Proteomic and Systems Biology Analysis of the Monocyte Response to <i>Coxiella burnetii</i> Infection

<div><p><i>Coxiella burnetii</i> is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and <i>C. burnetii</i> is considered a significant bioterror threat. <i>C. burnetii</i> occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of <i>C. burnetii</i> and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.</p></div

DAVID analysis of the linkages of transaldolase 1 (TAL1).

<p>Diagram from the KEGG pathway analysis database produced by DAVID⁶¹. The red star indicates TAL1 (EC 2.2.1.2). The diagram shows the involvement of transaldolase 1 in the pentose phosphate pathway.</p

Soluble protein fraction, 96 hr postinfection.

<p>Progenesis master image. Where outlined, blue numbered spots were determined to be differentially expressed in a statistically significant manner, following image analysis and offline statistical analysis. The analysis included 3 biological replicates, with 6 technical replicates per biological replicate, including reciprocal Zdye color labeling, as described in the text. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069558#pone-0069558-t001" target="_blank">tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069558#pone-0069558-t002" target="_blank">2</a> for statistical analyses and protein identifications.</p

DAVID analysis of the linkages of aldehyde dehydrogenase 2 (Aldh2).

<p>Diagram from the KEGG pathway analysis database produced by DAVID⁶¹. The red star indicates Aldh2 (EC 1.2.1.3). The diagram shows the involvement of aldehyde dehydrogenase 2 (Aldh2) in Valine, Leucine, and Isoleucine degradation.</p

Bioinformatic data for differentially expressed monocyte proteins.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069558#s3" target="_blank">Results</a> of the bioinformatic analysis for protein identified as differentially expressed. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069558#pone-0069558-t001" target="_blank">table 1</a> for statistical analysis of the changes in individual protein spots from the gel data. The scores for the various bioinformatic tools were calculated by their respective software, as was %FDR for X!Hunter and X!Tandem P3 (referred to as P3 in the table). The number of unique peptides and the %coverage was calculated, following manual validation of the peptides from each of the tools. Accession numbers for Mascot are for the NCBInr database, the accession numbers for X!Hunter and P3 are for the IPI database.</p

Detail of regulation and proposed Rab7 functions from figure S3.

<p>Detail from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069558#pone.0069558.s002" target="_blank">figure S2</a>, showing the regulation of Rab7 GTP activity and the various potential roles of Rab7GTP. <i>C. burnetii</i> infection alters cellular cholesterol synthesis and alters lipid raft composition. Altered lipid raft composition impairs hydrolase delivery and bacterial lysis, as well as favoring recruitment of Rab7 to the phagolysosomal membrane. Proposed functional consequences of maintaining Rab7 recruitment to the phagosomal membrane include maintenance of vATPase at the phagolysosomal membrane, which would act to maintain acidification of the phagolysosome, required for metabolic and replicative processes of <i>C. burnetii</i>. RILP =  Rab Interacting Lysosomal Protein. GDI = Guanine nucleotide Dissociation Inhibitor.</p

Differentially regulated protein identifications and statistical data.

<p>Regulated proteins from <i>C. burnetii</i> infected monocyte samples compared to uninfected monocytes. Spot numbers indicate the spot numbers in the gel images. Selection criteria were p<0.05 and power score >0.7 for the differences in protein abundance in control compared to infected conditions. Fold change refers to the direction and magnitude of protein expression changes in the infected samples compared to the uninfected controls.</p