Bacterial endosymbionts of ticks are of interest due to their close evolutionary relationships with tick-vectored patho- gens. For instance, whereas many ticks contain Francisella-like endosymbionts (FLEs), others transmit the mammalian pathogen Francisella tularensis. We recently sequenced the genome of an FLE present in the hard tick Amblyomma maculatum (FLE-Am) and showed that it likely evolved from a pathogenic ancestor. In order to expand our under- standing of FLEs, in the current study we sequenced the genome of an FLE in the soft tick Ornithodoros moubata and compared it to the genomes of FLE-Am, Francisella persica—an FLE in the soft tick Argus (Persicargas) arboreus, Francisella sp. MA067296—a clinical isolate responsible for an opportunistic human infection, and F. tularensis,the established human pathogen. We determined that FLEs and MA067296 belonged to a sister taxon of mammalian pathogens, and contained inactivated versions of virulence genes present in F. tularensis, indicating that the most recent common ancestor shared by FLEs and F. tularensis was a potential mammalian pathogen. Our analyses also revealed that the two soft ticks (O. moubata and A. arboreus) probably acquired their FLEs separately, suggesting that the virulence attenuation observed in FLEs are not the consequence of a single acquisition event followed by speci- ation, but probably due to independent transitions of pathogenic francisellae into nonpathogenic FLEs within separate tick lineages. Additionally, we show that FLEs encode intact pathways for the production of several B vitamins and cofactors, denoting that they could function as nutrient-provisioning endosymbionts in ticks. Key words: tick, endosymbiont, Coxiella, Francisella, Coxiella-like, Francisella-like. Introduction aphidicola in aphids that feed exclusively on phloem Ticks (order Ixodida) are ectoparasites of reptiles, birds, and (Baumann 2005; Bennett and Moran 2015)—endosymbiotic mammals. Recent molecular clock estimates indicate that they bacteria present within tick cells are thought to compensate originated in the Carboniferous period (320 million years for the dearth of B vitamins and cofactors in blood (Smith ago, Ma) and diverged in the early Permian (300 Ma) into et al. 2015; Gottlieb et al. 2015; Gerhart et al. 2016). two major families: 1) Ixodidae, or hard ticks that possess a However, unlike most primary insect endosymbionts, tick hardened chitinous scutum, and 2) Argasidae, or soft ticks endosymbionts are not maintained within specialized cells that lack scutum (Jeyaprakash and Hoy 2009; Mans et al. called bacteriocytes, but are instead found in several tissues, 2016). Ticksdependon animbalanceddiet consistingentirely including salivary glands (Klyachko et al. 2007; Budachetri of vertebrate blood. Similar to endosymbiotic bacteria in et al. 2014), which could explain their propensity to be hori- insects with specialized diets—for example, Buchnera zontally transmitted between hosts (Duron et al. 2017). The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Genome Biol. Evol. 10(2):607–615. doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 607 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gerhart et al. GBE Ticks carry endosymbionts mostly from the genera Coxiella, adult female O. moubata obtained from a laboratory colony Rickettsia,or Francisella. In addition to these putative primary (Rego et al. 2005). Out of the 180 million sequencing reads, endosymbionts (i.e. those necessary for host survival), second- the vast majority were of host origin, with <5% of reads ary symbionts that are not strictly necessary, including contributing to the assembly of the eight FLE-Om contigs Midichloria, Arsenophonus, Rickettsiella,and Wolbachia (supplementary table S1, Supplementary Material online). In species, are found in varying rates in hard and soft tick pop- addition, low quantities of reads that correspond to ulations (Ahantarig et al. 2013; Duron et al. 2017). Clostridium spp. and Burkholderia spp. were also present Understanding the biology and evolution of tick endosym- (<0.5% of reads). No other bacterium was detected at sig- bionts is of special interest because the three primary niﬁcant levels, including the previously described Coxiella-like endosymbionts share close evolutionary relationships with “Symbiont A” (Noda et al. 1997). By screening several male tick-borne pathogens Coxiella burnetii, Rickettsia parkeri, and female O. moubata adults using both bacterial and and Francisella tularensis, respectively, that cause human Coxiella-speciﬁc 16S rDNA PCR primers, we conﬁrmed that and animal diseases (Petersen et al. 2009; Ahantarig et al. FLE-Om but not the Coxiella-like “Symbiont A” has been 2013; Duron et al. 2015). Previous studies indicate that retained in the O. moubata laboratory colony. As shown pre- pathogen-symbiont transitions could occur in either direction: viously in insects, this observation suggests that whereas FLE- The mammalian pathogen C. burnetii probably originated Om is likely a primary endosymbiont that provides a critical from a tick-associated nonpathogenic ancestor (Smith et al. function to O. moubata,the Coxiella-like “symbiont A” is a 2015; Duron et al. 2015; Moses et al. 2017), whereas the FLE secondary symbiont or a transient bacterium (McCutcheon in the hard tick Amblyomma maculatum (FLE-Am) likely and Moran 2010; Hall et al. 2016). evolved from a mammalian pathogen (Gerhartetal. 2016). The 1.56 Mb genome of FLE-Om is similar in size to that In this study, in order to better understand the evolution of other FLEs (FLE-Am and F. persica), but considerably smaller and functions of FLEs in ticks, we sequenced the genome of than the 1.90 Mb genome of the human pathogen F. tular- an FLE present in the soft tick Ornithodoros moubata (FLE- ensis (table 1). Concordantly, the Gþ C content and number Om). While this project was in progress, the genome of of protein-coding genes are also lower in the three FLEs in Francisella persica—an FLE in the soft tick Argus comparison to F. tularensis, signifying a more intimate host (Persicargas) arboreus (previously referred to as Argas persi- associationinFLEs, whichleads to small, Aþ T-biased cus) was published (Hinrichs et al. 2016). By comparing the genomes with fewer functional genes (McCutcheon and genomes of FLE-Om, FLE-Am, and F. persica, we show that Moran 2010). Intriguingly, while FLE-Om and FLE-Am have FLE genomes contain intact pathways for the synthesis of similar GC%, coding density, average gene length, pseudo- several B vitamins and cofactors lacking in vertebrate blood, gene content, rRNA gene content, etc., these characteristics suggesting that FLEs function as nutrient-provisioning endo- are very distinct in F. persica, indicating that its evolutionary symbionts in ticks. We also found that a clinical isolate history is different from that of the other two FLEs. (Francisella sp. MA067296), which caused an opportunistic human infection, is more closely related to FLEs than to the Francisella persica Has Higher Virulence Potential than established human pathogen F. tularensis. Both FLEs and Other FLEs MA067296 contain pseudogenized versions of virulence genes present in F. tularensis, indicating that the common All three FLEs encode several genes shown to be critical to the ancestor of FLEs and mammalian pathogens was equipped virulence of F. tularensis (Rowe and Huntley 2015; Meibom to function as a pathogen. Furthermore, FLEs and ticks do and Charbit 2010). However, in FLE-Am and FLE-Om, as not have corresponding phylogenies, suggesting that the expected in nutrient-provisioning endosymbionts, most viru- FLEs in the two soft ticks were derived independently, either lence genes have been inactivated or appear to have been from environmental sources or through horizontal transfer deleted (table 2, supplementary table S2, Supplementary from other hosts. Material online). Francisella persica, however, contains a con- siderably higher number of potentially functional virulence- associated genes, suggesting that this endosymbiont could FLE-Om Shares More Genome Characteristics with FLE-Am be pathogenic to mammals. In fact, F. persica (previously than with F. persica misidentiﬁed as Wolbachia persica) was found to cause fe- Intracellular “coccoid” bacteria in the malpighian tubules and ver and death in guinea pigs (Suitor and Weiss 1961). ovaries of O. moubata were ﬁrst described more than four Similarly, MA067296, a Francisella strain that caused an decades ago using microscopy (Reinhardt et al. 1972). opportunistic human infection (Kugeler et al. 2008; Recently, 16S rDNA PCR was used to conﬁrm that the verti- Challacombe et al. 2017) also encodes a larger complement cally transmitted bacterium was closely related to the human of intact virulence genes than FLE-Om and FLE-Am, sug- pathogen F. tularensis (Noda et al. 1997). Using Illumina high- gesting that the presence of full-length virulence genes cor- throughput sequencing, we analyzed DNA isolated from an responds to the ability of a bacterium to cause disease in 608 Genome Biol. Evol. 10(2):607–615 doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Francisella-like Endosymbionts GBE Table 1 Genome Features of the Human Pathogen F. tularensis and FLEs Feature F. tularensis FLE-Am F. persica FLE-Om Length (bp) 1,892,772 1,556,255 1,540,154 1,564,197 GþC% 32.30 31.80 31.39 31.80 No. of coding genes 1,556 1,001 1,096 989 Coding density (%) 92 57 68 56 Average gene length (bp) 937 886 964 881 No. of pseudogenes 227 484 205 543 No. of single copy genes 106/111 106/111 106/111 106/111 16S rDNA 3 2 3 2 23S rDNA 3 2 3 2 5S rDNA 4 3 3 3 tRNAs 38 32 38 34 IS elements 72 3 2 3 Accession # NC_006570 LNCT00000000 NZ_CP013022 LVCE00000000 Albertsen et al. (2013). Both coding and pseudogenized versions of IS elements have been included. Table 2 Intact and Pseudogenized Virulence-Associate Genes in F. tularensis, Francisella sp. MA067296, and FLEs Genes F. tularensis MA067296 F. persica FLE-Am FLE-Om a b Intact U Intact U Intact U Intact U Intact U LPS and capsule 22 0 16 2 13 0 8 5 9 2 Antigen synthesis 11 0 10 0 7 0 5 2 5 2 TypeIVpili 17 0 12 3 7 4 2122 10 Outer membrane 8 0 8 0 7 0 6 2 5 3 Inner membrane 10 0 10 0 8 0 8 0 9 0 Type VI sec. system 17 0 16 1 16 0 2 6 3 9 Number of full-length genes. Number of pseudogenes. mammals. Accordingly, while it has not yet been tested, the virulence-associated genes from F. persica and six genes inactivation of most virulence genes in FLE-Om and FLE-Am from FLE-Om and FLE-Am (ﬁg. 1). is assumed to have rendered these tick endosymbionts non- pathogenic to mammals. FLEs May Provide B Vitamins and Cofactors to Ticks Mobile elements may have played a signiﬁcant role in path- ogen attenuation through the disruption and removal of Vertebrate blood lacks several B vitamins and cofactors; there- virulence-associated genes. Past studies have demonstrated fore, vertically inherited intracellular bacteria that are present that association with hosts leads to a proliferation of mobile across tick lineages are thought to provide these essential elements in bacterial genomes, followed by an extensive nutrients to the host. We examined the metabolic pathways purge as the genome is minimized to its essentials with the present in FLEs and found that they encode complete path- development of an obligate host–bacterium relationship ways for the syntheses of biotin (B7), folate (B9), lipoic acid, (Moran and Plague 2004; McCutcheon and Moran 2010). riboﬂavin (B2), and FAD (ﬁg. 2). All lack phoA gene in the In accordance with this trend, the genome of F. tularensis, folate synthesis pathway and a phosphoric monoester hydro- which has a less intimate relationship with its host, is replete lase (EC 3.1.3.-) in the riboﬂavin pathway. However, previous with insertion sequences (IS), whereas the genomes of host- studies have shown that phoA might not be required to syn- restricted FLEs contain very few IS elements (table 1). Type VI thesize folate (Bermingham and Derrick 2002), and these two secretion system gene clusters in FLEs appear to have been genes have not been retained in most mutualists, suggesting particularly disrupted by these selﬁsh genetic elements, as a that their functions could be compensated by other genes concentration of partial and pseudogenized IS elements are (Klein et al. 2013; Smith et al. 2015; Gottlieb et al. 2015; found in this gene cluster. It is likely that insertion and Manzano-Marın et al. 2015; Boyd et al. 2017). The three transposition of IS elements resulted in the loss of two FLEs displayed somewhat different capabilities to synthesize Genome Biol. Evol. 10(2):607–615 doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 609 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gerhart et al. GBE FIG.1.—Mobile element invasion of Francisella Type VI secretion system gene cluster. The positions of the identiﬁed elements suggest that virulence- associated genes M-R were lost as a consequence of composite transposon excision in FLE-Am and FLE-Om. The movement of these elements also appears to have caused a 4.5 kb inversion in these species. The pictured region occurs twice in the genome of Francisella tularensis, but only once in each of the FLEs, which may also be attributable to IS duplication and transposition. Genes A-R correspond to F. tularensis FTT_1344–FTT_1361c, respectively. the remaining B vitamins and cofactors. Whereas FLE-Am and blood meals, thereby improving tick ﬁtness and providing the FLE-Om could use aspartate to produce pantothenate (B5) impetus for ticks to maintain FLEs through generations via and convert it to Coenzyme A (CoA), F. persica lacks the ability vertical transmission. Furthermore, unlike most other arthro- to make pantothenate but could synthesize CoA if provided pod endosymbionts, F. persica has been cultured in laboratory with B5. Conversely, only F. persica encodes the pathway re- media (Hinrichs et al. 2016), and it should be feasible to de- quired for producing NADþ/NADPþ from aspartate (except velop growth media for FLE-Am and FLE-Om using our geno- nadB, which contains an internal stop site), whereas FLE-Am mic data, as previously done for other fastidious bacteria and FLE-Om could only make the two cofactors from nicotin- (Renesto et al. 2003; Omsland et al. 2009). amide. None of the FLEs has the ability to synthesize pyridox- ine (B6), but they could make the cofactor PLP (pyridoxal FLEs Evolved from a Pathogenic Ancestor and Soft Ticks phosphate) from glyceraldehyde-3-phosphate and ribulose- Have Acquired FLEs Multiple Times 5-phosphate, which are byproducts of the pentose phosphate pathway present in the bacteria. Similarly, none of the FLEs Our phylogenetic estimation using 404 genes in 49 fully se- can synthesize thiamine (B1) from either cysteine or quenced Francisella genomes revealed that FLEs and the op- glyceraldehyde-3-phosphate, but have the enzyme thiamine portunistic human pathogen Francisella sp. MA067296 are diphosphokinase to produce the cofactor TPP (thiamine pyro- closely related to each other and belong to a sister taxon of phosphate) from vitamin B1. mammalian pathogens (ﬁg. 3). This evolutionary relationship, Along with FLEs, Coxiella and Rickettsia are the two most combined with the presence of homologous virulence genes widespread tick-associated bacteria (Duron et al. 2017). Our in both the FLE and mammalian-pathogen branches of the reconstruction of biosynthetic pathways present in the Francisella tree, indicate that the common ancestor of FLEs Rickettsia endosymbiont of Ixodes scapularis (REIS; Gillespie and F. tularensis was armed with potentially pathogenic char- et al. 2012) showed that it only has the ability to produce acteristics (table 2, supplementary table S2, Supplementary biotin and folate, indicating that REIS is likely a parasite and Material online). The ability of MA067296 and F. persica to not a nutrient-providing endosymbiont. In contrast, the two cause morbidity in humans and guinea pigs, respectively, fur- Coxiella-like endosymbionts (CLEs) have complete pathways ther supports the conclusion that FLEs likely evolved from a for the synthesis of several vitamins and cofactors, including pathogenic ancestor (Suitor and Weiss 1961; Kugeler et al. for those missing in FLEs, suggestive of their roles as primary 2008; Challacombe et al. 2017). However, it should be noted cofactor-provisioning endosymbionts in ticks. In addition to that we cannot completely rule out the possibility that each cofactor production, FLEs—but not CLEs—have retained the Francisella acquired its complement of virulence genes via heme biosynthesis pathway, and have the ability to recycle horizontal gene transfer or that some of the apparently pseu- nitrogen by incorporating ammonia, a metabolic waste prod- dogenized genes in FLEs are actually functional. uct, into the synthesis of glutamine, as shown in several insect Although soft ticks O. moubata and A. arboreus host FLE- endosymbionts (e.g. Zientz et al. 2006; Lopez-Sanchez et al. Om and F. persica, respectively, FLE-Om is more closely related 2009; Sabree et al. 2009; Hansen and Moran 2011). In sum, to FLE-Am in the hard tick A. maculatum than to F. persica FLEs have the potential to supply several vitamins and cofac- (ﬁg. 4). This incongruence between tick and FLE phylogenies is tors that are present in very low concentrations in their hosts’ not entirely surprising because host–endosymbiont 610 Genome Biol. Evol. 10(2):607–615 doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Francisella-like Endosymbionts GBE FIG.2.—Cofactor and B vitamin biosynthetic pathways in tick endosymbionts. Pathways for the production of B vitamins and cofactors in FLE-Om, FLE- Am, Francisella persica,CLEAA (Coxiella-like endosymbiont of Amblyomma americanum), CRt (Coxiella-like symbiont in Rhipicephalus turanicus), and REIS (Rickettsia endosymbiont of Ixodes scapularis) are shown. Gene names and EC numbers of enzymes that catalyze each step in each bacterium are shown. No color indicates that no functional copy of the gene was present. Cofactors and B vitamins are in bold. coevolution is rare in ticks, possibly due to horizontal transfer in hard ticks than to each other, suggesting that FLEs have of symbionts between unrelated tick species (Duron et al. been exchanged between hard and soft ticks (Duron et al. 2015). While a few CLE and FLE strains have coevolved with 2017). On the basis of these observations, the two FLEs in soft their tick hosts (Duron et al. 2017; Azagi et al. 2017), a com- ticks were probably acquired from independent sources: FLE- prehensive survey of 81 species of hard and soft ticks showed Om from a hard tick, and F. persica from an as yet unknown that FLEs in several soft ticks were more closely related to FLEs (possibly environmental) source. Horizontal transfer of Genome Biol. Evol. 10(2):607–615 doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 611 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gerhart et al. GBE FIG.3.—Phylogeny estimation of FLEs. A phylogenetic tree using 404 orthologous genes in 49 fully sequenced Francisella genomes is shown. Bootstrap (Maximum Likelihood) and posterior probability (Bayesian) values are shown on top and bottom, respectively, at each node as colored dots. Francisella sp. MA067296 is an opportunistic human pathogen; Francisella sp. TX077308 was isolated from seawater (Challacombe et al. 2017). endosymbionts is more prevalent among ticks than among et al. 2007; Budachetri et al. 2014). Since tick-borne patho- insects possibly because tick endosymbionts are not restricted gens are typically found in salivary glands and are spread via to specialized host cells, but instead are usually present in tick saliva while the arthropod is feeding on hosts, the pres- several tissues including in the salivary glands (Klyachko ence of FLEs in salivary glands is likely a holdover from their 612 Genome Biol. Evol. 10(2):607–615 doi:10.1093/gbe/evy021 Advance Access publication January 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/607/4828087 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Ticks Francisella-like Endosymbionts GBE FLE-Om O. moubata FLE-Am A. arboreus F. persica A. maculatum Francisella tularensis Pholcus phalangioides bootstrap/ post. prob > 0.90 FIG.4.—FLEs and hosts have incongruent evolutionary histories. FLE phylogeny (left; same as in ﬁg. 3) does not correspond to the host phylogeny (right; based on an 18S rDNA sequences). Branch lengths are not representative of evolutionary distances. Soft ticks and their FLEs are highlighted in green. previous pathogenic lifestyle, and could facilitate their hori- DNA was sequenced in a single lane of Illumina Hi-Seq zontal transfer to other ticks or even to other blood-feeding 2500 (100 bp, paired-end) at Oregon Health and Science arthropods such as keds, while cofeeding on the same verte- University’s Massively Parallel Sequencing Shared Resource, brate host (Wright et al. 2015; Lee et al. 2016). yielding 180 million read pairs. Low conﬁdence reads In conclusion, we show that while all three FLEs likely orig- were removed using Trimmomatic (Bolger et al. 2014)and inated from pathogenic francisellae and could function as assembled into contigs with IDBA (Peng et al. 2012), using a nutrient-provisioning endosymbionts, the FLEs in two soft k-mer range of 49:121, a step size of two, and a minimum ticks have dissimilar genome characteristics, virulence poten- contig length of 1 kb. All contigs were searched using BlastN tials, and evolutionary histories, indicative of independent ac- against a library of Francisella genome sequences obtained quisition events. The soft ticks either gained preexisting FLEs from NCBI to identify putative FLE contigs. All trimmed reads from other hosts, or acquired pathogenic Francisella strains were mapped to these contigs using bowtie2 and Samtools that evolved in parallel into FLE-Om and F. persica with varying (Langmead and Salzberg 2012; Li et al. 2009). Reads that degrees of virulence loss within their respective hosts. Similar mapped to the preliminary FLE contigs along with 20% of to FLEs, previous studies suggest that Sodalis-allied endosym- original reads were reassembled into a ﬁnal set of eight FLE- bionts of insects probably evolved from a pathogenic progen- Om contigs, which have been submitted to NCBI (accession: itor, indicating that arthropods could gain new symbionts by LVCE00000000.1). The completeness of the assembled ge- domesticating potentially pathogenic bacteria (Clayton et al. nome was examined using a single-copy gene database 2012). Because of the potential to utilize insights gained from (Albertsen et al. 2013), yielding identical results for FLE-Om, studying FLEs to control tularemia and other tick-borne dis- FLE-Am, F. persica,and F. tularensis (106/111 genes). The eases, it is critical that further studies are conducted to 1) identities of bacteria present in the tick microbiome were de- delineate the evolutionary histories of all FLEs in hard and termined by binning BlastN results using MEGAN5’s taxon- soft ticks, 2) deﬁne the evolutionary relationships between omy browser (Huson et al. 2007). To verify the absence of the FLEs and environmental Francisella (Barns et al. 2005; Keim Coxiella-like “Symbiont A” (Noda et al. 1997), DNA was et al. 2007), 3) understand the functional consequences of extracted from ﬁve adult male and female ticks and PCR the genetic differences observed between FLEs, and 4) vali- was performed using both bacterial and Coxiella-speciﬁc date the nutrients potentially provided by FLEs to ticks. 16S rDNA primers (Klyachko et al. 2007; Klindworth et al. 2013). Materials and Methods Sequencing, Genome Assembly, and Bacterial Genome Annotation and Assessment of Metabolic and Identiﬁcation Virulence Potential Afemale O. moubata from a laboratory colony maintained at FLE-Om contigs were annotated using NCBI’s prokaryotic ge- the Institute of Parasitology, Czech Republic was used to se- nome annotation pipeline. Cofactor synthesis pathways in quence the FLE-Om genome. The tick’s outer surface was FLEs, CLEs, and REIS were identiﬁed with BlastKOALA sterilized using 70% ethanol, and midgut tissue was extracted (Kanehisa et al. 2016), which maps protein sequences to and subjected to Protease K digestion, followed by DNA ex- the Kyoto Encyclopedia of Genes and Genomes (Manyam traction using a DNeasy kit and protocol (Qiagen). Puriﬁed et al. 2015). In addition, biosynthesis genes present in Genome Biol. 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