Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Shyamasree De Majumdar, Jing Yu, Maria Fookes, Sean P. McAteer, Enrique Llobet, Sarah Finn, Shaun Spence, Avril Monaghan, Adrien Kissenpfennig, Rebecca J. Ingram, José Bengoechea, David L. Gally, Séamus Fanning, Joseph S. Elborn, Thamarai Schneiders, Alan Hauser

    Research output: Contribution to journalArticle

    36 Citations (Scopus)

    Abstract

    Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.
    LanguageEnglish
    Pagese1004627
    JournalPLoS Pathogens
    Volume11
    Issue number1
    DOIs
    Publication statusPublished - 29 Jan 2015

    Fingerprint

    Regulon
    Klebsiella pneumoniae
    Lipid A
    Citrobacter
    Colistin
    Polymyxin B
    Enterobacter
    Surface Properties
    Multiple Drug Resistance
    Infection
    Salmonella
    Genes
    Lipopolysaccharides
    Macrophages
    Phenotype
    Pharmaceutical Preparations
    Proteins

    Keywords

    • RamA
    • Klebsiella

    Cite this

    De Majumdar, S., Yu, J., Fookes, M., McAteer, S. P., Llobet, E., Finn, S., ... Hauser, A. (2015). Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation. PLoS Pathogens, 11(1), e1004627. https://doi.org/10.1371/journal.ppat.1004627
    De Majumdar, Shyamasree ; Yu, Jing ; Fookes, Maria ; McAteer, Sean P. ; Llobet, Enrique ; Finn, Sarah ; Spence, Shaun ; Monaghan, Avril ; Kissenpfennig, Adrien ; Ingram, Rebecca J. ; Bengoechea, José ; Gally, David L. ; Fanning, Séamus ; Elborn, Joseph S. ; Schneiders, Thamarai ; Hauser, Alan. / Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation. In: PLoS Pathogens. 2015 ; Vol. 11, No. 1. pp. e1004627.
    @article{18a9b78f42c84b008f5b51d53f22afdc,
    title = "Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation",
    abstract = "Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.",
    keywords = "RamA, Klebsiella",
    author = "{De Majumdar}, Shyamasree and Jing Yu and Maria Fookes and McAteer, {Sean P.} and Enrique Llobet and Sarah Finn and Shaun Spence and Avril Monaghan and Adrien Kissenpfennig and Ingram, {Rebecca J.} and Jos{\'e} Bengoechea and Gally, {David L.} and S{\'e}amus Fanning and Elborn, {Joseph S.} and Thamarai Schneiders and Alan Hauser",
    note = "Reference text: 1. Barbosa TM, Levy SB (2000) Differential Expression of Over 60 Chromosomal Genes in Escherichia coli by Constitutive Expression of MarA. J Bacteriol 182: 3467–3474. doi: 10.1128/JB.182.12.3467-3474.2000 PMID: 10852879 2. Pomposiello PJ, Bennik MH, Demple B (2001) Genome-wide Transcriptional Profiling of the Escherichia coli Responses to Superoxide Stress and Sodium Salicylate. J Bacteriol 183: 3890–3902. doi: 10.1128/JB.183.13.3890-3902.2001 PMID: 11395452 3. Bennik MH, Pomposiello PJ, Thorne DF, Demple B (2000) Defining a rob regulon in Escherichia coli by using transposon mutagenesis. J Bacteriol 182: 3794–3801. doi: 10.1128/JB.182.13.3794-3801.2000 PMID: 10850996 4. Bailey AM, Ivens A, Kingsley R, Cottell JL, Wain J, et al. (2010) RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol 192:1607–1616. doi: 10.1128/JB.01517-09 PMID: 20081028 5. De Majumdar S, Veleba M, Finn S, Fanning S, Schneiders T (2013) Elucidating the regulon of multidrug resistance regulator RarA in Klebsiella pneumoniae. Antimicrob Agents Chemother 57: 1603–1609.doi: 10.1128/AAC.01998-12 PMID: 23318802 6. Martin RG, Rosner JL (2001) The AraC transcriptional activators. Curr Opin Microbiol 4: 132–137. doi:10.1016/S1369-5274(00)00178-8 PMID: 11282467 7. Piddock LJ (2006) Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol 4:629–636. doi: 10.1038/nrmicro1464 PMID: 16845433 8. Dangi B, Pelupessey P, Martin RG, Rosner JL, Louis JM, et al. (2001) Structure and Dynamics of MarA-DNA Complexes: An NMR Investigation. J Mol Biol 314: 113–127. doi:10.1006/jmbi.2001.5106 PMID: 11724537 9. Griffith KL, Shah IM, Myers TE, O’Neill MC, Wolf RE Jr. (2002) Evidence for “pre-recruitment” as a newmechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cellrelative to the number of SoxS molecules per cell. Biochem Biophys Res Commun 291: 979–986. doi:10.1006/bbrc.2002.6559 PMID: 11866462 10. Alekshun MN, Levy SB (1997) Regulation of Chromosomally Mediated Multiple Antibiotic Resistance: the mar Regulon. Antimicrob Agents Chemother 41: 2067–2075. PMID: 9333027 11. Zheng J, Tian F, Cui S, Song J, Zhao S, et al. (2011) Differential gene expression by RamA in ciprofloxacin-resistant Salmonella Typhimurium. PLoS One 6: e22161. doi: 10.1371/journal.pone.0022161 PMID: 21811569 12. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ (2005) Small-molecule inhibitor of Vibrio cholera virulence and intestinal colonization. Science 310: 670–674. doi: 10.1126/science.1116739 PMID:16223984 13. Casaz P, Garrity-Ryan LK, McKenney D, Jackson C, Levy SB, et al. (2006) MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis. Microbiology152: 3643–3650. doi: 10.1099/mic.0.2006/000604-0 PMID: 17159217 14. Chollet R, Chevalier J, Bollet C, Pages JM, Davin-Regli A (2004) RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 48:2518–2523. doi: 10.1128/AAC.48.7.2518-2523.2004 PMID: 15215103 15. Yassien MA, Ewis HE, Lu CD, Abdelal AT (2002) Molecular cloning and characterization of the Salmonella enterica Serovar Paratyphi B rma Gene, which confers multiple drug resistance in Escherichia coli. Antimicrob Agents Chemother 46: 360–366. doi: 10.1128/AAC.46.2.360-366.2002 PMID:11796342 16. Rosenblum R, Khan E, Gonzalez G, Hasan R, Schneiders T (2011) Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae. Int J Antimicrob Agents 38:39–45. doi: 10.1016/j.ijantimicag.2011.02.012 PMID: 21514798 17. Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M (2010) ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to Tigecycline. Antimicrob Agents Chemother 54: 2720–2723. doi: 10.1128/AAC.00085-10 PMID: 20350947 18. Abouzeed YM, Baucheron S, Cloeckaert A (2008) ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 52:2428–2434. doi: 10.1128/AAC.00084-08 PMID: 18443112 19. Yamasaki S, Nikaido E, Nakashima R, Sakurai K, Fujiwara D, et al. (2013) The crystal structure of multidrug-resistance regulator RamR with multiple drugs. Nat Commun 4: 2078. doi: 10.1038/ncomms3078 PMID: 23800819 20. Martin RG, Bartlett ES, Rosner JL, Wall ME (2008) Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration. 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    De Majumdar, S, Yu, J, Fookes, M, McAteer, SP, Llobet, E, Finn, S, Spence, S, Monaghan, A, Kissenpfennig, A, Ingram, RJ, Bengoechea, J, Gally, DL, Fanning, S, Elborn, JS, Schneiders, T & Hauser, A 2015, 'Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation', PLoS Pathogens, vol. 11, no. 1, pp. e1004627. https://doi.org/10.1371/journal.ppat.1004627

    Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation. / De Majumdar, Shyamasree; Yu, Jing; Fookes, Maria; McAteer, Sean P.; Llobet, Enrique; Finn, Sarah; Spence, Shaun; Monaghan, Avril; Kissenpfennig, Adrien; Ingram, Rebecca J.; Bengoechea, José; Gally, David L.; Fanning, Séamus; Elborn, Joseph S.; Schneiders, Thamarai; Hauser, Alan.

    In: PLoS Pathogens, Vol. 11, No. 1, 29.01.2015, p. e1004627.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

    AU - De Majumdar, Shyamasree

    AU - Yu, Jing

    AU - Fookes, Maria

    AU - McAteer, Sean P.

    AU - Llobet, Enrique

    AU - Finn, Sarah

    AU - Spence, Shaun

    AU - Monaghan, Avril

    AU - Kissenpfennig, Adrien

    AU - Ingram, Rebecca J.

    AU - Bengoechea, José

    AU - Gally, David L.

    AU - Fanning, Séamus

    AU - Elborn, Joseph S.

    AU - Schneiders, Thamarai

    AU - Hauser, Alan

    N1 - Reference text: 1. Barbosa TM, Levy SB (2000) Differential Expression of Over 60 Chromosomal Genes in Escherichia coli by Constitutive Expression of MarA. J Bacteriol 182: 3467–3474. doi: 10.1128/JB.182.12.3467-3474.2000 PMID: 10852879 2. Pomposiello PJ, Bennik MH, Demple B (2001) Genome-wide Transcriptional Profiling of the Escherichia coli Responses to Superoxide Stress and Sodium Salicylate. J Bacteriol 183: 3890–3902. doi: 10.1128/JB.183.13.3890-3902.2001 PMID: 11395452 3. Bennik MH, Pomposiello PJ, Thorne DF, Demple B (2000) Defining a rob regulon in Escherichia coli by using transposon mutagenesis. J Bacteriol 182: 3794–3801. doi: 10.1128/JB.182.13.3794-3801.2000 PMID: 10850996 4. Bailey AM, Ivens A, Kingsley R, Cottell JL, Wain J, et al. (2010) RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol 192:1607–1616. doi: 10.1128/JB.01517-09 PMID: 20081028 5. De Majumdar S, Veleba M, Finn S, Fanning S, Schneiders T (2013) Elucidating the regulon of multidrug resistance regulator RarA in Klebsiella pneumoniae. Antimicrob Agents Chemother 57: 1603–1609.doi: 10.1128/AAC.01998-12 PMID: 23318802 6. Martin RG, Rosner JL (2001) The AraC transcriptional activators. Curr Opin Microbiol 4: 132–137. doi:10.1016/S1369-5274(00)00178-8 PMID: 11282467 7. Piddock LJ (2006) Multidrug-resistance efflux pumps—not just for resistance. Nat Rev Microbiol 4:629–636. doi: 10.1038/nrmicro1464 PMID: 16845433 8. Dangi B, Pelupessey P, Martin RG, Rosner JL, Louis JM, et al. (2001) Structure and Dynamics of MarA-DNA Complexes: An NMR Investigation. J Mol Biol 314: 113–127. doi:10.1006/jmbi.2001.5106 PMID: 11724537 9. Griffith KL, Shah IM, Myers TE, O’Neill MC, Wolf RE Jr. (2002) Evidence for “pre-recruitment” as a newmechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cellrelative to the number of SoxS molecules per cell. Biochem Biophys Res Commun 291: 979–986. doi:10.1006/bbrc.2002.6559 PMID: 11866462 10. Alekshun MN, Levy SB (1997) Regulation of Chromosomally Mediated Multiple Antibiotic Resistance: the mar Regulon. Antimicrob Agents Chemother 41: 2067–2075. PMID: 9333027 11. Zheng J, Tian F, Cui S, Song J, Zhao S, et al. (2011) Differential gene expression by RamA in ciprofloxacin-resistant Salmonella Typhimurium. PLoS One 6: e22161. doi: 10.1371/journal.pone.0022161 PMID: 21811569 12. Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ (2005) Small-molecule inhibitor of Vibrio cholera virulence and intestinal colonization. Science 310: 670–674. doi: 10.1126/science.1116739 PMID:16223984 13. Casaz P, Garrity-Ryan LK, McKenney D, Jackson C, Levy SB, et al. (2006) MarA, SoxS and Rob function as virulence factors in an Escherichia coli murine model of ascending pyelonephritis. Microbiology152: 3643–3650. doi: 10.1099/mic.0.2006/000604-0 PMID: 17159217 14. Chollet R, Chevalier J, Bollet C, Pages JM, Davin-Regli A (2004) RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 48:2518–2523. doi: 10.1128/AAC.48.7.2518-2523.2004 PMID: 15215103 15. Yassien MA, Ewis HE, Lu CD, Abdelal AT (2002) Molecular cloning and characterization of the Salmonella enterica Serovar Paratyphi B rma Gene, which confers multiple drug resistance in Escherichia coli. Antimicrob Agents Chemother 46: 360–366. doi: 10.1128/AAC.46.2.360-366.2002 PMID:11796342 16. Rosenblum R, Khan E, Gonzalez G, Hasan R, Schneiders T (2011) Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae. Int J Antimicrob Agents 38:39–45. doi: 10.1016/j.ijantimicag.2011.02.012 PMID: 21514798 17. Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M (2010) ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to Tigecycline. Antimicrob Agents Chemother 54: 2720–2723. doi: 10.1128/AAC.00085-10 PMID: 20350947 18. Abouzeed YM, Baucheron S, Cloeckaert A (2008) ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 52:2428–2434. doi: 10.1128/AAC.00084-08 PMID: 18443112 19. Yamasaki S, Nikaido E, Nakashima R, Sakurai K, Fujiwara D, et al. (2013) The crystal structure of multidrug-resistance regulator RamR with multiple drugs. Nat Commun 4: 2078. doi: 10.1038/ncomms3078 PMID: 23800819 20. Martin RG, Bartlett ES, Rosner JL, Wall ME (2008) Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration. 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    PY - 2015/1/29

    Y1 - 2015/1/29

    N2 - Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

    AB - Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

    KW - RamA

    KW - Klebsiella

    U2 - 10.1371/journal.ppat.1004627

    DO - 10.1371/journal.ppat.1004627

    M3 - Article

    VL - 11

    SP - e1004627

    JO - PLoS Pathogens

    T2 - PLoS Pathogens

    JF - PLoS Pathogens

    SN - 1553-7366

    IS - 1

    ER -

    De Majumdar S, Yu J, Fookes M, McAteer SP, Llobet E, Finn S et al. Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation. PLoS Pathogens. 2015 Jan 29;11(1):e1004627. https://doi.org/10.1371/journal.ppat.1004627