Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Shi-qi An, Delphine L. Caly, Yvonne McCarthy, Sarah L. Murdoch, Joseph Ward, Melanie Febrer, J. Maxwell Dow, Robert P. Ryan (Lead / Corresponding author)

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    Abstract

    Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (Kd~2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.
    Original languageEnglish
    Article numbere1004429
    Number of pages12
    JournalPLoS Pathogens
    Volume10
    Issue number10
    DOIs
    Publication statusPublished - 16 Oct 2014

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    Virulence
    Proteins
    Stenotrophomonas maltophilia
    Biofilms
    Xanthomonas campestris
    bis(3',5')-cyclic diguanylic acid
    Calorimetry
    Mutation
    Second Messenger Systems
    Transcriptome
    Pseudomonas aeruginosa
    Transcription Factors
    Yeasts
    Genes

    Cite this

    An, S., Caly, D. L., McCarthy, Y., Murdoch, S. L., Ward, J., Febrer, M., ... Ryan, R. P. (2014). Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence. PLoS Pathogens, 10(10), [e1004429]. https://doi.org/10.1371/journal.ppat.1004429
    An, Shi-qi ; Caly, Delphine L. ; McCarthy, Yvonne ; Murdoch, Sarah L. ; Ward, Joseph ; Febrer, Melanie ; Dow, J. Maxwell ; Ryan, Robert P. / Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence. In: PLoS Pathogens. 2014 ; Vol. 10, No. 10.
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    abstract = "Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (Kd~2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.",
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    An, S, Caly, DL, McCarthy, Y, Murdoch, SL, Ward, J, Febrer, M, Dow, JM & Ryan, RP 2014, 'Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence', PLoS Pathogens, vol. 10, no. 10, e1004429. https://doi.org/10.1371/journal.ppat.1004429

    Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence. / An, Shi-qi; Caly, Delphine L.; McCarthy, Yvonne; Murdoch, Sarah L.; Ward, Joseph; Febrer, Melanie; Dow, J. Maxwell; Ryan, Robert P. (Lead / Corresponding author).

    In: PLoS Pathogens, Vol. 10, No. 10, e1004429, 16.10.2014.

    Research output: Contribution to journalArticle

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    An S, Caly DL, McCarthy Y, Murdoch SL, Ward J, Febrer M et al. Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence. PLoS Pathogens. 2014 Oct 16;10(10). e1004429. https://doi.org/10.1371/journal.ppat.1004429