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

Abstract

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ϳ28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a ϳ28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.

Genetic Diversity of the Q Fever Agent,Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

“…One of these, CBUA0016 (cbhEЈ), was previously thought to be specific to isolates causing acute disease (42), an idea later discounted upon analysis of a larger group of isolates (65). A second polymorphic ORF (CBU0952) encodes a 28-kDa immunodominant outer membrane protein termed AdaA that was previously found to be synthesized only by acute disease isolates in genomic groups I and II (82). A third polymorphic ORF (CBU0953) encodes an amino acid permease.…”

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

“…To address the hypothesis, we used three different methods. First, we prepared a Sarkosylinsoluble fraction of Coxiella, a method commonly used to purify omps of Gram-negative bacteria, previously employed for this purpose with C. burnetii (Zhang et al, 2005). Analysis of Sarkosyl fractions of a mixed-cell population of Coxiella by FW blots and immunoblotting with anti-rLimB antibodies showed that: (i) both molecular mass forms of LimB were present in the insoluble fraction but absent in the Sarkosyl-soluble fraction, (ii) Ni-HRP was bound by both LimB isoforms and (iii) both protein isoforms were recognized by anti-LimB antibodies (Fig.…”

Coxiella burnetii Infects Primary Bovine Macrophages and Limits Their Host Cell Response

“…Gene mapping (20) and sequencing of the C. burnetii genome unveiled potential virulence genes based on sequence homologies. For example, the acute-disease antigen A (adaA) was described to be primarily present in strains isolated from acutely diseased patients (21). Despite an increasing number of effector proteins identified, their role as virulence factors during C. burnetii infection remains to be proven (22)(23)(24)(25).…”