The IS1111 insertion sequence used for detection of Coxiella burnetii is widespread in Coxiella-like endosymbionts of ticks (original) (raw)

* Corresponding author: Laboratoire MIVEGEC (Maladies Infectieuses et Vecteurs : Ecologie, Génétique, Evolution et Contrôle), Centre National de la Recherche Scientifique (UMR5290) – Université de Montpellier – Institut pour la Recherche et le Développement (UR224), F-34394 Montpellier, France. Tel: +33 (0)4 6741 6158;

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17 July 2015

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12 August 2015

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Olivier Duron, The IS1111 insertion sequence used for detection of Coxiella burnetii is widespread in _Coxiella_-like endosymbionts of ticks, FEMS Microbiology Letters, Volume 362, Issue 17, September 2015, fnv132, https://doi.org/10.1093/femsle/fnv132
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Coxiella is a genus of obligate intracellular bacteria engaged in a variety of interactions with eukaryotes. The type species, Coxiella burnetii, infects several vertebrate species, including humans, and is the causative agent of Q fever. Multiple copies of a specific transposable element, the insertion sequence IS1111, are present in the genome of C. burnetii and are routinely used for confirmation of Q fever cases. Recently, many _Coxiella_-like bacteria that are closely related but genetically distinct to C. burnetii have been found in ticks. These _Coxiella_-like bacteria are maternally inherited endosymbionts, present at high prevalence in tick populations and engaged in mutualistic interactions with their arthropod hosts. In this study, the presence of IS1111 was examined in the _Coxiella_-like endosymbionts and in bacteria of the Coxiella sister-genus, Rickettsiella. This screening reveals that a wide range of IS1111 copies were present in the _Coxiella_-like endosymbionts of ticks. DNA sequencing further identified genetically divergent IS1111 copies, including degraded copies that constitute an important genomic fossil record of past IS1111 expansions. These results show that IS1111 is not specific to C. burnetii, suggesting that Q fever detection assays based only on this element may lead to misidentification with _Coxiella_-like endosymbionts.

INTRODUCTION

Obligate intracellular bacteria of the genus Coxiella (Gammaproteobacteria: Legionellales: Coxiellaceae) are widespread and biologically diverse. The Coxiella type species, Coxiella burnetii, is the causative agent of Q fever, a zoonotic disease of vertebrates, including humans (Madariaga et al.2003; Raoult, Marrie and Mege 2005; van Schaik et al.2013; Vanderburg et al.2014). The common ways of infection are inhalation of contaminated barnyard dust and contact with excreta of infected animals as birthing products which harbor high titration of C. burnetii (Madariaga et al.2003; Raoult, Marrie and Mege 2005; Vanderburg et al.2014). Other infection pathways (e.g. sexual, oral or congenital) are thought to be rare although some studies produce clear evidence that ticks can carry C. burnetii (Duron et al. in press). For instance, in the case study by Pacheco et al. (2013), C. burnetii infection in two Amblyomma tick species was confirmed using an impressive array of detection methods, including hemolymph tests, isolation in Vero cells and multilocus DNA sequencing. It is also noteworthy that the highly virulent reference strain, Nine Mile, was primarily isolated from a guinea pig upon which Dermacentor andersoni ticks had fed (McDade 1990). Although Q fever is actually far more frequently transmitted via the airborne route, ticks probably act as major drivers of the heterospecific transmission and spatial dispersal of Q fever among vertebrates (Duron et al. in press).

Ticks do however not only harbor C. burnetii. _Coxiella_-like bacteria, closely related but genetically distinct to C. burnetii, were recently found common in ticks with approximately three quarters of tick species being infected (Noda, Munderloh and Kurtti 1997; Reeves et al.2006; Jasinskas, Zhong and Barbour 2007; Klyachko et al.2007; Clay et al.2008; Reeves 2008; Machado-Ferreira et al.2011; Almeida et al.2012; Lalzar et al.2012; Duron, Jourdain and McCoy 2014; Wilkinson et al.2014; Duron et al.2015). These _Coxiella_-like endosymbionts seem confined to ticks and, to the current knowledge, they pose a much lower infection risk to vertebrates than C. burnetii (Duron et al. in press). These bacteria substantially differ from C. burnetii in their biological traits, behaving as subtle endosymbionts engaged in intricate interactions with ticks. In many cases, the _Coxiella_-like endosymbionts were found to massively infect ovaries and to be maternally inherited through the egg cytoplasm (Klyachko et al.2007; Machado-Ferreira et al.2011; Lalzar et al.2012; Lalzar, Friedmann and Gottlieb 2014; Duron et al.2015). Their concomitant presence in the Malpighian tubules further suggests a possible role in nutrition, osmoregulation and excretion (Klyachko et al.2007; Machado-Ferreira et al.2011; Lalzar, Friedmann and Gottlieb 2014). Indeed, their elimination with an antibiotic treatment was shown to negatively impact the fitness of the lone star tick Amblyomma americanum (Zhong, Jasinskas and Barbour 2007). Accordingly, no recognizable virulence genes were found in two _Coxiella_-like endosymbionts of ticks that were recently sequenced (Gottlieb, Lalzar and Klasson 2015; Smith et al.2015), indicating that these bacteria are likely non-pathogenic. In contrast, their genomes encode major vitamin and cofactor biosynthesis pathways, suggesting that these bacteria are vitamin-provisioning endosymbionts. The ubiquity of _Coxiella_-like endosymbionts in many tick groups—in which infection is often at fixation—corroborates the hypothesis of an obligate mutualist bacterium (Duron et al.2015). Such patterns have been found in other exclusive blood-feeding species like bedbugs (Hosokawa et al.2010) and tsetse flies (Akman et al.2002), two insect groups which rely on a single food source throughout their developmental cycle and harbor beneficial microbes that provide nutrients absent from their restricted diets.

The discovery that ticks can carry both C. burnetii and _Coxiella_-like endosymbionts underscores the need to be able to clearly distinguish between the two. Nowadays, numerous C. burnetii detection methods are in use (Duron et al. in press; Sidi-Boumedine and Rousset 2011). Among them, polymerase chain reaction (PCR) and quantitative PCR (qPCR) targeting the IS1111 element are usually thought to be the most sensitive methods for specific detection and quantification of C. burnetii (Klee et al.2006; Denison, Thompson and Massung 2007; Sidi-Boumedine and Rousset 2011). The IS1111 element is a typical insertion sequence: a genetically compact bacterial transposable element encoding no functions other than a transposase (Tnp), i.e. an enzyme which catalyzes its own transposition (Siguier, Filee and Chandler 2006; Cerveau et al.2011a). Insertion sequences as IS1111 are fundamentally selfish DNA parasites, with the capacity to autonomously replicate, proliferating and jumping to other loci within, but also between, bacterial genomes (Siguier, Filee and Chandler 2006; Cerveau et al.2011a). All C. burnetii genomes contain multiple IS1111 copies dispersed along the bacterial chromosome (Hoover, Vodkin and Williams 1992; Seshadri et al.2003; Klee et al.2006; Denison, Thompson and Massung 2007; Beare et al.2009; Sidi-Boumedine et al.2014). These copies have led to an accelerated genomic evolution: recombination between IS1111 copies induces important plasticity in the C. burnetii genomes with chromosomal rearrangement of syntenic blocks and massive DNA insertions/deletions (Beare et al.2009). Comparison of the IS1111-Tnp shows that they are 99% identical in DNA sequence within and between C. burnetii genomes, suggesting a recent acquisition and expansion of IS1111 in this bacterial species (Seshadri et al.2003; Beare et al.2009). However, although IS1111 is considered only present in C. burnetii, an IS1111-like element showing 90% nucleotide identity with the IS1111-Tnp of C. burnetii was recently identified in the _Coxiella_-like endosymbiont of the Wombat tick Bothriocroton auruginans (Vilcins, Old and Deane 2009). Its discovery shows that some IS1111 diversity may exist in other bacteria than C. burnetii.

Here, the distribution and the diversity of IS1111 were examined beyond C. burnetii. This topic was approached by undertaking an extensive screening for the presence of IS1111 in a collection of bacterial endosymbionts from ticks. This collection includes endosymbionts belonging to Coxiella and to its sister genus, Rickettsiella, which was also recently found widespread in ticks (Kurtti et al.2002; Vilcins, Old and Deane 2009; Leclerque and Kleespies 2012; Anstead and Chilton 2014; Duron et al.2015). Sequencing of the Tnp gene was further used to characterize the diversity and the putative functionality of different IS1111 copies found in the endosymbionts of ticks.

MATERIALS AND METHODS

Tick collection

A collection of 115 DNA templates from 42 tick species was used (Table 1). Within each examined tick species, there were one to four specimens separately investigated for analysis. For each tick DNA template, bacterial infection had been formally characterized in previous studies through multilocus DNA sequencing (Duron, Jourdain and McCoy 2014; Duron et al.2015), as detailed below. Of the 42 tick species used here, 37 species, including 15 species of soft ticks (Argasidae) and 22 of hard ticks (Ixodidae), are infected by _Coxiella_-like endosymbionts, as detailed in Table 1. The five other tick species (three soft tick species and two hard tick species) are infected by Rickettsiella endosymbionts (Table 1). Each of the 42 examined tick species harbored a specific Coxiella or Rickettsiella multilocus genotype (on the basis of five housekeeping gene sequences: 16S rRNA, 23S rRNA, rpoB, GroEL and dnaK; for more details, see Duron et al.2015): as a result, 37 Coxiella and 5 Rickettsiella different genetic strains are thus present in this collection. Each of the Coxiella strains belongs to one of the four phylogenetic clades (A–D) recently described within the Coxiella genus (Duron et al.2015), as reported in Table 1. None of these tick DNA templates was found infected by C. burnetii on the basis of multilocus DNA sequencing (Duron, Jourdain and McCoy 2014; Duron et al.2015).

Table 1.

Distribution of IS1111 in _Coxiella_-like and Rickettsiella endosymbionts of ticks.

Table 1.

Distribution of IS1111 in _Coxiella_-like and Rickettsiella endosymbionts of ticks.

Screening and sequencing for IS1111

To detect genotype IS1111, a new detection method based on a nested PCR to amplify a 570 base pair (bp) fragment of the IS1111-Tnp gene was developed here. The IS1111-Tnp sequence of the reference C. burnetii strain RSA 493/Nine Mile I (GenBank accession number AE016828) was used as reference to design specific PCR primers. The efficiency of this IS1111 typing method was ascertained through positive PCR amplification and clear IS1111 sequences from a DNA template of the RSA 493/Nine Mile I isolate of C. burnetii.

Nested PCR amplifications for IS1111-Tnp were performed as follows. The first PCR run with the external primers IS1111_F1 (5′-CGTCCTTAACATCACATTSCCGCG) and IS1111_R2 (5′-GTGTGGRGGARCGAACCATTGG) to amplify a 670-bp Tnp fragment. It was performed in a 10 μL volume containing 20–50 ng of genomic DNA, 3 mM of each dNTP (Thermo Scientific), 8 mM of MgCl2 (Thermo Scientific), 3 μM of each primer, 1 μL of 10× PCR buffer (Thermo Scientific) and 0.5 U of Taq DNA polymerase (Thermo Scientific). A 1 μL aliquot of the PCR product from the first reaction was then used as a template for the second round of amplification. The second PCR was run with the internal primers IS1111_F2 (5′-CCCCAACAASACCTCCTTATTCC) and IS1111_R1 (5′-CCGCAGCACSTCAAACCGTATG) to amplify a 570-bp Tnp fragment. It was performed in a total volume of 25 μL and contained 8 mM of each dNTP (Thermo Scientific), 10 mM of MgCl2 (Thermo Scientific), 7.5 μM of each of the internal primers, 2.5 μL of 10× PCR buffer (Thermo Scientific) and 1.25 U of Taq DNA polymerase (Thermo Scientific). All PCR amplifications were performed under the following conditions: initial denaturation at 93°C for 3 min, 30 cycles of denaturation (93°C, 30 s), annealing (56°C, 30 s), extension (72°C, 1 min) and a final extension at 72°C for 5 min. An aliquot of the PCR products of the second amplification round was electrophoresed in a 1.5% agarose gel to check for IS1111 presence. All positive PCR products were sequenced directly in both directions (Eurofins, France). The chromatograms were manually inspected and cleaned with CHROMAS LITE (http://www.technelysium.com.au/chromas_lite.html) and sequence alignment was performed using CLUSTALW (Thompson, Gibson and Higgins 2002) implemented in MEGA (Tamura et al.2007). All IS1111-Tnp sequences obtained in this study were deposited in GenBank (accession numbers: KT345175–KT345185).

RESULTS

Distribution of IS1111

The IS1111-Tnp was successfully amplified from 25 of the 115 DNA templates (21.7%; Table 1). It was found in association with 11 of the 37 tick species infected by _Coxiella-_like endosymbionts (29.7%), while none of the 5 species infected by Rickettsiella was IS1111-positive. The IS1111-positive Coxiella DNA templates are from the two main tick families, i.e. soft ticks (six species of the Ornithodoros genus and one species of Argas) and hard ticks (three species of Rhipicephalus and one species of Haemaphysalis) (Table 1). For the 7 IS1111-positive soft tick species, IS1111 was observed in all tested DNA templates within each species (21 positive templates out of 21). Conversely, for the 4 IS1111-positive hard tick species, IS1111 was only observed in one DNA template per species (4 positive templates out of 10) (Table 1).

Overall, IS1111-Tnp was found in association with 11 of the 37 _Coxiella_-like genetic strains examined (29.7%). Worthy of note is that most of the IS1111-positive _Coxiella_-like endosymbionts (7/11) belong to the A Coxiella clade (Table 1). Remarkably, the IS1111 was actually found in seven of the eight A _Coxiella_-like endosymbionts, suggesting that IS1111 is largely widespread in the A Coxiella clade. In contrast, the four other IS1111-positive _Coxiella_-like endosymbionts belong to the C (3 IS1111-positive C _Coxiella_-like endosymbionts of 16 examined) and to the D (one of six) Coxiella clades. None of the seven _Coxiella_-like endosymbionts of the B clade was found positive for IS1111.

Diversity of IS1111 copies

While an expected 570 bp IS1111-Tnp amplicon was obtained from the C. burnetii RSA 493/Nine Mile I isolate, the examination of the IS1111-Tnp amplicons from _Coxiella_-like endosymbionts revealed variable lengths ranging from 289 to 582 bp, a pattern resulting of nucleotide insertions or deletions (indels) along the DNA sequence. On the basis of Tnp DNA sequences, 10 genetically distinct haplotypes of IS1111 were characterized from the 25 IS1111-positive DNA templates. Each of the IS1111-positive tick species harbored a different IS1111 haplotype with the exception of two species, Rhipicephalus sp.1 and Rhipicephalus sp.2, which harbored genetically identical IS1111 copies. None of these IS1111 haplotypes was strictly identical to the IS1111 copies found in C. burnetii or in the _Coxiella_-like endosymbiont of B. auruginans.

As the variable sizes of IS1111 copies should result in different transpositional activity, the analysis was further refined by examining amino-acid sequences (Fig. 1). Alignments of the IS1111-Tnp amino-acid sequences revealed the presence of many conserved sites with the IS1111-Tnp of C. burnetii but only IS1111 of the _Coxiella_-like endosymbiont of Rhipicephalus decoloratus has an intact sequence (99.6% amino-acid identity with the IS1111-Tnp of C. burnetii). The nine other IS1111 haplotypes found in _Coxiella_-like endosymbionts have degraded sequences, and likely are non-functional: seven contain internal stop codon(s), terminating the Tnp-coding sequences, and all show several frameshift mutations caused by indels in a number of sites. Similarly, the _Coxiella_-like endosymbiont of B. auruginans described by Vilcins, Old and Deane (2009) has also an IS1111 degraded copy harboring several frameshift mutations (Fig. 1) and may be non-functional.

Figure 1.

Alignment of amino-acid sequences deduced from IS1111-Tnp. Sequences obtained from this study are underlined. Conserved amino-acid residues, variable sites and insertions/deletions are provided relatively to a functional copy of IS1111-Tnp from the C. burnetii RSA 493 RSA 493/Nine Mile I genome and are shown in black, grey and white, respectively. Numbers indicate amino-acid positions along the IS1111-Tnp sequence. Asterisk: stop codon; Dash: gap; ?: absence of amino-acid residues due to frameshift mutations.

DISCUSSION

When originally described, IS1111 had only been found in C. burnetii (Hoover, Vodkin and Williams 1992; Seshadri et al.2003; Beare et al.2009), but the present work and the early work of Vilcins, Old and Deane (2009) further show that a wide range of IS1111 copies exists in other bacteria, the _Coxiella_-like endosymbionts of ticks. Divergent IS1111 copies were found in _Coxiella_-like endosymbionts hosted by distantly related tick species, as shown with soft ticks (Ornithodoros, Argas) and hard ticks (Riphicephalus, Haemaphysalis). Through _Coxiella_-like endosymbionts, IS1111 has thus a broad distribution across the entire Coxiella genus and is not specific to C. burnetii. Conversely, no IS1111 copy was found among strains of the _Coxiella_-sister genus, Rickettsiella.

An examination of IS1111 nucleotide diversity shows that this element is not of recent origin. First, the presence of putatively functional copies in both C. burnetii and _Coxiella_-like endosymbionts and, second, the identification of many degraded copies that constitute an important genomic fossil record of past IS1111 expansions both indicate a long evolutionary history within the Coxiella genus. The presence of degraded copies in _Coxiella_-like endosymbionts lacking functional copies of IS1111 further shows that the IS1111 transpositional activity was probably not constant over evolutionary time. These degraded copies have been generated in an ancient period of transpositional activity of IS1111 before to be inactivated and pseudogenized. A very similar pattern has evidenced for diverse IS in other endosymbionts found in arthropods as well exemplified by Wolbachia: after an IS enters a genome, its copy number expands rapidly through transposition genera but, through natural selection acting on the whole genome, the IS may further become virtually extinct (Cerveau et al.2011b; Duron 2013). In this scenario, periodic lateral transfers of IS1111 from one Coxiella strain to another are thus crucial for the long-term maintenance of this IS in bacterial genomes (Cerveau et al.2011b; Duron 2013). The presence of almost identical and putatively functional IS1111 copies in C. burnetii and in the _Coxiella_-like endosymbiont of R. decoloratus is precisely the typical signature of such lateral transfer.

The presence of IS1111 in both C. burnetii and in the _Coxiella_-like endosymbionts raises into question the specificity of the diagnostic tests used in field studies of Q fever epidemiology: the detection of IS1111 in ticks may reveal infection by a _Coxiella_-like endosymbiont rather than by C. burnetii, leading to an overestimation of pathogen prevalence. Furthermore, the intense intrinsic transposition activity of IS1111 also poses additional questions on its distribution beyond the Coxiella genus. The presence of so many IS1111 copies in C. burnetii genomes is the consequence of this activity (Seshadri et al.2003; Beare et al.2009), but this may also allow IS1111 to experience lateral transfers to non-Coxiella bacteria, and especially those present in ticks. This transfer pattern was recently described for another insertion sequence, ISRpe1, which was first found in the tick endosymbiont Rickettsia peacockii (Simser et al.2005) but was further found in several other intracellular bacteria, often distantly phylogenetically related as Wolbachia and Cardinium (Felsheim, Kurtti and Munderloh 2009; Duron 2013). A remarkable example of this process is the presence of nearly identical ISRpe1 copies in Rickettsia and Cardinium endosymbionts coinfecting the deer tick Ixodes scapularis, a pattern suggesting that IS can readily transfer between the genomes of the intracellular bacteria concomitantly hosted by ticks. In this context, the detection of IS1111 in tick DNA templates may be not sufficient to definitely conclude that a Coxiella bacterium is present.

In conclusion, an important consideration for future surveys of C. burnetii is the specificity of detection methods used for Q fever epidemiology. In recent years, remarkable progress has been made in designing new PCR-based techniques to detect C. burnetii, including IS1111 detection assays but also multiple-locus variable number tandem repeat analysis and multispacer sequence typing (Duron et al. in press; Sidi-Boumedine and Rousset 2011). However, the ability of these techniques to distinguish between C. burnetii and _Coxiella_-like endosymbionts remains entirely unknown and needs to be further tested before they can be applied to tick samples. The examination and the sequencing of additional genetic markers, as 16S rRNA or rpoB, are clearly required to avoid any misidentification.

I am deeply grateful to C. Chevillon, E. Jourdain, K. Sidi-Boumedine and S. Moutailler, E. Rousset for helpful discussion, to V. Noël for technical help and to K. Sidi-Boumedine for providing DNA templates of the RSA 493/Nine Mile I C. burnetii isolate. I would also like to thank C. Arnathau, L. Beati, A. S. Biguezoton, C. Chevillon, D. González Acuña, F. Dantas-Torres, G. Diatta, P. Durand, A. Guglielmone, K. Hansford, E. Jourdain, M. Labruna, M. S. Latrofa, J. Liu, K. D. McCoy, J. Medlock, S. Nava, F. Renaud, U. E. Schneppat, D. Spratt and J.-F. Trape for providing tick samples.

FUNDING

This work was supported by the CNRS-INEE (Centre National de la Recherche Scientifique – Institut Ecologie et Environnement, Programme PEPS-Ecologie de la Santé 2014 ‘SYMPATTIQUES’).

Conflict of interest. None declared.

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