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Research Article

Microevolution of the Chromosomal Region of Acute Disease Antigen A (adaA) in the Query (Q) Fever Agent Coxiella burnetii

  • Dimitrios Frangoulidis mail,

    DimitriosFrangoulidis@Bundeswehr.org

    Affiliation: Bundeswehr Institute of Microbiology, Munich, Germany

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  • Wolf D. Splettstoesser,

    Affiliation: Bundeswehr Institute of Microbiology, Munich, Germany

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  • Olfert Landt,

    Affiliation: TIB MOLBIOL GmbH, Berlin, Germany

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  • Jasmin Dehnhardt,

    Affiliation: TIB MOLBIOL GmbH, Berlin, Germany

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  • Klaus Henning,

    Affiliation: Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Wusterhausen, Germany

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  • Angela Hilbert,

    Affiliation: Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Wusterhausen, Germany

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  • Tilman Bauer,

    Affiliation: Freiburg, Germany

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  • Markus Antwerpen,

    Affiliation: Bundeswehr Institute of Microbiology, Munich, Germany

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  • Hermann Meyer,

    Affiliation: Bundeswehr Institute of Microbiology, Munich, Germany

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  • Mathias C. Walter equal contributor,

    equal contributor Contributed equally to this work with: Mathias C. Walter, Johannes K.-M. Knobloch

    Affiliations: Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, Department of Genome-Oriented Bioinformatics, Center of Life and Food Science Weihenstephan, Technische Universität München, Freising, Germany

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  • Johannes K.-M. Knobloch equal contributor

    equal contributor Contributed equally to this work with: Mathias C. Walter, Johannes K.-M. Knobloch

    Affiliation: University of Lübeck, Institute for Medical Microbiology and Hygiene, Lübeck, Germany

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  • Published: January 03, 2013
  • DOI: 10.1371/journal.pone.0053440

Abstract

The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.

Introduction

Coxiella (C.) burnetii is an obligate intracellular bacterium causing Q fever (query fever), a zoonotic disease which is ubiquitous throughout the world with exception of New Zealand. Primary reservoirs of C. burnetii are cattle, goats, sheep, and ticks. Other species including fish, birds, rodents, cats, and even arthropods are known to become infected. Predominantly in small ruminants, infection of adult animals is asymptomatic. However, it can lead to abortion in female ruminants and dispersion of large concentrations of C. burnetii by amniotic fluids and the placenta. The bacteria may also be excreted in milk, urine, and feces of infected animals. The main source of human infections is contact with infected sheep during lambing or the inhalation of dried tick feces [1], [2].

Human infections with C. burnetii are usually self-limiting and are associated with fever, fatigue, headache, as well as myalgia. In cases of acute Q fever, atypical pneumonia and hepatitis have been reported. Infections persisting for more than six months are regarded as chronic Q fever. The clinical symptoms are of more severe nature including endocarditis, chronic hepatitis, osteomyelitis, and septic arthritis, infection of aneurysm or vascular grafts [3][5]. Risk factors for the development of chronic Q fever are underlying vascular or cardiac disease, immunosuppression or pregnancy. During pregnancy, Q fever may cause premature birth, abortion, or neonatal death [6].

Several studies have tried to identify molecular markers related to the different clinical manifestations of acute and chronic Q fever [7]. One such approach was a classification based on the presence of different plasmid DNA [8]. Of the four plasmids QpH1, QpRS, QpDG and QpDV [8][11] and the plasmid DNA-derived sequence integrated into the chromosome (“plasmidless” strains [12]), only QpH1 was reported to be associated with acute and QpRS with chronic Q fever [8], [10]. However, this correlation was not confirmed by other researchers. Instead, host-dependent risk factors were determined to be responsible for an acute or chronic outcome of infection with C. burnetii [13], [14]. Furthermore, plasmid-related classification was found to correlate well with the genomic groups I to VI postulated after RFLP- and microarray based experiments [15], [16]. QpH1 forms the group I-III, QpRS represents group IV, the plasmidless isolates could be assigned to group V, and the special Dugway group, with the QpDG plasmid, forms cluster VI. Until now, the QpDV plasmid could not be associated with a distinct genomic group.

In 1998, To and colleagues [17] identified a 28 kDa protein (P28) that was immunodominant in isolates from patients with acute Q fever. The same group identified the gene and proposed to use the chromosomal acute disease antigen A (adaA) as a diagnostic marker for acute Q fever [18]. The gene has an open reading frame (ORF) consisting of 684 bp, coding for the hypothetical protein CBU_0952 (NP_819961), which was identified in a recent study [19].

The experimental basis for the correlation between adaA and the progress of disease were ten human isolates only: four of them from acute and six originating from chronic cases, as well as 11 from animals [18], [20]. In this study we investigated 23 strains from human patients and 86 strains of animal origin. We also included nucleotide sequences of seven published genomes [19], [21], [22] resulting in an improved classification of the adaA region. Phylogenetic analysis showed a good correlation to published whole genome data comparison including genomic grouping providing new information on microevolutionary events.

Materials and Methods

Strains and DNA Extraction

All but one C. burnetii isolates from different hosts and geographical regions (Table 1 and 2) were propagated in Buffalo Green Monkey (BGM) cell cultures under biosafety level 3 (BSL-3) conditions. Heat-inactivation was performed as described elsewhere [14], [23]. All strains were identified as C. burnetii by amplifying regions of the insertion sequence IS1111a [19], [24] in a real-time PCR assay as described previously [25]. DNA was extracted with the MagNA-Pure-Compact-System (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. C burnetii DNA from a pregnant woman with chronic Q fever was extracted from paraffin-embedded placental tissue, as described previously [26].

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Table 1. adaA genotypes of human Coxiella burnetii isolates from acute and chronic Q fever.

doi:10.1371/journal.pone.0053440.t001
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Table 2. adaA genotypes in 86 animal-derived Coxiella burnetii strains.

doi:10.1371/journal.pone.0053440.t002

Fifty different bacterial species used as negative controls were obtained from the German national strain collection (DSMZ, Braunschweig, Germany) or from different clinical sources [27].

PCR Assays

To generate a reliable tool for the detection and identification of the adaA gene, a real-time PCR assay with HybProbe-technology (Roche) was designed. For this, the adaA ORF (684 bp) was cloned from the reference strain (Nine Mile RSA493; NC_002971) into E. coli and confirmed by DNA sequencing (GenExpress, Berlin, Germany). Seven different primers and three different sets of hybridization probes were selected and tested with the cloned target as template (data not shown). The best results were obtained with primers adaA_S and adaA_R, which generated a fragment of 127 bp in length (NC_002971 position: 902656-782), using 0.5 µM of primers and 0.15 µM of probes adaA_FL and adaA_LC with FastStart DNA Master PLUS (Roche, Mannheim, Germany) in a volume of 20 µl. After 10 min at 95°C for polymerase activation, 45 amplification cycles were performed, each with 10 s denaturation at 95°C, 8 s annealing at 55°C and 15 s elongation at 72°C on a LightCycler 1.2 instrument (Roche, detection in channel F2). Melting curve analysis comprised of 20 s denaturation at 95°C and 20 s incubation at 40°C by continuous heating to 85°C with a slope of 0.2°C/s was performed afterwards.

The plasmids QpH1 and QpRS were determined by conventional PCR as described elsewhere [28]. Two new PCR assays were designed to identify the QpDV plasmid and the “plasmidless” type (Table 3). The PCR conditions of these assays are as follows: After 10 min at 95°C for FastStart Taq DNA polymerase activation, 45 amplification cycles were performed, each with 10 s denaturation at 95°C, 10 s annealing at 55°C and 11 s elongation at 72°C on a LightCycler 1.2 instrument. Again, a melting curve analysis was completed after 20 s denaturation at 95°C and 30 s incubation at 40°C by continuous heating to 95°C with a slope of 0.2°C/s.

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Table 3. Primers and probes used in this study.

doi:10.1371/journal.pone.0053440.t003

For analyzing the region flanking the adaA gene deletion, an extended PCR-primer pair system comprising primer R4 and L4 was designed. The PCR was performed on a GeneAmp PCR-system 9700 (PE Applied Biosystems, USA) with the Expand Long Template Mix, buffer 2 (Roche, Mannheim, Germany) and Clontech Polymerase BD-Advantage (Becton-Dickinson, Heidelberg, Germany). The cycling protocol encompassed 150 s at 95°C, 35 cycles with 30 s at 95°C, 30 s at 53°C, 8 min at 68°C and a final extension for 7 min at 68°C. Chromosomal DNA of Nine Mile wild type was used as positive control for all PCR reactions (amplicon size of approx. 8 kb). For further amplification of the specific PCR product, a nested PCR was performed with the primers L4 nested and R4 nested using the same instruments and chemistry.

Determination of Sequence Polymorphisms

After identification of adaA positive strains, we performed a second PCR using the primers Cox adaA_* and Cox adaA_ATG with BigDye Terminator chemistry (Applied Biosystems/Life Technologies, Darmstadt, Germany) on a 3100 ABI sequencer (Applied Biosystems/Life Technologies, Darmstadt, Germany) in order to identify sequence variations in the entire adaA gene. To analyze the adaA deletion regions, PCR-products generated with primers L4 nested and R4 nested were cloned into Plasmid pAlli10 according to the kit manual (Alligator cloning kit; Trenzyme, Constance, Germany). Sequencing reactions in an ABI 3300 sequencer (Applied Biosystem, Darmstadt, Germany) were performed with primers M13-FP, M13-RP, Pa3, and Pa4 to cover the entire region of the deletion mutants. Sequence analysis was performed using the Software packages of Vector NTI (version 9.0; Invitrogen, Carlsbad, CA, USA) and MEGA (version 5.03, Center for Evolutionary Medicine and Informatics, Tempe, USA [http://www.megasoftware.net]).

Phylogenetic Analysis

An in silico analysis of the seven reference genomes was performed using the genome aligner Mauve [29]. Based on the computed multiple sequence alignment of the adaA chromosomal region, long collinear blocks (LCBs) were used to identify syntenic regions across two or more genomes (see Figure 1). The comparison of deletion types presented in strains from our collection, shown in Figure 2, was performed on an end-trimmed multiple sequence alignment computed with MAFFT [30]. Phylogenetic trees were calculated based on the multiple sequence alignments using the maximum parsimony criterion as implemented in PAUP* [31]. Thereby, deletions were encoded as additional states according to [32] using SeqState (version 1.4.1, [33]). To root the trees, Dugway was set as outgroup. Bootstrap values were computed based on 1,000 samples and were higher than 90% for all branches.

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Figure 1. Polymorphisms of the adaA genetic region demonstrated using seven C. burnetii reference genomes.

Three deletion events resulting in two main deletion types (Q154 and Q212) and three adaA gene variants (adaA, adaASNP and adaArep) resulting from independent mutation processes were identified in silico. The organization of the adaA flanking region of the Dugway genome (displayed in the center) is compared to the other six reference genomes. Just for the purpose of visual compactness, the light orange region was pruned between the ORFs CBUD_1100 and CBUD_1122. Gene annotations are obtained from GenBank. Coding genes are drawn in blue, pseudogenes in white and the adaA gene in red. Arrow direction represents the location of the ORFs at the forward and backward strand. Locus tags are shortened by its common prefix and written above the ORFs. If gene names are known, they are written next to their locus tag and in parentheses. Long collinear blocks (LCBs) are shown as colored rectangles at the backbone of the Dugway genome. The pairwise syntenic regions between the genomes are drawn in lighter color, accordingly. The phylogenetic relationship is shown at the left side. Just for visualization purposes, the Dugway genome was rotated to the middle. The length of the branches encodes the number of mutation events. Three different deletions were identified and its affected LCBs are drawn to the phylogenetic branch where the deletion events may have occurred. Dugway and RSA331 were chosen as representative strains to show the intact adaA gene and adaASNP gene, respectively. A fragment of strain F10 is exposed in the figure to show the inserted repeat variant (adaArep). The figure was drawn using genoPlotR [37]. *Q321 replaces RSA334, which was accidently assigned to the published whole genome data.

doi:10.1371/journal.pone.0053440.g001
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Figure 2. Sequence-based discrimination of the Q154 adaA gene deletion type.

This special deletion type is defined by deletion 1 (see Figure 1). Differential bases (SNPs and insertion) of analyzed strains are shown in reference to the published genome of Q154, position 834,296 to 836,007. Vertical numbers display the respective position relative to position in the genome of strain Q154. The large deletion 1 is indicated with an asterix (*). Three conserved distinctions defining the subgroup B are marked in dark grey. The insertion of a ‘T’ is located between position 834,978 and 834,979 of the Q154 genome. Subgroup B can be further divided into two groups B1 and B2 (light grey lines) by 5 conserved SNPs (marked light grey).

doi:10.1371/journal.pone.0053440.g002

Results

Specificity and Reproducibility of the Real Time PCR Assay

The adaA_S and adaA_R primers and probes were specific for all 94 adaA positive C. burnetii strains investigated. The C. burnetii reference strain (Nine Mile RSA493) was adaA positive whereas the well-known strain Priscilla Q177 containing the QpRS plasmid displayed no PCR signal. DNAs from 50 other bacterial species were negative, indicating that the assay is specific (data not shown). The limit of detection was determined by a serial dilution of photometrically quantified plasmid and genomic DNA and was found to be one plasmid copy per reaction. Melting curve analysis of C. burnetii-derived PCR products revealed a single distinct melting peak at approximately 61°C for all investigated strains. The size of the amplicons was confirmed by gel electrophoresis and determined as 127 bp (data not shown) with the exception of 4 strains, which showed a larger product.

Distribution of the adaA Gene in C. burnetii Isolates

Ten out of the fourteen strains obtained from human patients with acute disease were positive in the adaA gene real-time PCR and all of them carried the plasmid QpH1. Four strains related to fever (n = 1), pneumonia (n = 1) and hepatitis (n = 2), were negative in the adaA PCR assay. In contrast to the other isolates, these strains showed the QpDV plasmid type.

Six isolates from chronic cases (one chronic hepatitis and five endocarditis patients) were negative for the adaA gene. The plasmid types were QpRS, QpDV or “plasmidless” (strain Scurry Q217). However, two C. burnetii isolates from French patients with endocarditis and one abortion-derived isolate from a chronically infected patient with endocarditis and valve replacement were adaA positive. All of these three strains harbored the QpH1 plasmid as well (Table 1).

Eighty-one out of the 86 investigated animal strains were adaA positive and carried plasmid type QpH1. Three of the five negative strains isolated from goat or sheep showed the QpRS plasmid type, while the two other strains derived from tick pools, were also QpH1 positive (Table 2).

Sequence Variations of the adaA Gene

Gel electrophoresis analysis of the adaA PCR products for some isolates showed a larger product of about 900 bp as opposed to the expected size of 684 bp. Sequence analysis of the 900 bp amplicon revealed a 226 bp duplication (base 107 to 332), resulting in a premature stop codon after 133 amino acids. Due to this observation, in all adaA positive strains the whole ORF was sequenced. Only one human (chronic Q fever endocarditis) and three animal isolates (two goat, one sheep) presented this variation. Further sequence analysis revealed a novel non-synonymous single nucleotide polymorphism at position A431T, resulting in an amino acid substitution of glutamic acid (E) by valine (V) – which was found in 49 strains. This included nine out of 14 isolates from acute Q fever patients and one out of eight isolates from chronic Q fever patients (Table 1). In animals, the SNP was present in all of the studied species (see Table 2).

adaA Deletion Sites

All 15 adaA negative strains were analyzed by sequencing the products of long-range PCR targeting flanking regions of the gene. The sequences were aligned with the chromosomal adaA flanking region of strain Dugway and lacking regions were designated as deletions (Figure 1). Analysis of these and comparison with published adaA negative genomes (Q154, NC_011528 and Q212, NC_011527) revealed two differing main deletion types (Figure 1). The adaA gene in the reference strain Nine Mile RSA493 is located close to a described insertion site (between CBU_0948 and CBU_0949; [21]) compared to the Dugway genome comprising more than 20 putative ORFs (CBUD_1100 to CBUD_1122; NC_009727). The more common deletion 1 resulted from a large 27.1 kb deletion of the adaA gene, an ORF designated CBU_0949 in strain Nine Mile, and the ORFs CBUD_1100 to CBUD_1122 (Figure 1, deletion 1). This resulted in the Q154-type of adaA negative strains (13 strains, Tables 1, 2).

CBU_0949 annotated in strain Nine Mile is the truncated version of ORF CBUD_1099 annotated in strain Dugway which was partially deleted by the large deletion comprising the ORFs CBUD_1100 to CBUD_1122 (deletion 2). Another deletion event is seen in the strain Q212 and is the consequence of a 5,9 kb deletion (Figure 1, deletion 2.1) in a genetic background homologous to strain Nine Mile [21] remaining 801 bp upstream of deletion 1 as well as from a truncated ORF CBU_0949. This type of deletion was only seen in one human chronic Q fever isolate with liver involvement and in one tick isolate from Germany. Only strains of the Q154-deletion type show a minor difference of 228 bp in the 3′-flanking chromosomal region of the deletion 1 and 2. Comparison of all sequenced Q154-type adaA deletions revealed several non-coding SNPs as well as a one base-pair insertion (Figure 2). Making use of these variations, the strains of the Q154-deletion type can be differentiated further into two subgroups (A+B) by two conserved SNPs as well as the one base-pair insertion (Figure 2, dark grey). Within subgroup B, 5 SNPs were used for further differentiation (subgroups B1 and B2, Figure 2, light grey).

Phylogenetic Analysis

The phylogenetic relationship based on adaA gene variants was studied using MAFFT and PAUP* and is shown in Figure 3. The analysis matches with the former RFLP-based genomic grouping I to VI [15] and the microarray-based whole-genome comparisons by Beare et al. [16]. Group I, represented by the Nine Mile/RSA493 strain is carrying the adaA wildtype, whilst group II represented by RSA331, carries the adaASNP variant. Both groups harbor the QpH1 plasmid. Group IV (QpRS associated) was formed by the Q154-deletion types and group V (“plasmidless” = Q212-deletion) and group VI (“Dugway/QpDG” = non-disrupted adaA region) could also be allocated to distinct adaA (deletion) variants. In our analysis, the genomic groups I and II are related to group V whereas IV and VI show a closer relationship to each other. In addition, another distinct group of isolates carrying the QpDV plasmid type (identical to the above described Q154_B1-type) also reveals a close relationship to group IV and VI as well as the newly defined Q154_B2 subcluster.

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Figure 3. Phylogenetic relationship of studied strains.

Maximum parsimony tree using 1,713 bases in the dataset computed with PAUP* and rooted using Dugway (bold) as outgroup. Plasmid type, genomic group (GG), adaA types and the subgroups of the Q154-deletion type are shown next to the tree. Branch lengths correspond to the number of mutations. Animal strains are written in italic. IP: integrated plasmid; WT: wildtype; D2.1: deletion 2.1; D1: deletion 1. The figure was drawn using APE [38].

doi:10.1371/journal.pone.0053440.g003

Discussion

The sequence variations of the Coxiella burnetii specific acute disease antigen A gene, postulated to be associated with acute human Q fever [18], were completely analyzed in silico and in vitro. In contrast to previous publications, this study used a large and well-defined collection of strains from human clinical entities and different animal species to resolve the heterogeneous structure of this gene region. Using a novel real-time PCR assay, we were able to demonstrate that 10 out of 14 strains isolated from acute human Q fever patients were adaA positive. The adaA negative isolates are derived from acute Q fever patients with hepatitis (n = 2), fever (n = 1), or pneumonia (n = 1). This supports a recently published observation from a Spanish study [34], in which all strains isolated from acute Q fever hepatitis patients were adaA negative and only one C. burnetii isolate from a pneumonic patient was adaA positive. In our study, in only three out of nine isolates from chronic Q fever endocarditis patients, including one isolate from paraffin-embedded abortion material, the adaA gene was detected. Thus, our results do not support a significant association of the adaA gene with the acute course of Q fever.

Plasmid Correlation

The correlation between plasmid type and adaA genotype present in human C. burnetii isolates was also observed in animal-derived strains with only very few exceptions. Sixty five out of 67 strains carrying QpH1 displayed an adaA positive genotype. Only two QpH1 strains, isolated from a tick pool from a region in south-western Germany, were negative. The type strain Dugway carrying the QpDG plasmid was also adaA positive [18]. The three strains of QpRS plasmid type were adaA negative, corroborating the results of the strains isolated from chronic human disease. In summary, we confirmed that in every adaA positive strain (except Dugway) we could detect the QpH1 plasmid as well. As no positional linkage was found between both, this cannot be sufficiently explained by molecular reasons and remains an open question.

adaA Gene Variants

For the first time, our in-depth analysis showed a quite heterogeneous appearance of the structure of this genetic region. We identified five different variants: the adaA wildtype, a second variant with a tandem repeat, a SNP type and two different deletion types. The adaA-repeat type (size: 226 bp, two copies, starting at position 107) was first described by a French group in 2009 [20], and were correlated to C. Burnetii isolates from goats. In contrast to this, our study identified the tandem repeat also in one sheep strain and one isolate from a chronic Q fever endocarditis patient. The SNP at position 431 resulted in an amino acid substitution (glutamic acid to valine). Remarkably, this is the predominant variant in strains from acute Q fever (10 out of 11) patients, predominantly the pneumonic variant. Only one chronic sample (“99/3”) also harbored this variant, indicating that there may be an acute respiratory disease pattern before chronification. In animals, the SNP was detected in more than 50% of the analyzed isolates (49 out of 81). Regarding to the species level, the ratio is as follows: the majority of sheep isolates (18/21) carried this genotype. In goats (4/8) and cows (6/32), the presence was more varied. This correlates well with the observations, that ewes are the predominant source for Q fever outbreaks infecting humans in the past.

Using the SNP, a rough trace back analysis of Q fever was performed with the two German human isolates. The “290/03” C. burnetii strain was epidemiologically allocated to a major German Q fever outbreak in Soest in 2003 [35] and two C. burnetii isolates from a sheep flock in the same geographical region also carried the adaA gene SNP. In the chronic Q fever case, dedicated to the isolated DNA “99/3”, all regional sheep strains isolated prior to the beginning of the disease (before 1999) were also presenting this variant. Other possible interpretations for our findings are that humans are not as susceptible for the “normal” adaA gene variant as for the SNP variant, or that both variants are linked to different chromosomal changes responsible for different phenotypes in the susceptible host.

Deletions in the adaA Gene Region May Reflect Phylogenetic Events in the Evolution of C. burnetii

We could show that the adaA negative status of isolates is much more complex than described to date. By comparing all of the published genomes, and the sequenced adaA deletion sites in this study, a clear discernible genome reduction is seen resulting in the following phylogenetic relationship placing Dugway as common ancestor (Figure 1): The 27.2 kb deletion 1 from Dugway resulted in the Q154 adaA deletion genotype, while the independent 21.4 kb deletion 2 in Dugway, not affecting the adaA gene region, resulted in Nine Mile and RSA331 (distinguishable from each other by the adaASNP). The subsequent deletion 2.1 of 5.9 kb within Nine Mile resulted in the Q212 adaA deletion genotype.

The first genomic variation divides the chronic disease branch (Q154) from the acute Q fever cluster (RSA331). Further, the Q154-deletion could be subdivided by several conserved non-coding SNPs and one insertion into three other clusters (see Figure 2). Cluster A presents QpRS strains from chronic Q fever endocarditis patients, whereas the second cluster B, with multiple plasmid types, is further differentiated into human-derived isolates. The QpDV plasmid (B1) and an animal-based group (B2) harboring QpH1 and QpRS plasmids. Deletion 2.1 (5.9 kb) correlates very well with findings from a recently published paper [21] that described the lack of ORFs in strains belonging to the QpRS and other plasmid types different from QpH1. Furthermore the identified Q154- respectively Q212-deletion type in two QpH1 strains from ticks (see above) may indicate that horizontal gene transfer is possible in ticks infected by multiple Coxiella clones.

By looking at the few single mutation events (SNPs, InDels) in the small genomic region encoding the adaA gene, we could reconstruct the same phylogenetic relationship as shown previously with whole genome analysis methods [16]. This adds strongly to the evidence of the clonal evolution of C. burnetii.

Genomic Grouping

Our results also correlate well with plasmid-related classification and the genomic groups I-VI postulated after RFLP and microarray based experiments [15], [16] resolving these groups further. The described distinction inside the genomic groups I to III could be confirmed for the groups I and II, where two adaA gene variants are seen (wildtype and SNP). In more detail, both clusters differ in 11 (RSA331) and 4 SNPs (RSA493), respectively. These groups share a hypothetical common ancestor with Q212 (group V) further divided by 14 additional SNPs and the deletion event described earlier. On the other hand, we could demonstrate a closer evolutionary relationship between group IV and VI, supporting C. burnetii Dugway isolate 5J108-111 as a common ancestor of all described genomic groups. Interestingly, Q321 clusters phylogenetically with a distinct group formed by QpDV-harboring strains exhibiting the adaA deletion type Q154_B1. Until today, this relevant C. burnetii strain cluster bearing the QpDV plasmid was not placed into a genomic group, most likely due to missing isolates in the former studies. We assume that all members of this special subdivision build the genomic group VII, formerly proposed by Beare et al. [16]. This additional evidence for the existence of the genomic group VII was confirmed by the above-mentioned Spanish study, where two isolates from our collection (F2 and F4) were placed together with a local tick cluster of 32 strains. Unfortunately, no plasmid type information was given. Hence, we can associate two of the above mentioned new Q154-deletion type subclusters with existing genomic groups IV (A) and VII (B1), whereas the other subcluster B2 can be postulated as one or two new genomic groups IX and X, depending on the occurrence of different plasmid types.

An additional analysis of the inter-species relationship of the AdaA protein with HHblits, a remote homology detection tool based on HMM-HMM [36], revealed a homologous protein in Legionella pneumophila, the taxonomically closest related species. Unfortunately, no function could be assigned to this protein in Legionella so far. Hence, the function and relevance of the AdaA protein is still unknown.

It seems that the microevolutionary process of C. burnetii is homogenously displaced and no clear allocation to distinct genes is possible. This could explain the observed phylogenetic relationships between strains independent of type and number of studied genes or gene regions. It fits within the thesis of a highly conserved genome in this species and is useful in molecular strain comparison studying various endemic regions and clinical entities.

Therefore, classification of adaA gene variants, if present in the genome, or determination of the deletion type, is an important tool for the investigation of molecular epidemiology and the evolution of Q fever.

Acknowledgments

Sequencing analysis has been done in part at GATC, Constance, Germany. We would like to thank Claudia Kahlhofer for excellent technical assistance and Georg Baljer (Gießen, Germany) for providing some of the C. burnetii strains. We are also deeply grateful to Lea Cleary, Heike Geringer and Eduardo Thüroff for proof reading the manuscript and helpful comments.

Author Contributions

Conceived and designed the experiments: DF. Performed the experiments: JD JK MA OL. Analyzed the data: DF HM JK MA MW OL WS. Contributed reagents/materials/analysis tools: AH KH MW TB WS. Wrote the paper: DF JK MW OL.

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