A modification in wild-type and mutant strains, respectively. Overall, this systems study is the first to elucidate the complex and dynamic interactions of multiple cellular pathways associated with the polymyxin mode of action against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has been highlighted by the recent WHO Global Priority Pathogen List due to multidrug resistance. Without new antibiotics, polymyxins remain a last-line therapeutic option for this difficult-to-treat pathogen. The emergence of polymyxin resistance highlights the growing threat to our already very limited antibiotic armamentarium and the urgency to understand the exact mechanisms of polymyxin activity and resistance. Integration of the correlative metabolomics and transcriptomics results in the present study discovered that polymyxin treatment caused significant perturbations in the biosynthesis of lipids, lipopolysaccharide, and peptidoglycan, central carbon metabolism, and oxidative stress. Importantly, lipid A modifications were surprisingly rapid in response to polymyxin treatment at clinically relevant concentrations. This is the first study to reveal the dynamics of polymyxin-induced cellular responses at the systems level, which highlights that combination therapy should be considered to minimize resistance to the last-line polymyxins. The results also provide much-needed mechanistic information which potentially benefits the discovery of new-generation polymyxins.

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Attaching and Effacing Bacterial Effector NleC Suppresses Epithelial Inflammatory Responses by Inhibiting NF-κB and p38 Mitogen-Activated Protein Kinase Activation

Infection and Immunity ◽

10.1128/iai.05033-11 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3552-3562 ◽

Cited By ~ 65

Author(s):

Ho Pan Sham ◽

Stephanie R. Shames ◽

Matthew A. Croxen ◽

Caixia Ma ◽

Justin M. Chan ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Intestinal Inflammation ◽

Inflammatory Responses ◽

Mitogen Activated Protein Kinase ◽

Loop Model ◽

Wild Type ◽

Content Type ◽

Mitogen Activated Protein ◽

The Impact

ABSTRACTEnteropathogenicEscherichia coli(EPEC) and enterohemorrhagicE. coliare noninvasive attaching and effacing (A/E) bacterial pathogens that cause intestinal inflammation and severe diarrheal disease. These pathogens utilize a type III secretion system to deliver effector proteins into host epithelial cells, modulating diverse cellular functions, including the release of the chemokine interleukin-8 (IL-8). While studies have implicated the effectors NleE (non-locus of enterocyte effacement [LEE]-encoded effector E) and NleH1 in suppressing IL-8 release, by preventing NF-κB nuclear translocation, the impact of these effectors only partially replicates the immunosuppressive actions of wild-type EPEC, suggesting another effector or effectors are involved. Testing an array of EPEC mutants, we identified the non-LEE-encoded effector C (NleC) as also suppressing IL-8 release. Infection by ΔnleCEPEC led to exaggerated IL-8 release from infected Caco-2 and HT-29 epithelial cells. NleC localized to EPEC-induced pedestals, with signaling studies revealing NleC inhibits both NF-κB and p38 mitogen-activated protein kinase (MAPK) activation. UsingCitrobacter rodentium, a mouse-adapted A/E bacterium, we found that ΔnleCand wild-typeC. rodentium-infected mice carried similar pathogen burdens, yet ΔnleCstrain infection led to worsened colitis. Similarly, infection with ΔnleCC. rodentiumin a cecal loop model induced significantly greater chemokine responses than infection with wild-type bacteria. These studies thus advance our understanding of how A/E pathogens subvert host inflammatory responses.

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Diverse Hap43-Independent Functions of the Candida albicans CCAAT-Binding Complex

Eukaryotic Cell ◽

10.1128/ec.00014-13 ◽

2013 ◽

Vol 12 (6) ◽

pp. 804-815 ◽

Cited By ~ 12

Author(s):

Po-Chen Hsu ◽

Chun-Cheih Chao ◽

Cheng-Yao Yang ◽

Ya-Ling Ye ◽

Fu-Chen Liu ◽

...

Keyword(s):

Candida Albicans ◽

Protein Kinase ◽

Ribosome Biogenesis ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

Virulence Traits ◽

Wide Range ◽

Independent Functions ◽

Low Nitrogen

ABSTRACTThe CCAAT motif is ubiquitous in promoters of eukaryotic genomes. The CCAAT-binding complex (CBC) is conserved across a wide range of organisms, specifically recognizes the CCAAT motif, and modulates transcription directly or in cooperation with other transcription factors. InCandida albicans, CBC is known to interact with the repressor Hap43 to negatively regulate iron utilization genes in response to iron deprivation. However, the extent of additional functions of CBC is unclear. In this study, we explored new roles of CBC inC. albicansand found that CBC pleiotropically regulates many virulence traitsin vitro, including negative control of genes responsible for ribosome biogenesis and translation and positive regulation of low-nitrogen-induced filamentation. In addition,C. albicansCBC is involved in utilization of host proteins as nitrogen sources and in repression of cellular flocculation and adhesin gene expression. Moreover, our epistasis analyses suggest that CBC acts as a downstream effector of Rhb1-TOR signaling and controls low-nitrogen-induced filamentation via the Mep2-Ras1-protein kinase A (PKA)/mitogen-activated protein kinase (MAPK) pathway. Importantly, the phenotypes identified here are all independent of Hap43. Finally, deletion of genes encoding CBC components slightly attenuatedC. albicansvirulence in both zebrafish and murine models of infection. Our results thus highlight new roles ofC. albicansCBC in regulating multiple virulence traits in response to environmental perturbations and, finally, suggest potential targets for antifungal therapies as well as extending our understanding of the pathogenesis of other fungal pathogens.

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Circadian Activation of the Mitogen-Activated Protein Kinase MAK-1 Facilitates Rhythms in Clock-Controlled Genes in Neurospora crassa

Eukaryotic Cell ◽

10.1128/ec.00207-12 ◽

2012 ◽

Vol 12 (1) ◽

pp. 59-69 ◽

Cited By ~ 33

Author(s):

Lindsay D. Bennett ◽

Phillip Beremand ◽

Terry L. Thomas ◽

Deborah Bell-Pedersen

Keyword(s):

Protein Kinase ◽

Circadian Clock ◽

Neurospora Crassa ◽

Cellular Growth ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

Cell Wall Integrity Pathway ◽

Wide Range ◽

Mitogen Activated Protein ◽

Significant Enrichment

ABSTRACTThe circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungusNeurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway inSaccharomyces cerevisiaeand the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted fromNeurosporacells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ∼500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e.,ccg-1and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.

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