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Molecular cloning of a Coxiella burnetii gene encoding a macrophage infectivity potentiator (Mip) analogue
Y Y Mo1, N P Cianciotto, L P Mallavia
1Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
Abstract:
The gene encoding a protein that reacted with antibodies specific for Legionella pneumophila macrophage infectivity potentiator (LpMip) was cloned from Coxiella burnetii, the obligate intracellular rickettsia that causes Q fever in humans. Nucleotide sequencing analysis revealed an ORF encoding a gene product of 230 amino acids with a molecular mass of 25.5 kDa and a predicted pI of 10.7. The predicted amino acid sequence from the ORF shows similarity with Mip/Mip-like proteins of Legionella (46%) and Chlamydia (30%). Moreover, like LpMip, the amino acid sequence of the C terminus of this protein has over 35% identity to prokaryotic and eukaryotic FK506-binding proteins (FKBPs) that belong to a superfamily of immunophilins and are peptidyl-prolyl cis-trans isomerases (PPIases). When overproduced in Escherichia coli, the C. burnetii protein also exhibited PPIase activity. Taken together, these results demonstrate that C. burnetii encodes a Mip analogue (CbMip). A putative leader peptide at the N terminus of CbMip was detected by computer analysis. Furthermore, TnphoA mutagenesis demonstrated that in E. coli CbMip was secreted. In view of the role of Mip/Mip-like proteins in the pathogenesis of Legionella and Chlamydia, CbMip may be a C. burnetii virulence factor.
Insights
Coxiella burnetii encodes a Mip analogue (CbMip) with peptidyl-prolyl cis-trans isomerase activity, similar to Legionella pneumophila Mip. This C. burnetii protein may function as a virulence factor in Q fever pathogenesis.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Coxiella burnetii causes Q fever, a significant human health concern.
- Legionella pneumophila macrophage infectivity potentiator (LpMip) is crucial for bacterial pathogenesis.
- Mip/Mip-like proteins are implicated in the virulence of intracellular pathogens.
Purpose of the Study:
- To identify and characterize a Mip analogue in Coxiella burnetii.
- To investigate the enzymatic activity and potential role of the C. burnetii Mip analogue (CbMip).
Main Methods:
- Gene cloning and nucleotide sequencing of the C. burnetii Mip analogue.
- Bioinformatic analysis of the predicted amino acid sequence.
- Overexpression of CbMip in Escherichia coli and assessment of its peptidyl-prolyl cis-trans isomerase (PPIase) activity.
- TnphoA mutagenesis to determine protein secretion.
Main Results:
- A gene encoding a C. burnetii Mip analogue (CbMip) was successfully cloned.
- CbMip shares sequence similarity with LpMip and possesses PPIase activity.
- Computer analysis predicted a leader peptide, and experimental data confirmed CbMip secretion in E. coli.
Conclusions:
- Coxiella burnetii possesses a Mip analogue (CbMip) with functional PPIase activity.
- CbMip exhibits characteristics similar to known virulence factors of other intracellular bacteria.
- CbMip is a potential virulence factor contributing to C. burnetii pathogenesis.