Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

Abstract

c Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Effectors have consequently evolved to contain ELFs in the form of domains or motifs that functionally mimic eukaryotic host cell proteins [113]. The higher frequency of ELFs in bacterial effectors versus noneffectors provides a powerful predictive tool for assembling candidate effector lists [73,88,[90][91][92][94][95][96]107,113]. Indeed, recognition of a cohort of Coxiella genes encoding 33 amino acid ankyrin repeat domains (ARDs) resulted in identification of the first class of Coxiella Dot/Icm effectors [90,92].…”

“…Four of ten dot/icm genes tested complemented the corresponding mutant in L. pneumophila, suggesting functional similarity between the two systems [85,86]. Consequently, and owing to the lack of genetic tools for Coxiella, L. pneumophila was extensively used as a surrogate host to identify Coxiella T4BSS substrates using both CyaA and BlaM cytosolic translocation reporter assays [87][88][89][90][91][92][93][94][95][96]. Indeed, of the 143 currently recognized Coxiella Dot/Icm effectors, 125 were initially identified in L. pneumophila.…”

“…At the nucleic acid level, a strong predictor of effector genes is the presence of an upstream PmrA regulatory element [88,95,114]. PmrA is a response regulator that couples with the sensory histidine kinase PmrB to form a twocomponent system.…”

“…Indicators of effector genes are upstream PmrA regulatory motifs and genomic location [73,[87][88][89][90][91][92][94][95][96]107]. When assembling candidate effector gene lists, the predictive power of these features is amplified when multiple criteria are combined in machine learning [95] and manual approaches [88,94]. Ultimately, Dot/Icm substrates must be empirically validated using translocation assays.…”

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

“…The targeted peptide sequences are present in the S. cerevisiae MAPKs HOG1, FUS3, and KSS1; in the regulatory proteins REG1 and MMF1; and in subunits of several complexes (Pat1p, RIF1, and NMD2). Although the presence of protein phosphatase activity has not yet been reported for E. coli HADs (25,40), this activity has been proposed for several microbial effector proteins with HAD domains from the pathogenic bacteria Porphyromonas gingivalis and Coxiella burnetii (76,77).…”