bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424491; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Ticks convert pathogenic Coxiella into endosymbionts

2

3 Amanda E. Brenner1, Sebastián Muñoz-Leal2, Madhur Sachan1, Marcelo B. Labruna3, Rahul

4 Raghavan1*

5

6 1Department of Biology and Center for Life in Extreme Environments, Portland State

7 University, Portland, Oregon, 97201, USA.

8 2Departamento de Patología y Medicina Preventiva, Facultad de Ciencias Veterinarias,

9 Universidad de Concepción, Chillán, Ñuble, Chile.

10 3Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina

11 Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brazil.

12

13

14 *Author for correspondence:

15 Rahul Raghavan | Phone: 503-725-3872 | Email: [email protected]

16

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424491; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

17 ABSTRACT

18 Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality

19 in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the

20 reproductive success of ticks — a major disease vector, while Coxiella burnetii is the etiological

21 agent of human Q fever and uncharacterized coxiellae cause infections in both animals and

22 humans. To better understand the evolution of pathogenesis and symbiosis in this group of

23 intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros

24 amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales.

25 Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen,

26 than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts

27 and pathogens, indicating that several CLE lineages have evolved independently from

28 pathogenic Coxiella. We also determined that the last common ancestor of CLEOA and C.

29 burnetii was equipped to infect macrophages, and that even though HGT contributed

30 significantly to the evolution of C. burnetii, most acquisition events occurred primarily in

31 ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution

32 of C. burnetii, which previously was assumed to have emerged when an avirulent tick

33 endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic

34 pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the

35 human pathogen but not the tick symbiont, and show that the use of heme analogs is a

36 promising approach to controlling C. burnetii infections.

37

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424491; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

38 INTRODUCTION

39 A bacterium’s genome size and gene content signal both the degree of its dependence on the

40 host and the length of the bacterium-host relationship. For example, a bacterium that has

41 established a long-term, obligate symbiosis would have a tiny genome filled with protein-coding

42 genes (Wernegreen et al. 2000). Conversely, the genome of a bacterium that is in early stages of

43 symbiosis is usually large and contains numerous pseudogenized genes, which, as the relationship

44 progresses, would eventually be lost, resulting in a tiny genome (Moran 2002; McCutcheon and

45 Moran 2011). The genomes of Coxiella-like endosymbionts (CLEs) found in ticks (Acari:

46 Ixodida) fall into both categories: Some ticks, e.g., Amblyomma americanum and A. sculptum,

47 contain small-genomed CLEs (~0.6 Mbp) that have few pseudogenes, indicating that they

48 represent an ancient lineage of tick endosymbionts (Smith et al. 2015). In contrast, CLEs in

49 Rhipicephalus turanicus (CRt), and R. sanguineus have large genomes (~1.7 Mbp) filled with

50 pseudogenes, denoting that the bacteria are in early stages of symbioses (Gottlieb et al. 2015;

51 Tsementzi et al. 2018). While most ticks contain CLEs, a few have Francisella-like

52 endosymbionts (FLEs) instead (Gerhart et al. 2016, 2018; Duron et al. 2017). All FLEs studied

53 to date have large genomes (~1.5 Mbp) with hundreds of pseudogenes, including inactivated

54 virulence genes, indicating that FLEs evolved recently from pathogenic ancestors (Gerhart et al.

55 2016, 2018; Duron et al. 2018b). Irrespective of their age, CLEs and FLEs improve the

56 reproductive fitness of their hosts by likely providing metabolites missing in vertebrate blood,

57 ticks’ sole nutritional source (Gottlieb et al. 2015; Smith et al. 2015; Gerhart et al. 2016, 2018;

58 Duron et al. 2017, 2018b; Tsementzi et al. 2018).

59 C. burnetii, the causative agent of human Q fever, has also been detected in ticks; in fact,

60 the intracellular pathogen was first isolated from hard ticks Dermacentor andersoni and

3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424491; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

61 Haemaphysalis humerosa (Cox 1938; Smith and Derrick 1940). In addition, transstadial

62 transmission and fecal excretion of C. burnetii occur in laboratory-raised ticks (Eldin et al. 2017;

63 Körner et al. 2020). However, it is not clear whether ticks play any meaningful role in the natural

64 spread of C. burnetii (Duron et al. 2015b); instead, Q fever generally occurs following inhalation

65 of C. burnetii-contaminated aerosols originating from infected farm animals (Maurin and Raoult

66 1999; Eldin et al. 2017). Within the human lungs, C. burnetii infects alveolar macrophages and

67 generates a large replicative vacuole, termed the Coxiella-containing vacuole (CCV), by

68 subverting host responses through a Dot/Icm Type IVB secretion system (T4BSS). This

69 secretion system is essential to the pathogenicity of both C. burnetii and Legionella pneumophila,

70 the two established pathogens in the order Legionellales (Segal et al. 1999; Chen et al. 2010;

71 Beare et al. 2011; Newton et al. 2014; Burstein et al. 2016). Genes for T4BSS, which evolved

72 from conjugation machinery, have spread across the bacterial kingdom via horizontal gene

73 transfer (HGT), a process through which organisms gain foreign genes, allowing them to quickly

74 adapt to a new environment (Ochman et al. 2000; Lerat et al. 2005).

75 The closest relatives of C. burnetii are CLEs present in ticks (Almeida et al. 2012; Duron

76 et al. 2015a; Smith et al. 2015), leading to the notion that the human pathogen emerged when

77 an avirulent tick endosymbiont gained pathogenicity genes, probably via HGT (Duron et al.

78 2015a; Gerhart et al. 2016). Contrary to this hypothesis, by sequencing the genome of a CLE in

79 Ornithodoros amblus (henceforth referred to as CLEOA), we show that a common virulent

80 ancestor gave rise to both C. burnetii and CLEOA. The potentially-pathogenic ancestor

81 contained genes for most virulence factors, including T4BSS, indicating that the erstwhile

82 bacterium was likely capable of infecting mammalian macrophages. In CLEOA, homologs of

83 most virulence-associated genes have been rendered non-functional, but its genome is enriched

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.29.424491; this version posted December 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

84 for B vitamin and cofactor biosynthesis pathways, suggesting that a virulent bacterium has

85 morphed into a nutrient-provisioning tick endosymbiont. Similar to this pathogen-to-

86 endosymbiont transformation, we found that several other tick endosymbionts likely evolved

87 from pathogenic ancestors, indicating that the blood-sucking arthropod is proficient at

88 “domesticating” virulent bacteria into endosymbionts. Finally, by inhibiting C. burnetii growth

89 using a synthetic analog of heme, a metabolite produced by C. burnetii but not by CLEOA, we

90 demonstrate how knowledge gained through comparative genomics could be applied to

91 developing novel strategies to control Q fever, which is difficult to treat with currently available

92 antibiotics.

93

94 RESULTS

95 CLEOA arose from a pathogenic ancestor

96 Phylogenetic trees based mainly on 16S rDNA have previously indicated that the closest relatives

97 of C. burnetii are CLEs present in Orniothodoros and Argas soft ticks (family Argasidae) (Almeida

98 et al. 2012; Duron et al. 2015a; Smith et al. 2015); however, all CLE genomes available to date

99 are from CLEs in hard ticks (family Ixodidae) (Gottlieb et al. 2015; Smith et al. 2015;

100 Tsementzi et al. 2018; Guizzo et al. 2017), stymieing earlier efforts to understand C. burnetii

101 evolution. Here, by sequencing the first soft-tick CLE genome, we were able to build a more

102 definitive phylogenomic tree, which confirmed that CLEOA is a sister taxon of C. burnetii (Fig.

103 1; Supplemental Table S1). In addition, the presence of pseudogenized T4BSS genes in

104 CLEOA indicates that the tick-symbiont evolved from a pathogenic ancestor with a functional

105 T4BSS (Fig. 2; Supplemental Table S2).

5 106 To resolve the ancestry of pathogenicity in Legionellales we determined the prevalence of

107 T4BSS, which is an essential virulence factors in this group of bacteria that includes human

108 pathogens (C. burnetii and L. pneumophila), opportunistic pathogens (Rickettsiella), and

109 symbionts (CLEs). Our analyses revealed that the secretion system is intact in all members of

110 this order with the exception of CLEs, which only contained remnants of the T4BSS (Figs. 1,

111 2). The most parsimonious explanation for this phyletic pattern is that T4BSS was present in the

112 common ancestor of all Legionellales and was later lost in lineages that gave rise to CLEs,

113 including CLEOA.

T4BSS Francisella tularensis FrancisellaFrancisellaFrancisella tularensis tularensistularensis FrancisellaFrancisellaFrancisellaFrancisella tularensis tularensistularensis tularensis ThiomicrospiraThiomicrospiraThiomicrospira aerophila aerophilaaerophila ThiomicrospiraThiomicrospiraThiomicrospiraThiomicrospira aerophila aerophilaaerophila aerophila BerkiellaBerkiellaBerkiella aquae aquaeaquae BerkiellaBerkiellaBerkiellaBerkiella aquae aquaeaquae aquae BerkiellaBerkiellaBerkiella cookevillensis cookevillensiscookevillensis BerkiellaBerkiellaBerkiellaBerkiella cookevillensis cookevillensiscookevillensis cookevillensis TatlockiaTatlockiaTatlockia micdadei micdadeimicdadei TatlockiaTatlockiaTatlockiaTatlockia micdadei micdadeimicdadei micdadei LegionellaLegionellaLegionella israelensis israelensisisraelensis LegionellaLegionellaLegionellaLegionella israelensis israelensisisraelensis israelensis LegionellaLegionellaLegionella pneumophila pneumophilapneumophila subsp. subsp.subsp. pascullei pasculleipascullei LegionellaLegionellaLegionellaLegionella pneumophila pneumophilapneumophila pneumophila subsp. subsp.subsp. subsp. pascullei pasculleipascullei pascullei LegionellaLegionellaLegionella pneumophila pneumophilapneumophila subsp. subsp.subsp. pneumophila pneumophilapneumophila str. str.str.Legionella ATCC ATCCATCCLegionellaLegionellaLegionella 43290 4329043290 pneumophila pneumophilapneumophila pneumophila subsp. subsp.subsp. subsp. pneumophila pneumophilapneumophila pneumophila str. str. str.ATCCstr. ATCCATCC ATCC 43290 4329043290 43290 LegionellaLegionellaLegionella pneumophila pneumophilapneumophila subsp. subsp.subsp. pneumophila pneumophilapneumophila str. str.str.Legionella Philadelphia1 Philadelphia1Philadelphia1LegionellaLegionellaLegionella pneumophila pneumophilapneumophila pneumophila subsp. subsp.subsp. subsp. pneumophila pneumophilapneumophila pneumophila str. str. str.Philadelphia1str. Philadelphia1Philadelphia1 Philadelphia1 FluoribacterFluoribacterFluoribacter bozemanae bozemanaebozemanae FluoribacterFluoribacterFluoribacterFluoribacter bozemanae bozemanaebozemanae bozemanae FluoribacterFluoribacterFluoribacter gormanii gormaniigormanii FluoribacterFluoribacterFluoribacterFluoribacter gormanii gormaniigormanii gormanii FluoribacterFluoribacterFluoribacter dumoffii dumoffiidumoffii str. str.str. NY23 NY23NY23 FluoribacterFluoribacterFluoribacterFluoribacter dumoffii dumoffiidumoffii dumoffii str. str. str.NY23str. NY23NY23 NY23 N1 FluoribacterFluoribacterFluoribacter dumoffii dumoffiidumoffii str. str.str. TexKL TexKLTexKL FluoribacterFluoribacterFluoribacterFluoribacter dumoffii dumoffiidumoffii dumoffii str. str. str.TexKLstr. TexKLTexKL TexKL AquicellaAquicellaAquicella lusitana lusitanalusitana AquicellaAquicellaAquicellaAquicella lusitana lusitanalusitana lusitana AquicellaAquicellaAquicella siphonis siphonissiphonis AquicellaAquicellaAquicellaAquicella siphonis siphonissiphonis siphonis DiplorickettsiaDiplorickettsiaDiplorickettsia massiliensis massiliensismassiliensis DiplorickettsiaDiplorickettsiaDiplorickettsiaDiplorickettsia massiliensis massiliensismassiliensis massiliensis RickettsiellaRickettsiellaRickettsiella viridis viridisviridis RickettsiellaRickettsiellaRickettsiellaRickettsiella viridis viridisviridis viridis RickettsiellaRickettsiellaRickettsiella grylli grylligrylli RickettsiellaRickettsiellaRickettsiellaRickettsiella grylli grylligrylli grylli RickettsiellaRickettsiella isopodorumisopodorum Rickettsiella isopodorum N2 RickettsiellaRickettsiella isopodorumisopodorum RickettsiellaRickettsiellaRickettsiella isopodorum isopodorumisopodorum CoxiellaCoxiella(CLEAA)Coxiella endosymbiont endosymbiontendosymbiont of ofof Amblyomma AmblyommaAmblyomma americanum americanumamericanum(CLEAA)(CLEAA)(CLEAA)(CLEAA) CoxiellaCoxiella(CRm)Coxiella endosymbiont endosymbiontendosymbiont of ofof Rhipicephalus RhipicephalusRhipicephalus microplus microplusmicroplus(CRm)(CRm)(CRm)(CRm) CoxiellaCoxiella(CRt)Coxiella endosymbiont endosymbiontendosymbiont of ofof Rhipicephalus RhipicephalusRhipicephalus turanicus turanicusturanicus(CRt)(CRt)(CRt)(CRt) Confidence N3 CoxiellaCoxiella(CLEOA)Coxiella endosymbiont endosymbiontendosymbiont of ofof Ornithodoros OrnithodorosOrnithodoros amblus amblusamblus(CLEOA)(CLEOA)(CLEOA)(CLEOA) 90-100 CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. Dugway DugwayDugway CoxiellaCoxiellaCoxiellaCoxiella burnetii burnetiiburnetii burnetii str. str. str.Dugwaystr. DugwayDugway Dugway 80-89 Coxiella burnetii str. CbuK Q154 70-79 N4 CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. CbuK CbuKCbuK Q154 Q154Q154 CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str. str.CbuK CbuKCbuK Q154 Q154Q154 CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. MSU MSUMSU Goat GoatGoat CoxiellaCoxiellaCoxiellaCoxiella burnetii burnetiiburnetii burnetii str. str. str.MSUstr. MSUMSU MSU Goat GoatGoat Goat N5 T4BSS Status CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. PE15890 PE15890PE15890 CoxiellaCoxiellaCoxiellaCoxiella burnetii burnetiiburnetii burnetii str. str. str.PE15890str. PE15890PE15890 PE15890 Coxiella burnetii str. ATCC 3262 Functional CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. ATCC ATCCATCC 3262 32623262 CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str. str.ATCC ATCCATCC 3262 32623262 Pseudogenized CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. NMII NMIINMII CoxiellaCoxiellaCoxiellaCoxiella burnetii burnetiiburnetii burnetii str. str. str.NMIIstr. NMIINMII NMII Absent CoxiellaCoxiellaCoxiella burnetii burnetiiburnetii str. str.str. RSA RSARSA493RSA 493 493493 CoxiellaCoxiellaCoxiellaCoxiella burnetii burnetiiburnetii burnetii str. str. str.RSA493str. RSA493RSA493 RSA493 114 115 Figure 1. CLEOA is the closet relative of C. burnetii. Maximum likelihood and Bayesian trees 116 built using 117 single-copy protein-coding genes were combined to generate the shown 117 phylogenomic tree. Bootstrap support and posterior probabilities agreed at all branchpoints and

6 118 are depicted as a single confidence value. The Dot/Icm Type IVB secretion system (T4BSS), 119 which is critical to pathogenesis, is found in all members of the order Legionellales, but has been 120 pseudogenized in CLEs. Nodes N1-N5 mark major branching points in the evolution of C. 121 burnetii. 122

CLEOA C. burnetti RSA493 CRt

IcmX IcmX IcmX Intact T4BSS Gene IcmW IcmW IcmW IcmV IcmV Pseudogenized DotA DotA T4BSS Gene

Intact Non-T4BSS Gene

DotB DotB Pseudogenized DotC DotC Non-T4BSS Gene DotD DotD IcmS IcmS IcmT Intact IcmQ Transposase Gene IcmP IcmQ IcmO Scale: 1.0 Kb IcmP IcmN IcmO IcmL2 IcmL1 IcmN IcmK IcmL2 IcmL IcmL1 IcmE IcmK IcmK IcmG IcmE IcmC

IcmG IcmC IcmD IcmD IcmJ IcmD IcmJ IcmB IcmB IcmB

IcmH IcmH 123

124 Figure 2. CLEs contain nonfunctional T4BSS. Comparison of T4BSS genes in CLEOA, C. 125 burnetii RSA493 and CRt indicate that the secretion system has been rendered nonfunctional in 126 tick endosymbionts. Filled blocks represent intact genes. Outlined blocks represent 127 pseudogenized genes. 128

129

7 130 Multiple CLEs have evolved from pathogens

131 Coxiella detected in ticks are classified into four clades, three of which contain intermingled

132 pathogens and endosymbionts (Fig. 3; Supplemental Table S3; Duron et al. 2015a). Clade A

133 includes C. burnetii — the human pathogen, and CLEOA, which arose from a pathogenic

134 ancestor, as discussed above. In Clade B, CLEs of Haemaphysalis ticks are present along with a

135 presumably pathogenic Coxiella that caused horse infection (Seo et al. 2016). Clade C has CRt,

136 a pathogen-derived endosymbiont, along with strains that caused opportunistic human skin

137 infections (Gottlieb et al. 2015; Angelakis et al. 2016; Guimard et al. 2017; Tsementzi et al.

138 2018; Ben-Yosef et al. 2020). Only Clade D, which contains small-genomed CLEs (e.g.,

139 CLEAA), has no known pathogenic representatives. This phylogenetic pattern of

140 endosymbionts clustering with pathogens indicate that, similar to the pathogenic ancestry of

141 CLEOA and CRt, CLEs of several other ticks have also evolved from pathogenic coxiellae.

142 Thus, based on phylogenetic and T4BSS distribution patterns, we surmise that Coxiella strains

143 that infect vertebrates (e.g., humans, horses, birds) and invertebrates (e.g., crayfish) are

144 widespread across the globe (Fig. 3), and ticks have converted several of them into beneficial

145 endosymbionts.

8 Legionella pneumophila subspecies pneumophila strain Philadelphia1 Coxiella endosymbiont of Ornithodoros marocanus isolate Omaro1 Coxiella endosymbiont of Amblyomma geayi isolate CoxAgeay1 Coxiella endosymbiont of Amblyomma naponense isolate CoxAnapo1 Coxiella endosymbiont of Haemaphysalis punctata isolate Haepun3 Coxiella species H-GG-175 Uncultured Coxiella species clone S40 Coxiella cheraxi strain TO-98 Coxiella endosymbiont of Ornithodoros erraticus isolate Oer1 CoxiellaLegionella endosymbiont pneumophila of subspecies Amblyomma pneumophila coelebs isolate strain CoxAcoel1 Philadelphia1 CoxiellaLegionella endosymbiont pneumophila of subspecies Amblyomma pneumophila calcaratum strain isolate Philadelphia1 CoxAcalca Coxiella endosymbiont of DermacentorOrnithodoros silvarummarocanus isolate isolate Dsilv1 Omaro1 Coxiella endosymbiont of AmblyommaDermacentorOrnithodoros geayi marginatusmarocanus isolate isolate CoxAgeay1 Dmar1Omaro1 Coxiella endosymbiont of Amblyomma naponensegeayiscalpturatum isolate isolate CoxAgeay1isolate CoxAnapo1 CoxAscalpt1 CoxiellaLegionellaLegionella endosymbiont pneumophilapneumophila of subspeciessubspecies AmblyommaHaemaphysalis pneumophilapneumophila naponenselatepunctatum punctata strainstrainisolate isolate isolate Philadelphia1Philadelphia1 CoxAnapo1 Haepun3 CoxAlate1* Human Pathogen LegionellaRickettsiellaCoxiella speciesendosymbiont pneumophila Clade H-GG-175 of subspecies OrnithodorosHaemaphysalis pneumophila sonrai punctata isolate strain isolate Oson1 Philadelphia1 Haepun3 LegionellaCoxiellaUnculturedCoxiella endosymbiontspeciesendosymbiont pneumophila Coxiella H-GG-175 species of ofsubspecies OrnithodorosOrnithodoros clone S40 pneumophila marocanusoccidentalismarocanus strain isolate isolateisolate Philadelphia1 Omaro1 Omaro1Oocci1 CoxiellaUnculturedCoxiella endosymbiontcheraxiendosymbiont Coxiella strain species ofTO-98of AmblyommaOrnithodorosAmblyomma clone* 1358S40 geayi geayimarocanus isolateisolate isolate CoxAgeay1CoxAgeay1 Omaro1 Crayfish Infection CoxiellaUnculturedCoxiella endosymbiont cheraxiendosymbiont Coxiella strain species ofTO-98of AmblyommaOrnithodorosAmblyomma clone 1357 geayinaponense naponensemarocanuserraticus isolate isolate isolate isolate isolateCoxAgeay1 Oer1 CoxAnapo1 CoxAnapo1Omaro1 CoxiellaCoxiella endosymbiont endosymbiont of of Amblyomma HaemaphysalisOrnithodorosHaemaphysalis naponensecajennensegeayicoelebs erraticus punctatapunctata isolate isolate isolate isolate isolate isolateisolateCoxAgeay1 CoxAcoel1 Oer1 CoxAnapo1 CoxAcaj1 Haepun3Haepun3 CoxiellaCoxiella endosymbiontspeciesspecies H-GG-175H-GG-175 of AmblyommaHaemaphysalis naponensecoelebssculptumcalcaratum punctata isolate isolate isolate isolateisolate CoxAcoel1 CeAS-UFV CoxAnapo1 CoxAcalcaHaepun3 CoxiellaUnculturedUncultured speciesendosymbiont CoxiellaCoxiella H-GG-175 speciesspecies of AmblyommaDermacentorHaemaphysalis* cloneclone S40S40 cajennense calcaratumsilvarum punctata isolate isolateisolate Dsilv1 CoxAcalcaAcajen1Haepun3 Equine Infection UnculturedCoxiellaCoxiella speciesendosymbiont cheraxicheraxi Coxiella strainH-GG-175strain species TO-98ofTO-98 DermacentorAmblyomma clone S40 americanummarginatussilvarum isolate isolateisolate Dsilv1 Dmar1 C904 CoxiellaUnculturedCoxiella endosymbiont cheraxiendosymbiont Coxiella strain species TO-98of of Rhipicephalus DermacentorOrnithodorosAmblyommaOrnithodoros clone S40 scalpturatummarginatus erraticus erraticusmicroplus isolateisolate isolateisolate isolate Oer1Oer1 Rhmicro1Dmar1 CoxAscalpt1 CoxiellaCoxiella endosymbiontcheraxiendosymbiont strain of TO-98of Rhipicephalus AmblyommaOrnithodorosAmblyomma coelebslatepunctatumscalpturatum coelebserraticus microplus isolateisolate isolate isolate isolate isolate CoxAcoel1CoxAcoel1 Oer1 CLERM CoxAscalpt1 CoxAlate1 CoxiellaCoxiella endosymbiont endosymbiont of ofof RhipicephalusAmblyommaOrnithodoros Amblyomma coelebs calcaratumlatepunctatumcalcaratumsonraierraticus australis isolateisolate isolateisolate isolateisolate isolateOson1CoxAcoel1 Oer1Rhaus1 CoxAcalcaCoxAcalca CoxAlate1 CoxiellaCoxiella endosymbiont endosymbiont of ofof Rhipicephalus AmblyommaDermacentorOrnithodorosDermacentor coelebs calcaratum silvarumsonrai occidentalissilvarumannulatus isolateisolate isolateisolate isolate isolate Oson1CoxAcoel1 Dsilv1Dsilv1 CoxAcalcaRhannu1 Oocci1 CoxiellaUnculturedCoxiella endosymbiont endosymbiont Coxiella species of of DermacentorRhipicephalus AmblyommaOrnithodorosDermacentor clone 1358 calcaratum silvarummarginatusoccidentalis marginatusdecoloratus isolate isolate isolate isolate isolateisolate Dsilv1 CoxAcalca Dmar1 Dmar1 Oocci1Rhdeco1 CoxiellaUnculturedCoxiella endosymbiont endosymbiont Coxiella species of of RhipicephalusDermacentor AmblyommaAmblyomma clone 13571358 scalpturatummarginatussilvarumscalpturatum evertsi isolateisolate isolate isolateisolate Rhever1Dsilv1 Dmar1 CoxAscalpt1CoxAscalpt1 * Avian Pathogen CoxiellaUnculturedCoxiella endosymbiont endosymbiont Coxiella species of of RhipicephalusAmblyommaDermacentor Amblyomma clone 1357 cajennense latepunctatumscalpturatummarginatuslatepunctatum *bursa isolate isolateisolate isolate isolateRhbursa1isolate CoxAcaj1Dmar1 CoxAscalpt1 CoxAlate1CoxAlate1 CoxiellaCoxiella endosymbiont endosymbiont of ofof RhipicephalusAmblyomma OrnithodorosOrnithodoros cajennenselatepunctatumsculptumscalpturatum sonraisonraisanguineus isolateisolate isolate isolate isolate isolate Oson1Oson1 CeAS-UFV CoxAcaj1 Rhsa1CoxAscalpt1 CoxAlate1 CoxiellaLegionellaCoxiellaceaeCoxiella endosymbiont endosymbiont pneumophila bacterium of ofAL07subspecies AmblyommaOrnithodoros Ornithodoros pneumophila cajennenselatepunctatum sculptumsonrai occidentalisoccidentalis isolate isolate strain isolate isolate isolateisolateOson1 Philadelphia1CeAS-UFV Acajen1 Oocci1 Oocci1CoxAlate1 CoxiellaUnculturedUncultured endosymbiont CoxiellaCoxiella speciesspecies ofof RhipicephalusAmblyommaOrnithodoros cloneclone 13581358 cajennense americanumsonraioccidentalis sanguineus isolate isolate isolateisolate Oson1 Acajen1 C904Oocci1CRS-CAT UnculturedCoxiellaCoxiellaceaeUncultured endosymbiont CoxiellaCoxiella bacterium speciesspecies of AL08 RhipicephalusOrnithodorosAmblyomma cloneclone 135713581357 americanummarocanusoccidentalis microplus isolate isolateisolate Rhmicro1Omaro1 C904Oocci1 UnculturedCoxiellaCoxiella endosymbiontendosymbiont Coxiella species ofof AmblyommaRhipicephalusAmblyomma clone 13571358 cajennensegeayicajennense microplusturanicus isolate isolate isolate isolateCoxAgeay1 Rhtura1 CoxAcaj1CLERMRhmicro1CoxAcaj1 CLADE D CoxiellaUnculturedCoxiella endosymbiont endosymbiont Coxiella species of of RhipicephalusAmblyomma Amblyomma clone 1357 cajennensenaponense sculptumsculptum australismicroplusturanicus isolate isolate isolate isolateisolate CeAS-UFV CeAS-UFV Rhaus1 CoxAnapo1CoxAcaj1CRtCLERM • CoxiellaCoxiella endosymbiontendosymbiont of of Rhipicephalus AmblyommaHaemaphysalisAmblyomma cajennensesculptumcajennense annulatusaustralispusillus punctata isolate isolate isolateisolate CeAS-UFVRhpusi1 Rhaus1 CoxAcaj1Acajen1Rhannu1Haepun3Acajen1 CLEs of Amblyomma CoxiellaCoxiella endosymbiontspecies endosymbiont H-GG-175 of of RhipicephalusAmblyommaOrnithodoros AmblyommaAmblyomma cajennensesculptum americanumamericanum peruvianusannulatusdecoloratus isolate isolate isolate isolate isolateisolate CeAS-UFV Acajen1Rhannu1 OpC2 C904 C904Rhdeco1 • No Known Pathogens CoxiellaUnculturedCoxiella endosymbiontendosymbiont Coxiella species of of AmblyommaRhipicephalus OrnithodorosRhipicephalus clone S40 cajennenseamericanum spheniscus evertsi microplusdecoloratusmicroplus isolate isolate isolateisolate isolate Rhever1 Acajen1 Rhmicro1 Rhmicro1Osphe1C904 Rhdeco1 CoxiellaCoxiella endosymbiontcheraxiendosymbiont strain ofTO-98ofof RhipicephalusAmblyommaOrnithodorosRhipicephalus americanum amblus bursamicroplusevertsimicroplus isolate isolate isolate isolateisolate Rhbursa1 AB428Rhever1 CLERMRhmicro1 CLERMC904 CoxiellaCoxiella endosymbiontendosymbiont ofof RhipicephalusOrnithodorosRhipicephalus capensiserraticus sanguineusaustralismicroplusbursaaustralis isolate isolate isolate isolateisolate isolate Rhbursa1 Oca1Oer1 Rhaus1 Rhaus1CLERMRhmicro1 Rhsa1 CoxiellaCoxiellaceaeCoxiella endosymbiont endosymbiont bacterium of ofAL07 Rhipicephalus AmblyommaOrnithodorosRhipicephalus coelebs maritimus australis annulatussanguineusmicroplusannulatus isolate isolate isolate isolateisolate isolate CoxAcoel1 Rhaus1 Omari1 Rhannu1CLERMRhannu1 Rhsa1 CoxiellaCoxiellaceaeCoxiella endosymbiont endosymbiont bacterium of ofAL07 Rhipicephalus ArgasAmblyommaRhipicephalus monachus calcaratum annulatusaustralissanguineus decoloratusdecoloratus isolate isolate isolateAmo01 isolate isolateisolate Rhaus1 CoxAcalcaRhannu1 CRS-CAT Rhdeco1Rhdeco1 CoxiellaCoxiellaceaeCoxiella endosymbiontburnetiiendosymbiont bacterium strain of Ammassalik ofAL08 Rhipicephalus DermacentorRhipicephalus silvarum annulatus evertsisanguineusdecoloratusevertsi isolateisolate isolate isolateisolate Rhever1Dsilv1Rhever1 Rhannu1 CRS-CATRhdeco1 Coxiella endosymbiontburnetii strain ofATCC RhipicephalusDermacentor 3262 marginatus evertsituranicus isolate isolate isolate Rhever1 Rhtura1 Dmar1 CLADE C CoxiellaCoxiellaceaeCoxiella endosymbiontendosymbiont bacterium of ofAL08 RhipicephalusRhipicephalus bursaevertsidecoloratusbursa isolate isolateisolate isolate Rhbursa1 Rhbursa1Rhever1 Rhdeco1 • CoxiellaCoxiella endosymbiontburnetiiendosymbiont strain ofNMIIof RhipicephalusAmblyommaRhipicephalus scalpturatum sanguineusbursaevertsituranicussanguineus isolate isolate isolate isolateisolate Rhbursa1 Rhever1 Rhtura1CRt Rhsa1CoxAscalpt1Rhsa1 CLEs of Rhipicephalus CoxiellaCoxiellaceaeCoxiellaceae endosymbiont bacteriumbacterium ofof AL07AL07 RhipicephalusAmblyommaRhipicephalusOrnithodoros* latepunctatum brasiliensis sanguineusbursapusillusturanicus isolate isolate isolate isolateisolate isolateRhbursa1 Rhpusi1 CRt Obr01Rhsa1 CoxAlate1 • Human Skin Infection Strains CoxiellaceaeCoxiellaCoxiella endosymbiont burnetiiendosymbiont bacterium strain of Dugwayof ofAL07 Rhipicephalus OrnithodorosRhipicephalus sonrai peruvianussanguineus pusillussanguineus isolate isolate isolate isolate isolateOson1 Rhpusi1 OpC2 Rhsa1 CRS-CATCRS-CAT CoxiellaCoxiellaceaeCoxiellaceae endosymbiontburnetii bacterium bacterium strain ofPE-15890 ofAL07 AL08AL08 RhipicephalusOrnithodoros* spheniscusoccidentalis peruvianussanguineus isolate isolate OpC2Osphe1 CRS-CATOocci1 CoxiellaceaeUnculturedCoxiellaCoxiella endosymbiontburnetiiendosymbiont Coxiella bacterium strain species of ofZ3055ofAL08 Rhipicephalus OrnithodorosRhipicephalus clone 1358 spheniscus amblussanguineus turanicusturanicus isolate isolate isolateisolate isolate AB428 Rhtura1 Rhtura1Osphe1 CRS-CAT CoxiellaUnculturedCoxiellaceaeCoxiella endosymbiontburnetii endosymbiont Coxiella bacterium strain species CbuKof of ofAL08 Rhipicephalus OrnithodorosRhipicephalus clone Q154 1357 capensis amblusturanicus turanicus isolate isolate isolate isolate AB428 Oca1 Rhtura1 CRtCRt CoxiellaCoxiella endosymbiontendosymbiont ofof RhipicephalusAmblyommaOrnithodorosRhipicephalus cajennense capensismaritimus rostratus pusillusturanicuspusillus isolateisolate isolateisolate Rhpusi1 Oca1Rhpusi1 Orost1 Omari1Rhtura1CRtCoxAcaj1 CoxiellaBacteriumCoxiella endosymbiont endosymbiont symbiont of Haemaphysalisof of RhipicephalusArgasAmblyomma OrnithodorosOrnithodoros monachus sculptumlagrangei maritimus peruvianusperuvianuspusillusturanicus isolate isolateclone isolateAmo01isolate isolateisolate HLSD3 CeAS-UFVRhpusi1 Omari1CRt OpC2OpC2 CoxiellaCoxiella endosymbiont burnetiispeciesendosymbiont strainH-JJ-10 ofAmmassalik of *RhipicephalusAmblyomma ArgasOrnithodorosOrnithodoros monachus cajennense spheniscusperuvianusspheniscuspusillus isolate isolate Amo01isolate isolate isolate Rhpusi1 Acajen1 OpC2 Osphe1Osphe1 CoxiellaUnculturedCoxiella endosymbiont burnetiiendosymbiont Coxiella strain species AmmassalikofATCC of OrnithodorosAmblyomma Ornithodoros clone 3262 CYP-1 americanum spheniscusperuvianus amblusamblus isolateisolate isolateisolate AB428AB428 OpC2Osphe1C904 CoxiellaBacteriumCoxiella endosymbiont burnetiiendosymbiont symbiont strain of HaemaphysalisofNMII ATCCof Rhipicephalus OrnithodorosOrnithodoros 3262 longicornis capensisspheniscusamblus capensismicroplus isolate isolateisolate isolateisolate AB428 Oca1Oca1 Rhmicro166Osphe1 CoxiellaUnculturedCoxiella endosymbiont burnetiiendosymbiont bacterium strain clone ofNMII ofof RhipicephalusOrnithodoros Ornithodoros CH1 capensis maritimusbrasiliensisamblusmaritimusmicroplus isolate isolate isolateisolate isolate AB428 Oca1 Omari1CLERMOmari1 Obr01 CoxiellaBacteriumCoxiella endosymbiont burnetiiendosymbiont symbiont strain of Haemaphysalisof Dugwayofof Rhipicephalus ArgasOrnithodorosArgas monachusmonachus shimogacapensismaritimus brasiliensisaustralis isolateisolate clone isolate isolate isolate Amo01 Amo01isolate HSKY-3 Oca1 Rhaus1Omari1 Obr01 CoxiellaCoxiella endosymbiont burnetiiburnetii strainstrain of AmmassalikPE-15890DugwayAmmassalik RhipicephalusHaemaphysalisArgasOrnithodoros monachus maritimus annulatus obesa isolate isolate isolateAmo01isolate TPSD8 Omari1Rhannu1 CoxiellaCoxiella endosymbiontburnetii burnetii strain strainstrain ofAmmassalik ATCCPE-15890Z3055ATCC IxodesRhipicephalusArgas 32623262 monachushexagonus decoloratus isolate isolate Amo01 Ihexa1 isolate Rhdeco1 CLADE B CoxiellaCoxiella endosymbiont burnetiiburnetii strainstrain ofCbuKNMIIAmmassalikATCCZ3055NMII RhipicephalusIxodes Q1543262 ricinus isolateevertsi Iric1isolate Rhever1 • CLEs of Haemaphysalis CoxiellaCoxiella endosymbiontburnetii endosymbiont strain ofNMIICbuK ATCCofof RhipicephalusIxodes OrnithodorosOrnithodoros Q1543262 uriae isolate brasiliensisrostratusbrasiliensisbursa Iuriae1 isolate isolate isolateisolate Rhbursa1 Orost1 Obr01Obr01 • Equine Infection Strain CoxiellaBacteriumCoxiella endosymbiontburnetii burnetii symbiont strain strain of HaemaphysalisofNMII DugwayofDugway RhipicephalusAmblyommaOrnithodoros splendidumlagrangei brasiliensisrostratussanguineus cloneisolate isolate isolateisolate HLSD3 Orost1 CoxAsplendi1 Obr01Rhsa1 CoxiellaCoxiellaceaeBacteriumCoxiella burnetiiendosymbiontspeciesburnetii symbiont bacterium strain H-JJ-10strain of HaemaphysalisofDugway PE-15890PE-15890AL07 AmblyommaOrnithodoros variegatumlagrangei brasiliensis clone isolate isolate HLSD3 Avar1 Obr01 CoxiellaUnculturedCoxiella endosymbiontburnetii speciesburnetii Coxiella strain H-JJ-10strain species ofPE-15890Dugway Z3055Z3055 IxodesRhipicephalus clone species CYP-1 sanguineus isolate Isp1iso1 isolate CRS-CAT CoxiellaCoxiellaceaeBacteriumUnculturedCoxiella burnetiiendosymbiontburnetii symbiont Coxiella bacterium strainstrainstrain speciesof CbuKHaemaphysalisofPE-15890 Z3055CbuKAL08 Amblyomma clone Q154Q154 CYP-1 thollonilongicornis isolate isolate CoxAthol1 66 Geographic Origin CoxiellaUnculturedBacteriumCoxiella burnetiiendosymbiont endosymbiont symbiont bacterium strainstrain of clone CbuKofHaemaphysalis Z3055of RhipicephalusAmblyomma OrnithodorosOrnithodoros CH1 Q154 loculosum longicornis rostratusrostratusturanicus isolate isolate isolateisolate Orost1Aloc1Orost1 Rhtura166 North America CoxiellaBacteriumUnculturedBacterium burnetiiendosymbiont symbiontsymbiontbacterium strain ofof clone HaemaphysalisCbuKofHaemaphysalis OrnithodorosRhipicephalus CH1 Q154 lagrangeishimoga kairouanensislagrangeirostratus turanicus clone cloneisolateclone isolate isolateHSKY-3 HLSD3HLSD3 Orost1 CRt Oka1 BacteriumCoxiellaCoxiella endosymbiont speciesspecies symbiont H-JJ-10H-JJ-10 of Haemaphysalisofof RhipicephalusHaemaphysalisOrnithodoros lagrangeishimoga rupestris rostratuspusillus obesa cloneisolate isolatecloneisolate HSKY-3 HLSD3 TPSD8 Rhpusi1Orupes1Orost1 South America CoxiellaBacteriumUnculturedUncultured endosymbiontspecies symbiont CoxiellaCoxiella H-JJ-10 speciesofspecies Haemaphysalisof IxodesHaemaphysalisOrnithodoros cloneclone hexagonus CYP-1CYP-1 lagrangei peruvianus obesa isolate isolateclone isolateIhexa1 HLSD3 TPSD8 OpC2 Europe UnculturedCoxiellaBacteriumBacterium endosymbiontspecies symbiontCoxiellasymbiont H-JJ-10 species ofof HaemaphysalisofHaemaphysalis IxodesOrnithodoros clone hexagonusricinus CYP-1 longicornisspheniscus longicornisisolate isolate Iric1 isolateisolateIhexa1 66Osphe166 Asia BacteriumCoxiellaUnculturedUncultured endosymbiont symbiont Coxiella bacteriumbacterium speciesof clone cloneHaemaphysalisof IxodesOrnithodoros CH1cloneCH1 uriaericinus CYP-1 isolatelongicornis amblusisolate Iuriae1 isolateIric1 isolate AB428 66 UnculturedCoxiellaBacteriumBacterium endosymbiont symbiontsymbiontbacterium ofof clone HaemaphysalisofHaemaphysalis IxodesOrnithodorosAmblyomma CH1 uriae isolatesplendidum shimogalongicorniscapensisshimoga Iuriae1 clone cloneisolate isolate HSKY-3HSKY-3 Oca1 CoxAsplendi166 Africa BacteriumCoxiellaUnculturedCoxiella endosymbiontendosymbiont symbiont bacterium of clone Haemaphysalis ofof HaemaphysalisAmblyommaOrnithodorosHaemaphysalis CH1 variegatumsplendidum shimogamaritimus obesaobesa clone isolateisolate isolate isolateisolate HSKY-3 TPSD8TPSD8 Omari1 Avar1CoxAsplendi1 Australia CoxiellaBacteriumCoxiella endosymbiontendosymbiont symbiont of Haemaphysalis ofof IxodesHaemaphysalisArgasAmblyommaIxodes monachus hexagonusspecieshexagonus variegatumshimoga isolateobesa isolate isolateisolate cloneisolateIsp1iso1 Amo01isolate Ihexa1Ihexa1 HSKY-3 TPSD8 Avar1 CoxiellaCoxiella endosymbiont burnetiiendosymbiont strain ofAmmassalik of IxodesHaemaphysalis AmblyommaIxodes hexagonusspecies ricinusricinus* tholloni isolate isolateobesa isolate isolate Iric1isolateIric1Isp1iso1 Ihexa1 CoxAthol1 TPSD8 CLADE A CoxiellaCoxiella endosymbiontburnetiiendosymbiont strain ofATCCof IxodesAmblyommaIxodes 3262 uriaehexagonusricinusuriae* isolateloculosumtholloniisolate isolate isolate Iuriae1 Iuriae1isolate Iric1 isolate Ihexa1 CoxAthol1 Aloc1 • CLEs of Ornithodoros Confidence CoxiellaCoxiella endosymbiontburnetii endosymbiont strain ofNMII of Ixodes OrnithodorosAmblyommaAmblyomma uriae isolate splendidumloculosumsplendidumkairouanensis Iuriae1 isolate isolateisolate isolate Aloc1 CoxAsplendi1CoxAsplendi1 Oka1 Coxiella endosymbiont of Ixodes* ricinus isolate Iric1 • Human Pathogen C. burnetii 90-100 CoxiellaCoxiella endosymbiont endosymbiont of of Ixodes AmblyommaOrnithodorosAmblyomma uriae isolate variegatumsplendidum variegatumkairouanensisbrasiliensisrupestris Iuriae1 isolate isolateisolate isolateisolate isolate Orupes1 Avar1CoxAsplendi1 Avar1Obr01 Oka1 CoxiellaCoxiella endosymbiontburnetiiendosymbiont strain ofDugwayofof IxodesAmblyommaOrnithodorosIxodes speciesspecies variegatumsplendidum rupestris isolateisolate Isp1iso1Isp1iso1isolate isolateisolate Orupes1 Avar1CoxAsplendi1 80-89 * CoxiellaCoxiella endosymbiont burnetiiendosymbiont strain of PE-15890of IxodesAmblyomma Amblyomma species* variegatum thollonitholloni isolate isolateisolate Isp1iso1 isolate CoxAthol1CoxAthol1 Avar1 70-79 CoxiellaCoxiella endosymbiont burnetiiendosymbiont strain of Z3055of Ixodes AmblyommaAmblyomma* species tholloniloculosumloculosum isolate isolate Isp1iso1 isolateisolate CoxAthol1 Aloc1Aloc1 CoxiellaCoxiella burnetii endosymbiontendosymbiont strain CbuK ofof OrnithodorosAmblyommaOrnithodoros Q154* loculosumtholloni kairouanensiskairouanensis isolate isolate isolateCoxAthol1isolate Aloc1 Oka1Oka1 Pathogen* CoxiellaCoxiella endosymbiont endosymbiont of of OrnithodorosAmblyomma Ornithodoros loculosum kairouanensis rupestrisrostratusrupestris isolateisolateisolate isolate Orupes1Orost1Aloc1Orupes1 Oka1 CoxiellaBacterium endosymbiont symbiont of Haemaphysalisof Ornithodoros lagrangeikairouanensisrupestris cloneisolate isolate HLSD3 Orupes1 Oka1 146 Coxiella endosymbiontspecies H-JJ-10 of Ornithodoros rupestris isolate Orupes1 Uncultured Coxiella species clone CYP-1 Bacterium symbiont of Haemaphysalis longicornis isolate 66 147 Figure 3. CLE clades contain both tickUncultured endosymbionts bacterium clone CH1 and pathogens. A 16S rDNA-based Bacterium symbiont of Haemaphysalis shimoga clone HSKY-3 Coxiella endosymbiont of Haemaphysalis obesa isolate TPSD8 148 phylogenetic tree is shown. Bootstrap Coxiellasupport endosymbiont and posterior of Ixodes hexagonus probabilities isolate Ihexa1 are labeled above and Coxiella endosymbiont of Ixodes ricinus isolate Iric1 Coxiella endosymbiont of Ixodes uriae isolate Iuriae1 149 below branchpoints, respectively. NodesCoxiella with endosymbiont ≤70% ofbootstrap Amblyomma splendidum support isolate were CoxAsplendi1 collapsed to Coxiella endosymbiont of Amblyomma variegatum isolate Avar1 Coxiella endosymbiont of Ixodes species isolate Isp1iso1 150 polytomies. Taxa colors represent the continentCoxiella endosymbiont from of Amblyommawhich the tholloni host isolate was CoxAthol1 derived. Established Coxiella endosymbiont of Amblyomma loculosum isolate Aloc1 Coxiella endosymbiont of Ornithodoros kairouanensis isolate Oka1 151 pathogens are marked with asterisks. CladesCoxiella endosymbiont A-D were of Ornithodoros originally rupestris defined isolate Orupes1 by Duron et al. 2015a. 152

153 HGT was a major contributor to gene accumulation in C. burnetii’s ancestors

154 In order to better understand the evolution of C. burnetii, we traced the ancestry of its protein-

155 coding genes by determining whether their orthologs — either functional or pseudogenized —

156 were present in other Legionellales members. Out of 1,530 protein-coding genes whose

157 ancestries we could trace, 790 were deemed to be ancestral, meaning it was present in the

9 158 ancestor that diverged from Legionella (Node 1), and an additional 585 genes originated in

159 Nodes 2-4 (Fig. 4; Supplemental Table S4). This data demonstrates that the common ancestor

160 of C. burnetii and CLEOA contained most of the genes, including virulence factors, present in

161 C. burnetii, and was hence well equipped to infect mammals.

162 A major impediment to unspooling the evolutionary history of C. burnetii is the sparse

163 availability of Coxiellaceae genomes, which makes it difficult to ascertain whether genes were

164 gained by C. burnetii’s ancestors at a Nodes 2-5 or were instead lost in other bacteria represented

165 at each node. To overcome this difficulty, we calculated each C. burnetii gene’s nucleotide

166 composition (%GC) and Codon Adaptation Index (CAI), two measures known to distinguish

167 foreign-origin genes from ancestral ones (Fig. 4; Supplemental Table S5; Sharp and Li 1987;

168 Lawrence and Ochman 1997; Jansen et al. 2003; Raghavan et al. 2012). Both %GC and CAI

169 values for genes that originated in Nodes 3-5 were significantly different from those of ancestral

170 (Node 1) genes, indicating that a considerable portion of these genes were acquired horizontally.

171 [Node 2 genes were excluded from this analysis due to small sample size (n=13).] Interestingly,

172 %GC and CAI values for Node 5 genes were not significantly different from those gained at

173 Node 4, suggesting that many of the genes currently found only in the human pathogen were

174 present in the common ancestor of C. burnetii and CLEOA, and were later lost in the tick

175 endosymbiont. However, it is clear that HGT has contributed to the accumulation of genes at

176 Node 5 as well because 88 out of 155 genes in this category showed phylogenetic patterns

177 consistent with HGT (Supplemental Fig. S1, Table S6). Cumulatively, our results validate the

178 important role HGT has played in assembling C. burnetii’s protein repertoire (Moses et al.

179 2017), and show that this process occurred principally in ancestors that preceded the C. burnetii-

180 CLEOA split.

10 Codon Adaptation Index %GC Codon Adaptation Index %GC A B C

0.8 0.8 N3 N2 0.5 N5 0.5

0.7 0.7 N1 CAI 0.4 CAI 0.4 %GC %GC N4 0.6 0.6 0.3 0.3 0.5 0.5 Model N1 N3 N4 N5 (n) N1 N3 N4 N5 (n) Model N1 N3 N4 N5 N1 N3 N4 N5 (790) N1 (22) Model (148) N3 **** (768) N1 ns P-Value Key **** <0.0001 **** * (148) N3 ns ** (424) N4 *** <0.001 (155) N5 **** ** ns (424) N4 **** **** **** ** <0.01 (155) N5 **** **** **** ns * <0.05 181

182 Figure 4. HGT was a major contributor to gene accumulation in C. burnetii’s ancestors. Nodes 183 N1-N5 (as labeled in Fig. 1) represent major branchpoints in the evolution of C. burnetii. (A) 184 and (B) depict %GC and Codon Adaptation Index (CAI) distributions, respectively, for genes 185 originating at each node. Boxes illustrate each distribution’s interquartile range while the black 186 line dividing the box represents the median. Whiskers represent minimum and maximum values, 187 excluding outliers (black diamonds) which were determined using the Tukey method. P-values 188 shown in tables are for pairwise T-tests (pooled SD, BKY adjusted). Genes gained at N2 were 189 excluded due to small sample size (n=13). (C) C. burnetii genome composition based on nodes of 190 gene origin: N1: 43.9%, N2: 0.7%, N3: 8.2%, N4: 22.6%, N5: 8.6%. The unlabeled portion 191 represents potentially spurious genes (n=224) as well as genes with undefined nodes of origin 192 (n=44). 193

194 CLEOA potentially provides O. amblus with nutrients missing in vertebrate blood

195 Similar to other hematophagic organisms (Duron and Gottlieb 2020), ticks likely obtain

196 nutrients missing in blood from endosymbiotic bacteria such as CLEs and FLEs (Gottlieb et al.

197 2015; Smith et al. 2015; Gerhart et al. 2016, 2018; Duron et al. 2018b; Tsementzi et al. 2018).

198 An analysis of the CLEOA genome revealed that a large number of genes have been inactivated

199 in the endosymbiont as part of its adaptation to an obligately host-dependent lifestyle (Table 1).

200 Nevertheless, the bacterium has retained complete pathways for the synthesis of several vitamins

201 and cofactors, including those for the biosynthesis of biotin (vitamin B7), folic acid (B9),

202 riboflavin (B2), FAD, CoA, and lipoic acid. These pathways are also conserved in all CLEs and

11 203 FLEs (Fig. 5), suggesting that CLEOA and other tick endosymbionts provide one or more of

204 these metabolites to their respective hosts.

205 In addition to the vitamin and cofactor biosynthesis genes, CLEOA contains 91 genes

206 that have been deactivated or lost in C. burnetii (Supplemental Table S7). It is likely that many

207 of these CLEOA-specific genes have functions in the tick ecosystem but are not useful during

208 mammalian infections. Collectively, based on its genomic features (Table 1), recent loss of

209 T4BSS (Fig. 2), and presence of intact pathways for synthesizing B vitamins and cofactors (Fig.

210 5), we conclude that a pathogenic Coxiella was recruited to function as a nutrient-provisioning

211 endosymbiont in O. amblus.

212

213

214 Figure 5. CLEs and FLEs encode B vitamin and cofactor biosynthetic pathways. CLEOA, 215 CRt, CLEAA, FLE-Om (Francisella endosymbiont of Ornithodoros moubata; LVCE00000000), 216 FLE-Am (Francisella endosymbiont of Amblyomma maculatum; LNCT00000000), and F. persica 217 (FLE in Argas arboreus; NZ_CP013022) contain intact pathways for the synthesis of B vitamins 218 and cofactors. Enzymes catalyzing each step are labeled with gene names and EC numbers. 219 Blocks with no color indicate that a functional copy of a gene was not detected in that genome. 220 Depictions of metabolic pathways modified from Gerhart et al. 2018.

12 221 Table 1. Genome characteristics of CLEOA and C. burnetii. 222 CLEOA C. burnetii RSA493 NCBI Accession VFIV00000000 AE016828.3 Length (bp) 1,561,173 1,995,488 %GC 40.6 42.7 rRNA (5S, 16S, 23S) 1,1,1 1,1,1 tRNA 42 42 Functional genes 889 1,798 Pseudogenes 660 197 Single copy genesa 106/111 106/111 223 a Albertson et al. 2013 224

225 CLEOA has not retained genes that allow C. burnetii to withstand the harsh environs of CCV

226 Loss of T4BSS and associated genes. Because C. burnetii diverged from CLEOA recently, it

227 provided us an opportunity to perform a thorough comparison of the human pathogen’s genome

228 to that of a closely-related non-pathogen. Our analysis identified 980 functional genes in C.

229 burnetii whose orthologs have been either pseudogenized or deleted in CLEOA (Supplemental

230 Table S8). Among them are genes for T4BSS and for effectors secreted through T4BSS (Fig. 2;

231 Supplemental Table S2) that enable C. burnetii to generate its intracellular replicative niche,

232 termed Coxiella-containing vacuole (CCV) (Chen et al. 2010; Beare et al. 2011; Martinez et al.

233 2014, 2020; Newton et al. 2014). In addition, genes for PmrAB and EirA that control T4BSS

234 activity, and for eight T4BSS effectors present on C. burnetii’s QpH1 plasmid have been

235 inactivated in CLEOA (Maturana et al. 2013; Beare et al. 2014; Kuba et al. 2020). Therefore,

236 the secretion system, which is critical to the intra-macrophage growth of C. burnetii, is clearly

237 not required for CLEOA to grow within tick cells.

238

13 239 Loss of transporters of antibacterial molecules. To protect itself from noxious molecules

240 produced by the host, C. burnetii likely depends on transport proteins that efflux harmful

241 substances out of its cytoplasm. For instance, macrophages increase Cu2+ concentration within

242 phagosomes to kill intracellular bacteria (Neyrolles et al. 2015), and C. burnetii probably utilizes a

243 P-1B type ATPase to export copper from its cytoplasm to sustain intracellular growth (Rowland

244 and Niederweis 2012). This ATPase, along with 18 others of unknown function, have been

245 pseudogenized or deleted in CLEOA (Supplemental Table S9). In addition, C. burnetii encodes

246 25 putative transporters that could facilitate the pathogen’s growth within CCV, out of which,

247 19 have become non-functional in CLEOA (Supplemental Table S9), suggestive of the mild

248 nature of the tick endosymbiont’s intracellular compartment where antibacterial molecules are

249 probably not a major threat.

250

251 Diminished pH regulation. A defining feature of CCV is its acidity (pH ~4.75) (Vallejo

252 Esquerra et al. 2017; Samanta et al. 2019). To compensate, C. burnetii utilizes several

253 mechanisms to maintain its cytoplasmic pH close to neutral (Hackstadt 1983), many of which

254 have been rendered non-functional in CLEOA. For example, the enzyme carbonic anhydrase

3- 255 that catalyzes the production of bicarbonate (HCO ) buffer from CO2 (Vallejo Esquerra et al.

256 2017; Bury-Moné et al. 2008) has been pseudogenized in CLEOA (Supplemental Table S10).

257 Another pH-regulating strategy used by C. burnetii is to remove excess protons in its cytoplasm

258 via proton-antiporters such as the multi-protein Mrp antiporter, a pair of Na+/H+ antiporters,

259 and a K+/H+ antiporter. C. burnetii also encodes a pair of glutamate/gamma-aminobutyrate

260 (GABA) antiporters that export GABA in exchange for glutamate, thereby reducing the

261 cytoplasmic proton content. The Mrp antiporter, one of the two Na+/H+ antiporters, and both

14 262 glutamate/GABA antiporters have been pseudogenized in CLEOA (Supplemental Table S10).

263 A hallmark of C. burnetii is its unusually high number of basic proteins (~46% of proteins have

264 pI values ³9; average pI 8.22) that could function as a “proton sink,” which allows the pathogen

265 to maintain its cytoplasmic pH close to neutral (Seshadri et al. 2003). In contrast, only ~39% of

266 proteins in CLEOA have pI values ³9 (average pI 8.0), again illustrating a lack of acidic stress

267 within CLEOA’s intracellular vacuole. Collectively, our data suggest that the endosymbiont does

268 not face the constant threat of excess protons entering its cytoplasm, probably because its

269 intracellular niche, unlike C. burnetii’s, has a pH closer to neutral.

270

271 Loss of cell membrane and cell wall genes. In gram-negative bacteria, inner and outer

272 membranes along with peptidoglycan play important roles in stress response (Rowlett et al.

273 2017). In CLEOA, the gene that encodes PlsC, which converts lysophosphatidic acid into

274 phosphatidic acid (PA), a universal intermediate in the biosynthesis of membrane phospholipids,

275 has been pseudogenized. The plsC gene is essential in Escherichia coli, and a transposon insertion

276 in this gene in C. burnetii caused severe intracellular growth defect (Coleman 1990; Martinez et

277 al. 2014); hence, it is not clear how CLEOA is able to build its membranes without a functional

278 plsC, but one possibility is that the endosymbiont utilizes PA obtained from its host. Another

279 membrane-associated loss of function in CLEOA is the pseudogenization of the pldA gene that

280 encodes phospholipase A (PldA), which is critical to C. burnetii’s outer membrane function and

281 for optimal growth within macrophages (Stead et al. 2018). As for its peptidoglycan, CLEOA

282 contains intact genes for D, D-transpeptidases (also known as penicillin-binding proteins) that

283 catalyze 4-3 peptide cross-links between D-alanine and diaminopimelate; however, all L, D-

15 284 transpeptidase genes (annotated as “enhanced entry proteins”) have been pseudogenized,

285 indicating that the tick symbiont does not have the ability to generate 3–3 cross-links between

286 diaminopimelate molecules in its peptidoglycan. These nonclassical cross-links contribute to C.

287 burnetii’s environmental stability (Sandoz et al. 2016), and are probably not critical to CLEOA

288 because the endosymbiont is passed vertically from one generation to next. Collectively, as

289 observed in other endosymbionts (Nakabachi et al. 2006; McCutcheon and Moran 2010; Chong

290 and Moran 2018), CLEOA lacks numerous proteins that are typically considered integral to the

291 optimal functioning of bacterial cell membrane and cell wall.

292

293 Loss of antioxidant genes. An intricate network of antioxidants allows C. burnetii to thrives in a

294 phagolysosome-derived intracellular vacuole (Mertens and Samuel 2012). In contrast to CCV,

295 oxidative stress appears to be minimal in CLEOA’s intracellular vacuole because the

296 endosymbiont contains only a streamlined version of C. burnetii’s antioxidant defense system. For

297 instance, C. burnetii contains two superoxide dismutases (SODs), but CLEOA has retained the

298 cytoplasmic Fe-containing SodB, but not SodC, the periplasmic Cu/Zn-SOD. OxyR, the

299 master regulator of peroxide stress, along with a catalase, a peroxidase (AhpC2), a methionine

300 sulfoxide reductase, a hemerythrin-like protein, and a glutathione transferase that together help

301 mitigate oxidative stress have also been deactivated in CLEOA (Supplemental Table S11). In

302 addition, C. burnetii, but not CLEOA, has the ability to synthesize queuine, a guanine analog,

303 found in the first anticodon position of several post-transcriptionally modified tRNAs (Iwata-

304 Reuyl 2003). The precise functions of queuine is not understood, but it is thought to promote

305 the activity of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione

16 306 transferase, most of which, as mentioned above, have lost their functionality in CLEOA (Koh

307 and Sarin 2018).

308 C. burnetii utilizes both cytochrome bd (encoded by genes cydABX) and cytochrome o

309 (encoded by genes cyoABCD) as terminal oxidases, but CLEOA has only retained cytochrome o

310 genes. Cytochrome bd, which also functions as a quinol peroxidase that prevents the buildup of

311 oxidative free radicals (Endley et al. 2001; Omsland and Heinzen 2011), has become

312 nonfunctional in the tick endosymbiont. In addition, CLEOA does not encode genes for an acid

313 phosphatase and two sterol reductases that likely modify host proteins and cholesterol,

314 respectively, to protect C. burnetii from host-induced oxidative stress (Seshadri et al. 2003; Gilk

315 et al. 2010; Hill and Samuel 2011; Gilk 2012). Finally, C. burnetii is thought to compensate for

316 the lack of the oxidative branch of Pentose Phosphate Pathway (PPP)— a major source of

317 NADPH, by utilizing alternative NADPH-regenerating enzymes such as short chain

318 dehydrogenases and sterol reductases, and by salvaging NAD+ from the host (Bitew et al. 2018,

319 2020). In CLEOA, all four short chain dehydrogenases, the two eukaryote-like sterol reductases,

320 and the nicotinate-salvaging protein have become nonfunctional. In total, while the human

321 pathogen contains several mechanisms to defend against oxidative stress, most of these

322 antioxidant systems have been lost in CLEOA, most likely due to minimal oxidative stress

323 experienced by the bacterium within tick cells. Collectively, the loss of T4BSS, transporters, pH

324 regulation, cell wall modification, and antioxidant defense in CLEOA show that its intracellular

325 vacuole is a less stressful place to live than the phagolysosome-derived CCV occupied by C.

326 burnetii.

327

328 Heme analog inhibits C. burnetii growth

17 329 Cytochromes require heme as a cofactor, but CLEOA does not contain a functional heme

330 biosynthesis pathway, which is present in C. burnetii (Supplemental Table S12). The only intact

331 heme biosynthesis gene in CLEOA is ctaB, which encodes an enzyme that converts heme b to

332 heme o, a component of cytochrome o — the sole terminal cytochrome oxidase present in

333 CLEOA (Saiki et al. 1992). Based on this evidence, the endosymbiont appears to import heme b

334 from the tick hemocoel (vertebrate hemoglobin contains heme b) and converts it to heme o using

335 the ctaB-encoded protoheme IX farnesyltransferase. Additionally, while C. burnetii can import

336 ferrous iron released from iron-containing host molecules such as ferritin and transferrin

337 (Sanchez and Omsland 2020), free Fe2+ does not seem to be important for CLEOA’s

338 intracellular growth because the iron transporter FeoB has been pseudogenized, suggesting that

339 host-derived heme b serves as the tick endosymbiont’s heme and iron source.

340 The heme biosynthesis pathway, while absent in CLEOA, is conserved in all strains of C.

341 burnetii, probably because the iron-protoporphyrin molecule is critical to the pathogen’s ability to

342 grow within human macrophages (Moses et al. 2017). We tested C. burnetii’s dependence on

343 heme by treating both axenically grown and intracellular C. burnetii with gallium protoporphyrin

344 IX (GaPPIX), which can replace heme in cytochromes and other heme-containing enzymes

345 (Hijazi et al. 2017, 2018). As shown in Figure 6, ³250 nM of GaPPIX caused significant

346 inhibition of C. burnetii growth in ACCM-2, and treatment with ³2µM of GaPPIX resulted in

347 significant growth impairment of C. burnetii within THP-1 cells. Reassuringly, only GaPPIX

348 concentrations of ³512µM caused cytotoxicity in THP-1 cells (Supplemental Fig. S2),

349 indicating that gallium compounds could potentially be used to treat C. burnetii infections.

350

18 A 1.0 1.0 * ***

0.8 *** ** **

1.0 *** *** *** ***

0.8 *** ***

0.6 *** 0.8 *** *** 0.6 *** *** ***

0.4 *** 0.4 0.6 0.2 Relative Flourescence Intensity 0.2 0.4 Relative Flourescence Intensity 0.0 0.0 0 125 250 500 1000 2000 4000 8000

Relative Flourescence Intensity 0.2 0 125 250 500GaPPIX (nM)1000 2000 4000 8000 GaPPIX (nM) 0.0 0 125 250 500 1000 2000 4000 8000 GaPPIXB (nM) 08h post-treatment 1.0

08h24h post-treatment post-treatment 1.0 ** P-Values24h48h post-treatment post-treatment08h post-treatment 1.0 ***

*** <0.0001 *** 48h72h post-treatment post-treatment24h post-treatment 0.5 ** <0.001 72h post-treatment48h post-treatment * <0.01 0.5

72h post-treatment Difference Fold 0.5

Fold Difference Fold 0.0 0 2 8 32 Fold Difference Fold 0.0 GaPPIX (uM) 0 2 8 32 0.0 351 GaPPIX (uM)0 2 8 32 GaPPIX (uM) 352 Figure 6. A heme analog reduces C. burnetii growth. (A) Bacteria growing in ACCM-2 were 353 exposed to concentrations of GaPPIX shown in x-axis and were quantified using PicoGreen at 354 8h, 24h, 48h, and 72h post-treatment. Data shown are mean fluorescence intensity (± SE) 355 compared to the vehicle control (0nM). Statistical significance was analyzed using two-way 356 repeated measures ANOVA followed by Dunnett's test (n=5). (B) At 72h post-treatment, 357 bacterial growth within THP-1 cells was quantified using qPCR. Data shown as mean fold 358 difference (± SE) compared to control. Statistical significance was analyzed using one-way 359 ANOVA followed by Dunnett's test (n=3). 360

361 DISCUSSION

362 Although symbiotic and pathogenic coxiellae associated with ticks are found across the globe, it

363 is not clear how pathogenesis and symbiosis evolved in this group of bacteria. Here we show that

364 CLEOA, a soft-tick symbiont, and C. burnetii, a human pathogen, evolved recently from a

365 common ancestor that contained genes necessary to infect macrophages. Additionally, while

366 HGT contributed significantly to the evolution of C. burnetii, it occurred in ancestors prior to

19 367 the divergence of CLEOA and C. burnetii lineages. These discoveries clarify the evolution of C.

368 burnetii, which previously was thought to have evolved from an avirulent tick endosymbiont by

369 gaining virulence factors via HGT. We further show that the pathogenic ancestry of C. burnetii

370 and CLEOA fits into a general pattern of tick endosymbionts originating from erstwhile

371 pathogens, thereby revealing the ability of ticks to convert pathogenic bacteria into beneficial

372 endosymbionts. Lastly, by comparing the genomes of C. burnetii and CLEOA, we were able to

373 gain new insights into the intracellular biology of both bacteria, and show that metabolic

374 pathways retained only in the human pathogen are promising targets for the development of new

375 treatments against Q fever.

376 Emergence of tick-symbionts from virulent ancestors. Coxiella species related to CLEs infect a

377 wide range of animals (Shivaprasad et al. 2008; Woc-Colburn et al. 2008; Angelakis et al. 2016;

378 Seo et al. 2016; Guimard et al. 2017; Elliman and Owens 2020; Needle et al. 2020), but these

379 infectious strains are not the closest relatives of C. burnetii; instead, the human pathogen’s closest

380 relative is the soft-tick symbiont CLEOA. Akin to the CLEOA-C. burnetii relationship, CRt,

381 the endosymbiont in R. turanicus, is closely related to pathogenic Coxiella (termed “Candidatus

382 Coxiella massiliensis”) isolated from human skin infections, and a strain of Coxiella isolated from

383 horse blood is closely related to CLEs present in Haemaphysalis ticks (Angelakis et al. 2016; Seo

384 et al. 2016; Guimard et al. 2017). In addition to these pathogens, bacteria related to CLEs have

385 repeatedly caused fatal bird and crayfish infections (Fig. 3; Shivaprasad et al. 2008; Woc-

386 Colburn et al. 2008; Elliman and Owens 2020; Needle et al. 2020). Microscopic and histological

387 data from avian infections demonstrated that the bacteria have the ability to generate CCV-like

388 compartments within macrophages, and both avian and human skin infection strains have

20 389 “small-cell” and “large-cell” morphologies — two distinct characteristics of C. burnetii —

390 suggesting that the bacteria are genuine vertebrate pathogens (Shivaprasad et al. 2008; Woc-

391 Colburn et al. 2008; Angelakis et al. 2016; Guimard et al. 2017; Needle et al. 2020). Further

392 research, including sequencing their genomes, is required to elucidate the biology and

393 pathogenicity of these infective strains and to understand why only one, i.e., C. burnetii, among

394 several virulent lineages have evolved into a bona fide human pathogen.

395 Tick endosymbionts are ephemeral. Phylogenies of only a few CLEs are congruent with those of

396 their hosts (Duron et al. 2017; Binetruy et al. 2020), probably because older CLEs get replaced

397 by newer CLEs derived from distantly-related coxiellae. In a similar fashion, FLEs seem to have

398 replaced older CLEs in several tick lineages (Gerhart et al. 2016, 2018; Duron et al. 2017,

399 2018b). This ephemeral nature of CLEs is surprising because hematophagic arthropods typically

400 need a reliable partner to gain nutrients that are scarce in vertebrate blood. Insects such as

401 bedbugs and body lice that face similar nutrient scarcity have evolved stable long-term

402 relationships with endosymbionts (Perotti et al. 2007; Hosokawa et al. 2010). It is not clear why

403 that is not the case in ticks, but one possibility is that ticks do not need to establish long-term

404 relationships because they frequently encounter bacteria that are predisposed to becoming

405 nutrient-provisioning endosymbionts. For instance, both Coxiella and Francisella have the

406 metabolic wherewithal to provide nutrients missing in vertebrate blood, thereby improving tick

407 fitness and promoting the establishment of symbiotic relationships. Additionally, both bacteria

408 are capable of infecting tick ovaries, which ensures their vertical transfer, and thus the

409 maintenance of symbiotic relationships. Furthermore, tick behavior such as hyperparasitism,

410 wherein an unfed tick steals blood by piercing the integument of a newly-fed tick, could promote

21 411 the horizontal spread and eventual establishment of a new endosymbiont in a population (Helmy

412 et al. 1983; Labruna et al. 2007; Llanos-Soto et al. 2019). The plentiful supply of potential

413 endosymbionts in its environment could also obviate the need for ticks to evolve specialized

414 symbiont-carrying organs (Perotti et al. 2007; Hosokawa et al. 2010, 2012; Rio et al. 2012;

415 Simonet et al. 2018; Husnik et al. 2020), thereby explaining the absence bacteriomes in ticks.

416 Alternatively, malpighian tubules, which extends into most parts of a tick’s body cavity, and to

417 which CLEs and FLEs show strong tissue tropism, could be functioning as a stand-in for

418 bacteriome (Klyachko et al. 2007; Machado-Ferreira et al. 2011; Lalzar et al. 2014; Duron et al.

419 2018b).

420 Another reason for the unstable nature of CLE-tick relationships could be that the

421 constant turnover of endosymbionts protects ticks from being dependent on an endosymbiont

422 with reduced nutrient-provisioning capability. For instance, when a free-living bacterium evolves

423 into an obligate endosymbiont, its genome undergoes massive gene loss due to a combination of

424 factors, including reduced selection to maintain superfluous genes, genetic drift and possibly

425 adaptive evolution (Moran 1996; Clayton et al. 2016). This degenerative evolution, as observed

426 in CLEOA, is a convergent feature of heritable symbionts and could result in the irreversible loss

427 of fitness-improving genes, including genes that form the primary basis for symbiosis (Lambert

428 and Moran 1998; Andersson and Andersson 1999; McCutcheon and Moran 2010). If no

429 compensatory mechanisms are undertaken, this progressive gene loss could result in the

430 extinction of both the tick and the endosymbiont. One strategy to avoid this fate is to

431 periodically replace resident endosymbionts with new bacteria that have the needed metabolic

432 capabilities (Russell et al. 2017; Bennett and Moran 2020).

433 Gaining new symbionts via horizontal transmission could also protect ticks from

22 434 becoming dependent on a degraded endosymbiont. While the mechanistic details of this process

435 are not understood, phylogenetic patterns of CLE and FLE distribution strongly indicate the

436 occurrence of horizontal transmission of bacteria between ticks (Gerhart et al. 2016, 2018;

437 Duron et al. 2017; Binetruy et al. 2020). It is not clear whether there is direct tick-to-tick

438 transfer or whether unrelated ticks acquire similar bacteria from common environmental sources

439 (Duron et al. 2018a). In either case, tick-endosymbiont relationships appear to be much more

440 short-lived and dynamic than most insect-endosymbiont relationships. It should be noted

441 however that not all tick-endosymbionts are short-lived. Ticks that carry CLEs belonging to

442 Clade D (Fig. 3) appear to have established long-term relationships with their endosymbionts.

443 For instance, CLEs in A. americanum and A. sculptum have highly reduced (~0.60 Mbp) genomes

444 that are similar in size to Buchnera, which established its symbiosis with aphids more than 200

445 million years ago (Moran et al. 1993). Putting all this information together, it appears that a

446 combination of vertical inheritance, horizontal transmission, and periodic replacement of old

447 symbionts with new pathogen-derived symbionts underlies the complex distribution pattern of

448 endosymbionts observed in ticks (Gottlieb et al. 2015; Smith et al. 2015; Gerhart et al. 2016,

449 2018; Duron et al. 2017; Tsementzi et al. 2018; Binetruy et al. 2020).

450

451 Functions of CLEs and FLEs. While the exact functions of FLEs and CLEs have not been fully

452 characterized, previous studies have shown that they are often the sole bacterium present in long-

453 term laboratory tick colonies, an indication that the bacteria are essential to ticks’ wellbeing. In

454 addition, removal of the resident endosymbiont via antibiotic treatment reduced tick fitness,

455 which was reversed when ticks were provided with B vitamins (Zhong et al. 2007; Smith et al.

456 2015; Gerhart et al. 2016; Guizzo et al. 2017; Zhang et al. 2017; Duron et al. 2018b; Li et al.

23 457 2018; Ben-Yosef et al. 2020). Our genome analyses support a B vitamin-provisioning role for

458 tick endosymbionts because the genes required to synthesize B7 (biotin), B9 (folic acid), and B2

459 (riboflavin), and three coenzymes (FAD, CoA, and lipoic acid) are conserved in all CLEs and

460 FLEs. Future experiments should clarify whether any or all of these nutrients form the basis for

461 the CLE-tick and FLE-tick symbioses.

462

463 Pathogen-specific metabolic processes are potential targets to control Q fever. Genetic and

464 physiological capabilities accumulated by a bacterium are critical to its ability to adapt to new

465 environments, especially ones such as intra-macrophage vacuoles that do not facilitate the gain of

466 new genes via HGT. In accordance with this idea, our analyses showed that virulence factors and

467 metabolic genes utilized by C. burnetii to grow within CCV were present in the common

468 ancestor of C. burnetii and CLEOA. Befitting its obligate endosymbiotic lifestyle, many of these

469 genes have become non-functional in CLEOA, allowing us to identify metabolic processes that

470 are likely critical to C. burnetii’s intracellular growth. One metabolite that is exclusively produced

471 by the pathogen is heme, the iron-protoporphyrin required for oxidative phosphorylation, among

472 other functions. To test the importance of heme to C. burnetii, we exposed the bacterium to

473 GaPPIX, a Ga(III) complex of protoporphyrin IX. Ga(III) inhibits bacterial growth because it

474 binds to biological complexes that normally binds to Fe(III), but under physiological conditions

475 Ga(III) is not reduced to Ga(II), thereby disrupting essential redox-driven biological processes

476 (Bernstein 1998). We chose GaPPIX over other gallium-based formulations because it could

477 replace heme in cytochromes, is known to be bactericidal, and is not toxic to primary human

478 fibroblasts and established cell lines (Stojiljkovic et al. 1999; Arivett et al. 2015; Hijazi et al.

479 2018). C. burnetii lacks homologs of known heme transporters (Moses et al. 2017), hence, it is

24 480 not clear how GaPPIX enters into the pathogen, but our growth assays clearly demonstrated that

481 the heme analog is very effective at inhibiting both axenic and intracellular growth of C. burnetii

482 (Fig. 6). Encouragingly, a recent human trial showed that Ga could improve lung function in

483 people with cystic fibrosis and chronic Pseudomonas aeruginosa lung infections, and that the

484 molecule worked synergistically with other antibiotics to inhibit bacterial growth (Goss et al.

485 2018). Thus, Ga, which has been approved by FDA for intravenous administration (Bonchi et al.

486 2014), and its derivatives such as GaPPIX, hold great promise as new therapeutic tools. In

487 conclusion, this study provide novel insights into the evolution and biology of pathogenesis and

488 symbiosis in Coxiella, and demonstrate that the new information could be applied to developing

489 innovative therapeutic strategies.

490

491 METHODS

492 Genome sequencing and assembly. An O. amblus female, collected from soil underneath rocks

493 near a Spheniscus humbolti (Humboldt penguin) nesting area in Isla Grande de Atacama, Chile,

494 was identified as described in Clifford et al. 1980. DNA was extracted from the tick using

495 DNeasy Blood & Tissue kit (Qiagen) and was submitted to Yale Center for Genome Analysis

496 for Illumina (NovaSeq) sequencing. The resulting 150 bp paired-end reads were trimmed using

497 Trimmomatic resulting in approximately 220 million read pairs of suitable quality (Bolger et al.

498 2014). The reads were assembled into contigs using metaSPAdes (Nurk et al. 2017), and open

499 reading frames (ORFs) were identified using Prodigal (Hyatt et al. 2010). Contigs containing

500 Coxiella genes were tentatively identified using BLASTn and BLASTp by comparing to a

501 database of all publicly available sequences from Coxiellacea members. CONCOCT (Alneberg

25 502 et al. 2014) was used for binning contigs based on coverage and k-mer composition, and these

503 finding were merged with BLAST-based binning results. Approximately 20 million paired reads

504 that mapped to contigs identified as containing Coxiella genes were used for a final metaSPAdes

505 assembly resulting in a total of 101 contigs. The final collection of contigs was verified using

506 hmmsearch (Potter et al. 2018) to identify essential single-copy genes (Albertsen et al. 2013), as

507 well as RNammer (Lagesen et al. 2007) and tRNAscan-SE (Chan and Lowe 2019) to identify

508 ribosomal and transfer RNAs, respectively. We were unable to stitch the contigs together into a

509 closed chromosome because the contigs were filled with repeat sequences; however, the presence

510 of 106 out of 111 highly conserved single-copy genes in both CLEOA and C. burnetii indicate

511 that most of the CLEOA genome is represented in the assembled contigs. The final set of 101

512 contigs were submitted to NCBI (accession VFIV00000000) and annotated using the

513 Prokaryotic Genome Pipeline.

514

515 Phylogenetic analysis. Orthofinder (Emms and Kelly 2015) was utilized to identify 205 single-

516 copy genes present in 52 representative species from the order Legionellales (Supplemental

517 Tables S1, S13) in order to build the comprehensive phylogenomic tree Supplemental Figure S3.

518 A subset of 117 genes conserved in 30 species (Supplemental Tables S1, S13) were used to

519 generate Figure 1. For both trees, nucleotide sequences were aligned individually using global

520 MAFFT (Katoh and Standley 2013) and were then concatenated. GBlocks (Talavera and

521 Castresana 2007) was used to cull ambiguously aligned regions and jModelTest2 (Darriba et al.

522 2012) was used to select the appropriate model (GTR+I+G). Maximum Likelihood trees were

523 generated using RaxML and Bayesian trees were produced using MrBayes (Ronquist et al. 2012;

524 Stamatakis 2014). The 16s rDNA trees were built using the same process as above, with the final

26 525 tree based on 1203 nucleotide positions, and nodes with less than 70% support collapsed. To

526 confirm the HGT origin of Node 5 genes, homologs were identified via BLASTp (NCBI nr

527 database, e-value ≤10e-5, identity ≥30% identity, coverage ≥70%). The nucleotide sequences of

528 the homologs were collected into a database, and the Phylomizer pipeline

529 (https://github.com/Gabaldonlab/phylomizer) was used to generate individual Maximum

530 Likelihood trees using the 75 most closely related homolog sequences. Each tree was then

531 compared to an NCBI Taxonomy-based tree to validate HGT (Supplemental Fig. S1).

532

533 Determination of nodes of gene origin. The presence of functional homologs of C. burnetii

534 RSA493 (AE016828.3) genes in other members of the order Legionellales was determined using

535 BLASTp (identity ≥30%, coverage ≥70%, e-value ≤10e-5), and pseudogenized homologs were

536 detected using tBLASTn (identity ≥30%, coverage ≥50%, e-value ≤10e-5). The presence/absence

537 profile was utilized in Gain and Loss Mapping Engine (GLOOME) (Cohen et al. 2010) to

538 determine the posterior probability of each gene’s presence at nodes N1-N5. For each C. burnetii

539 gene, the node of origin was marked as the oldest node at which posterior probability was ≥0.7,

540 with all subsequent nodes also having posterior probability of ≥0.7, as described previously (Peer

541 and Margalit 2014).

542

543 Calculation of CAI and pI. We identified 22 highly conserved single-copy protein-coding genes

544 in C. burnetii that were highly expressed in both ACCM-2 and within human macrophages

545 based on previous RNA-seq data (Supplemental Table S5; Warrier et al. 2014; Wachter et al.

546 2019). CodonW (http://codonw.sourceforge.net) was used to generate CAI values for the 22

547 genes in order to generate a model for optimal codon usage in C. burnetii, which was then

27 548 compared to CAI values of sets of genes acquired at each node. All 22 genes used to build the

549 model belonged to Node 1, and were not included in this analysis. Potentially spurious genes

550 (n=224) that did not have any detectable homologs outside of C. burnetii, as well as genes with

551 undetermined nodes of origin (n=44) were excluded from this analysis (Supplemental Table

552 S14). Isoelectric points (pI) for all proteins in CLEOA and C. burnetii RSA493 (AE016828.3)

553 were calculated using IPC (Kozlowski 2016).

554

555 GaPPIX susceptibility assay

556 A 10mM GaPPIX (Frontier Scientific) solution was prepared in dimethyl sulfoxide (DMSO)

557 and was stored at 4°C under dark conditions until further use. C. burnetii was cultured in

7 558 ACCM-2 for 2 days at 37°C, 5% CO2 and 2.5% O2, and ~2x10 genome equivalents were

559 resuspended in fresh ACCM-2 containing 125nM, 250nM, 500nM, 1mM, 2mM, 4mM, or

560 8mM GaPPIX in 96-well black-bottom microplates (Greiner Bio-One). Bacterial growth was

561 measured using PicoGreen (Invitrogen) as described previously (Moses et al. 2017).

562 THP-1 human monocytes (ATCC, TIB-202) were cultured in sterile RPMI-1640

563 medium (Gibco) supplemented with 1mM sodium pyruvate, 0.05 mM beta-mercaptoethanol,

564 1% Pen-Strep, and 4500 mg/L glucose with 10% heat-inactivated fetal bovine serum (FBS) at

565 37°C, 5% CO2 in 6-well tissue culture plates. Prior to infection, cells were differentiated into

566 macrophages by treating with 30 nM phorbol 12-myristate 13-acetate (PMA) for 24h, followed

567 by resting in PMA-free RPMI for 24h. Infection of THP-1 cells with C. burnetii was carried out

568 using a 7d bacterial culture at a multiplicity of infection of 25. After briefly washing the cells

569 with PBS, bacteria-containing medium was added to each well and gently centrifuged for 10

570 minutes followed by incubation at 37°C, 5% CO2 for two hours. To remove extracellular

28 571 bacteria, cells were washed three times with PBS, and replaced with antibiotic-free RPMI and

572 were incubated for 48h before treating with GaPPIX- (2uM, 8uM, and 32uM) or DMSO- (as

573 control) containing media. After 72h, cells were washed three times with PBS and intracellular

574 bacterial load was measure using qPCR, as we described previously (Moses et al. 2017). Potential

575 cytotoxicity of GaPPIX was determined by measuring the levels of released lactate

576 dehydrogenase (LDH) in cell supernatants using an LDH Cytotoxicity Assay Kit (Invitrogen).

577

578 DATA ACCESS

579 The CLEOA genome generated in this study has been submitted to the NCBI Assembly

580 database (https://www. ncbi.nlm.nih.gov/assembly/) under Whole Genome Shotgun (WGS)

581 accession prefix VFIV, BioSample SAMN12040594, and BioProject PRJNA548565.

582

583 ACKNOWLEDGEMENTS

584 We thank Samantha Fancher, Andrew Ashford and Jim Archuleta for technical help, and Daniel

585 González-Acuña for assisting in tick collection. This project was supported in part by NIH

586 grants AI123464, AI126385, and AI133023 to R.R.

587

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895

39 896 SUPPLEMENTAL FIGURE LEGENDS

897 Supplemental Figure S1. HGT contributed to C. burnetii evolution. 88 Node 5 genes have

898 phylogenies (left) inconsistent with established taxonomic relationships (right), indicating that

899 they were acquired via HGT. Branches and species titles are colored by taxonomy as specified on

900 Page 1. (A) Maximum likelihood gene trees with bootstrap values labeled at branchpoints. C.

901 burnetii RSA493 genes are indicated by their locus tags and are highlighted in blue. Duplicate

902 genes are shown together in single trees. (B) Taxonomy-based trees depict the established

903 relationships among the taxa featured in A. Taxa sharing the same NCBI taxonomic identifier

904 number are displayed only once even if multiple representatives are present in A.

905

906 Supplemental Figure S2. GaPPIX is not toxic to macrophages at levels that significantly inhibit

907 C. burnetii growth. At 24h post-GaPPIX treatment of THP-1 derived macrophages, lactate

908 dehydrogenase (LDH) activity was determined by measuring the level of resorufin formation

909 using an LDH cytotoxicity assay. The cytotoxicity was reported as the percentage LDH released

910 compared to the maximum LDH activity. Data are shown as mean ± SEM percentage LDH

911 released, and statistical significance was analyzed using one-way ANOVA followed by Dunnett's

912 test (n=3).

913

914 Supplemental Figure S3. Legionellales phylogenomic tree. Maximum likelihood and Bayesian

915 trees were generated using 205 single-copy protein-coding genes conserved in all represented

916 taxa (Supplemental Tables S1, S13). Bootstrap support and posterior probabilities are labeled

917 above and below each node respectively. This tree was used in GLOOME (Cohen et al. 2010) to

918 determine the nodes of gene origin.

40