Cloning and Porin Activity of the Major Outer Membrane Protein P1 from Coxiella burnetii

Abstract

Coxiella burnetii, the etiological agent of Q fever, is a gram-negative obligate intracellular bacterium. Two striking characteristics of this microorganism are its ability to thrive within a phagolysosome and its ability to persist in the environment outside a host cell. These abilities have been attributed to the existence of C. burnetii developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC). Variants differ in protein profiles, including differential expression of a major outer membrane protein (MOMP) of C. burnetii, designated P1. The ϳ29-kDa MOMP is highly expressed in LCV, downregulated in SCV, and not apparent in SDC. We sought to characterize P1 through purification of native protein for N-terminal analysis, cloning, and functional studies. Highly purified P1, extracted from C. burnetii membranes by using the zwitterionic detergent Empigen, allowed the determination of N-terminal and internal peptide sequences. The entire P1 coding locus was cloned by PCR amplification based upon these peptide sequences, followed by inverse PCR. Comparison of the predicted P1 amino acid sequences among the C. burnetii isolates Nine Mile, Koka, Scurry, and Kerns indicated a high degree of conservation. Structural prediction suggests that the peptide has a predominantly ␤-sheet conformation, consistent with bacterial porins. Typical porin characteristics were observed for native P1, including detergent solubilization properties, heat modification of purified protein, and channel formation in a planar lipid bilayer. Characterization of differentially expressed P1 as a porin increases our understanding of the function of morphological variants and their role in pathogenesis.

Temporal Analysis of Coxiella burnetii Morphological Differentiation

“…The peak expression levels in exponential-phase C. burnetii organisms of genes encoding the porin protein P1 (CBU0311) and the alternative sigma factor RpoS (rpoS) are in agreement with published results showing differential synthesis of these proteins by LCV (35,39). Upregulation of P1 during exponential phase is presumably an adaptation by LCV to acquire nutrients from the lysosomal milieu (39).…”

“…We next conducted an evaluation of gene expression during the C. burnetii developmental cycle using TaqMan QPCR. Genes selected for this analysis included those that encode ScvA (scvA) and Hq1 (hcbA), basic proteins preferentially expressed by SCV and likely involved in chromatin condensation (18,20), and the stationary-phase sigma factor RpoS (rpoS) and the porin P1 (CBU0311), proteins preferentially expressed by LCV (35,39). Expression of the presumed housekeeping genes encoding 16S rRNA (rrs) and citrate synthase (gltA) was also assessed, in addition to that of genes encoding DotA (dotA) and EnhC (enhC), a structural protein of the C. burnetii type IV secretory apparatus and a possible type IV effector protein, respectively (9,34).…”

“…Two highly basic proteins, ScvA and Hq1, are DNA binding proteins specific to the SCV that likely play roles in chromatin condensation (18,20). Elongation factors EF-Tu and EF-Ts, the stationary-phase sigma factor RpoS, and a protein with porin activity termed P1 are preferentially expressed by LCV (33,35,39).…”

Is Q Fever an emerging or re-emerging zoonosis?

“…These differentially expressed antigens could allow the bacteria to escape the immune response by a switch of the exposed proteins on the surface of the bacteria and to survive in the acid vacuole. For example, unlike SDC, there are more P1 major outer membrane proteins in the LCV than in the SCV [103,173]. This protein is able to form a pore within a planar lipid bilayer and functions as an anion-selective porin that permits the substrate to enter into LCV and SCV [136].…”

Identification and Characterization of an Immunodominant 28-Kilodalton Coxiella burnetii Outer Membrane Protein Specific to Isolates Associated with Acute Disease

“…Due to the difficulties in cultivation and purification of C. burnetii, only a limited group of OMP-encoding genes have been characterized (4,16,28). Candidates for OMPs include the QpH1 plasmid-specific gene cbhEЈ for a 42-kDa surface protein (15) and the QpRS plasmid-specific gene cbbEЈ for a 55-kDa surface protein (12)(13)(14), which have been speculated to be virulence related and associated with acute or chronic Q fever in humans.…”