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C. burnetii protein and ATP synthesis are permissive to suboptimal iron availability. The requirement for iron to initiate and/or sustain C. burnetii replication and metabolism was assessed by performing downshift (D.S.) experiments where at 24, 48, and 72 h, C. burnetii cultures grown under optimal iron conditions were subcultured into ACCM-2 FeSO4 , and final yields were measured by absorbance after 8 days of incubation (a), and by quantifying levels of protein synthesis in C. burnetii cultures containing suboptimal concentrations of iron or supplemented with Bpdl (b). The negative control for protein synthesis (Control -) was ACCM-2, pH 7.0. To correlate energy requirements of replication and protein synthesis with iron availability, bacterial ATP pools were measured following incubation with suboptimal levels of iron or supplementation of Bpdl to the medium (c). All conditions were compared to ACCM-2 (Control ). Bars represent the average from 3 to 5 independent experiments. Error bars indicate SEM. *, P 0.05; ***, P 0.0001 (one-way ANOVA with Dunnett's posttest applied only to panels a and b; unpaired Student's t test used for starting versus final OD in panel a).
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Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viab...
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... 5 sustain replication, resulting in significantly lower final yields (Fig. 4a). In comparison, cultures that were downshifted to ACCM-2 FeSO4 at 48 h were able to initiate but not sustain replication (Fig. 4a). Cultures downshifted at 72 h, however, were capable of overcoming the iron limitation during the last 5 days of culture and reached final yields that were significantly different from starting turbidities ...
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... 5 sustain replication, resulting in significantly lower final yields (Fig. 4a). In comparison, cultures that were downshifted to ACCM-2 FeSO4 at 48 h were able to initiate but not sustain replication (Fig. 4a). Cultures downshifted at 72 h, however, were capable of overcoming the iron limitation during the last 5 days of culture and reached final yields that were significantly different from starting turbidities (i.e., day 3) and comparable to that of the positive control on day 8 (Fig. 4a). Thus, iron is required to initiate and sustain C. ...
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... at 48 h were able to initiate but not sustain replication (Fig. 4a). Cultures downshifted at 72 h, however, were capable of overcoming the iron limitation during the last 5 days of culture and reached final yields that were significantly different from starting turbidities (i.e., day 3) and comparable to that of the positive control on day 8 (Fig. 4a). Thus, iron is required to initiate and sustain C. burnetii replication in this axenic model. ) experiments where at 24, 48, and 72 h, C. burnetii cultures grown under optimal iron conditions were subcultured into ACCM-2 FeSO4 , and final yields were measured by absorbance after 8 days of incubation (a), and by quantifying levels of ...
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... FeSO 4 ), or ACCM-2 supplemented with 100 M iron chelator bipyridyl (Bpdl) for 24 h and then transferred to a labeling medium, and the amount of incorporated [ 35 S]Cys-Met was measured after 3 h. Bacteria incubated under positive control conditions had high relative incorporation of [ 35 S]Cys-Met and vice versa under negative control conditions (Fig. 4b). In contrast to what was observed for analysis of C. burnetii replication, suboptimal levels of FeSO 4 (i.e., both 0 and 5 M FeSO 4 ) did not correlate with a reduction in [ 35 S]Cys-Met incorporation (Fig. 4b). Nevertheless, incorporation of [ 35 S]Cys-Met was reduced in the presence of 100 M Bpdl to levels similar to the negative ...
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... under positive control conditions had high relative incorporation of [ 35 S]Cys-Met and vice versa under negative control conditions (Fig. 4b). In contrast to what was observed for analysis of C. burnetii replication, suboptimal levels of FeSO 4 (i.e., both 0 and 5 M FeSO 4 ) did not correlate with a reduction in [ 35 S]Cys-Met incorporation (Fig. 4b). Nevertheless, incorporation of [ 35 S]Cys-Met was reduced in the presence of 100 M Bpdl to levels similar to the negative control; however, this trend was not statistically significant (P 0.0694) (Fig. 4b). Overall, these data show that sustained C. burnetii protein synthesis requires less iron than sustained axenic replication, ...
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... replication, suboptimal levels of FeSO 4 (i.e., both 0 and 5 M FeSO 4 ) did not correlate with a reduction in [ 35 S]Cys-Met incorporation (Fig. 4b). Nevertheless, incorporation of [ 35 S]Cys-Met was reduced in the presence of 100 M Bpdl to levels similar to the negative control; however, this trend was not statistically significant (P 0.0694) (Fig. 4b). Overall, these data show that sustained C. burnetii protein synthesis requires less iron than sustained axenic replication, suggesting a role for iron in triggering pathogen ...
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... containing 5 M FeSO 4 , or ACCM-2 containing 100 M Bpdl. Conditions were maintained for 48 h before the relative bacterial ATP pools were quantified. Similar to data obtained for protein synthesis, there was no significant difference between control cultures and bacteria incubated with suboptimal levels of iron (i.e., both 0 and 5 M FeSO 4 ) (Fig. 4c). While there was less relative ATP in bacteria incubated with 100 M Bpdl compared to the positive control, the difference was not significant. Therefore, suboptimal ATP pools do not explain the discrepancy observed between C. burnetii replication and iron ...
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... genome suggests the bacterium harbors few genes related to iron acquisition, especially for complexed or bound forms of iron. Therefore, we assessed the ability of C. burnetii to utilize sequestered and bound forms of iron during axenic replication. We confirmed that C. burnetii is unable to replicate axenically when iron is sequestered with Bpdl (Fig. S4a), a phenomenon that can be reversed with supplementation of FeSO 4 at 25 M (Fig. S4b). These data are consistent with C. burnetii dependence on free iron. Moreover, in APCM FeSO4 , inhibition of C. burnetii replication occurred with Bpdl when supplemented at a 10-fold-lower concentration than in ACCM-2 cultures (Fig. S4c). Therefore, ...
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... for complexed or bound forms of iron. Therefore, we assessed the ability of C. burnetii to utilize sequestered and bound forms of iron during axenic replication. We confirmed that C. burnetii is unable to replicate axenically when iron is sequestered with Bpdl (Fig. S4a), a phenomenon that can be reversed with supplementation of FeSO 4 at 25 M (Fig. S4b). These data are consistent with C. burnetii dependence on free iron. Moreover, in APCM FeSO4 , inhibition of C. burnetii replication occurred with Bpdl when supplemented at a 10-fold-lower concentration than in ACCM-2 cultures (Fig. S4c). Therefore, even in APCM iron must be unsequestered to permit C. burnetii replication. To compare ...
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... is sequestered with Bpdl (Fig. S4a), a phenomenon that can be reversed with supplementation of FeSO 4 at 25 M (Fig. S4b). These data are consistent with C. burnetii dependence on free iron. Moreover, in APCM FeSO4 , inhibition of C. burnetii replication occurred with Bpdl when supplemented at a 10-fold-lower concentration than in ACCM-2 cultures (Fig. S4c). Therefore, even in APCM iron must be unsequestered to permit C. burnetii replication. To compare the effects of iron sequestration via Bpdl on C. burnetii replication to that of a different bacterium, E. coli was cultured in ACCM-2 FeSO4 -conditions permissible to growth of this bacteriumsupplemented with Bpdl. Obtained data indicate ...
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... To compare the effects of iron sequestration via Bpdl on C. burnetii replication to that of a different bacterium, E. coli was cultured in ACCM-2 FeSO4 -conditions permissible to growth of this bacteriumsupplemented with Bpdl. Obtained data indicate that E. coli has a greater capacity to replicate when iron is both limited and sequestered (Fig. S4c), compared to C. ...
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... phosphorylation, a process greatly reduced by chemical inhibition in C. burnetii (34), is another key process dependent on iron as a cofactor. While we show that ATP pools were not significantly reduced following a 48-h period under suboptimal iron conditions, there was an overall trend for reduced ATP when iron was sequestered using Bpdl (Fig. 4c). These observations were similar to that for overall protein synthesis with no significant difference observed under conditions of optimal versus suboptimal iron availability or iron sequestration (Fig. 4b). Therefore, it appears that C. burnetii ATP and protein synthesis are permissive to iron conditions that are insufficient for ...
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... following a 48-h period under suboptimal iron conditions, there was an overall trend for reduced ATP when iron was sequestered using Bpdl (Fig. 4c). These observations were similar to that for overall protein synthesis with no significant difference observed under conditions of optimal versus suboptimal iron availability or iron sequestration (Fig. 4b). Therefore, it appears that C. burnetii ATP and protein synthesis are permissive to iron conditions that are insufficient for replication of C. burnetii. Nevertheless, the addition of the iron chelator Bpdl resulted in a reduction in overall protein and ATP levels, Sanchez and Omsland July/August 2020 Volume 5 Issue 4 ...
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... iron uptake systems would be crucial for C. burnetii biological success and could explain the current absence of a C. burnetii feoAB mutant within current transposon libraries (58-60). Using both citrate-based (ACCM-2) and phosphate-based (APCM) axenic media, we determined that, indeed, C. burnetii is limited in its ability to acquire sequestered (Fig. S4) and citrate-bound (Fig. 2) forms of iron. These data are consistent with the inability of C. burnetii to acquire such iron species via-for example-a TonB-like protein, which would be required for active uptake of siderophores (61, 62) and ferric citrate complexes (63,64). When we evaluated the oxidation state of iron in ACCM-2 and ...
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Coxiella burnetii is a zoonotic bacterial obligate intracellular parasite and the cause of Query (Q) fever. During natural infection of female animals, C. burnetii shows tropism for the placenta and is associated with late-term abortion, at which time pathogen titer in placental tissue can exceed one billion bacteria per gram tissue. During later s...
Citations
... Thus, iron limitation triggers egress of the evolutionarily related intracellular pathogen Legionella pneumophila (O'Connor et al., 2016). Whether iron limitation also triggers C. burnetii egress has to be determined, as conflicting roles for iron in C. burnetii pathogenesis have been reported (Briggs et al., 2008;Howe & Mallavia, 1999;Sanchez & Omsland, 2020). Since C. burnetii is auxotroph for 11 amino acids (Sandoz et al., 2016), amino acid limitation might also be a possible trigger of egress. ...
Intracellular bacteria have evolved mechanisms to invade host cells, establish an intracellular niche that allows survival and replication, produce progeny, and exit the host cell after completion of the replication cycle to infect new target cells. Bacteria exit their host cell by (i) initiation of apoptosis, (ii) lytic cell death, and (iii) exocytosis. While bacterial egress is essential for bacterial spreading and, thus, pathogenesis, we currently lack information about egress mechanisms for the obligate intracellular pathogen C. burnetii, the causative agent of the zoonosis Q fever. Here, we demonstrate that C. burnetii inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Only at later stages of infection did we observe C. burnetii egress, which depends on previously established large bacteria‐filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. In summary, our data argue that C. burnetii egress in a non‐synchronous way at late stages of infection. Apoptosis‐induction is important for C. burnetii egress, but other pathways most likely contribute.
... Further, there is relatively little known about the molecular mechanisms of C. burnetii persistence. Diverse stimuli can induce C. burnetii persistence such as treatment with IFN-g or TNF-a cytokines, the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bisethanamine (DETA/NONOate) (Howe et al., 2002), and iron deprivation (Sanchez and Omsland, 2020). In murine L-929 cells infected by C. burnetii, IFN-g and TNF-a treatment induces increased expression of the nitric oxide synthase iNOS, suggesting a contribution of this enzyme to the Coxiella persistence state (Howe et al., 2002). ...
... The development of citrate cysteine ACCM-1 and ACCM-2 axenic media allowing C. burnetii growth outside of a host cell has facilitated this type of study in C. burnetii (Sandoz et al., 2014). In ACCM-2 medium supplemented with a range of iron sulfate FeSO 4 (from 1 to 250 mM), results indicate that C. burnetii tolerates molecular iron over a broad concentration range and undergoes loss of viability upon iron deprivation (Sanchez and Omsland, 2020). Moreover, chelation of host iron pools by different concentrations of Bpdl treatment for 3 days inhibited C. burnetii replication during infection of Vero cells. ...
... Moreover, chelation of host iron pools by different concentrations of Bpdl treatment for 3 days inhibited C. burnetii replication during infection of Vero cells. Other experiments are necessary to determine if iron-replete medium rescues Coxiella replication in the host cell, which would be consistent with persistence (Sanchez and Omsland, 2020). ...
In adapting to the intracellular niche, obligate intracellular bacteria usually undergo a reduction of genome size by eliminating genes not needed for intracellular survival. These losses can include, for example, genes involved in nutrient anabolic pathways or in stress response. Living inside a host cell offers a stable environment where intracellular bacteria can limit their exposure to extracellular effectors of the immune system and modulate or outright inhibit intracellular defense mechanisms. However, highlighting an area of vulnerability, these pathogens are dependent on the host cell for nutrients and are very sensitive to conditions that limit nutrient availability. Persistence is a common response shared by evolutionarily divergent bacteria to survive adverse conditions like nutrient deprivation. Development of persistence usually compromises successful antibiotic therapy of bacterial infections and is associated with chronic infections and long-term sequelae for the patients. During persistence, obligate intracellular pathogens are viable but not growing inside their host cell. They can survive for a long period of time such that, when the inducing stress is removed, reactivation of their growth cycles resumes. Given their reduced coding capacity, intracellular bacteria have adapted different response mechanisms. This review gives an overview of the strategies used by the obligate intracellular bacteria, where known, which, unlike model organisms such as E. coli, often lack toxin-antitoxin systems and the stringent response that have been linked to a persister phenotype and amino acid starvation states, respectively.
... For intracellular pathogens of eukaryotic cells, acquiring iron is a balancing act, as the acquisition must not damage or kill the host cells until pathogen proliferation reaches a critical level. Well-known strategies for this are as follows: 1) siderophores, which are high-affinity iron-chelating compounds that capture and transport iron across cell membranes; siderophores are utilized by many facultative intracellular bacteria, for example, Mycobacterium, Brucella, Legionella, Salmonella, Yersinia, Burkholderia, Listeria, and Shigella (56-61); 2) hemin binding/uptake (Legionella, Yersinia, Burkholderia) (62, 63); 3) iron released from Tf in endosomes is taken up (Chlamydia) (64,65); and 4) iron released from host iron-binding proteins in the acidic degradative Coxiella-containing compartment (Coxiella) (66). The present study reveals an unprecedented mechanism of iron acquisition, namely, ferritinophagy, which evolved in Ehrlichia. ...
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Ehrlichia chaffeensis infects and proliferates inside monocytes and macrophages by acquiring iron from the cellular labile iron pool and causes a potentially fatal disease called human monocytic ehrlichiosis. This report reveals a unique bacterial iron hijacking mechanism. Ehrlichia deploys a protein Etf-3 via the bacteria type IV secretion system. Etf-3 binds ferritin and induces ferritinophagy, thereby increasing the cellular labile iron pool for Ehrlichia to acquire iron for intracellular proliferation. This, in concert with coregulation of bacterial and host cell superoxide dismutases with type IV secretion system, enables Ehrlichia to prevent reactive oxygen species–induced host cell damage mediated by labile iron. This finding unifies current concepts of intracellular bacterial infection, ferritinophagy, and ROS, which may be exploited to inhibit Ehrlichia infection.
... As bacterial cells require iron for basic cellular processes such as respiration, defects in cellular iron uptake and utilization would decrease bacterial viability (25). In this study, the chicken-derived macrophages were infected with the WT and its derivative mutants strains. ...
Avian pathogenic Escherichia coli (APEC), widely spread among poultry, is well-known to cause colibacillosis in chickens, which results in significant losses in poultry industry. The ability to uptake iron in the extra-intestinal environment is prerequisite for APEC survival. For adaptation to the low-iron environments, the bacteria have evolved multiple iron acquisition systems to ensure optimal iron uptake. However, many components of these iron acquisition pathways are still not clearly known. An in silico analysis of the genome of a septicemic APEC O1 strain E516 identified two putative iron transport genes homologous to the c2515 and c2516 genes from uropathogenic E. coli CFT073. In this study, we constructed the single and double gene deletion mutants, and studied their biological characteristic and pathogenic traits through in vitro and in vivo assays. Reverse transcriptase PCR (RT-PCR) analyses demonstrated that the mutations destroying the reading frame of the target genes abolished their transcription. Deletion of the single or double genes of c2515 and c2516 in APEC E516 weakened its ability to produce siderophore. Consistently, the mutants exhibited growth defect under iron-depleted conditions and the intracellular iron levels in the mutants were decreased in comparison with that of the wild-type (WT). Cell infection assays showed that the iron uptake defective mutants were more easily eliminated by the macrophage. Inactivation of the c2515 and c2516 genes affected bacterial colonization of chicken tissues, as well as the 50% lethal dose levels compared with the WT strain. Moreover, the expression levels of several iron uptake-related genes were significantly decreased in the double-deletion mutant. In total, the c2515 and c2516 may involve in siderophore-mediated iron uptake and participate in the pathogenesis of APEC O1 strain E516.
... Based on this evidence, the endosymbiont appears to import heme b 333 from the tick hemocoel (vertebrate hemoglobin contains heme b) and converts it to heme o using 334 the ctaB-encoded protoheme IX farnesyltransferase. Additionally, while C. burnetii can import 335 ferrous iron released from iron-containing host molecules such as ferritin and transferrin 336(Sanchez and Omsland 2020), free Fe 2+ does not seem to be important for CLEOA's 337 intracellular growth because the iron transporter FeoB has been pseudogenized, suggesting that 338 host-derived heme b serves as the tick endosymbiont's heme and iron source. 339The heme biosynthesis pathway, while absent in CLEOA, is conserved in all strains of C. 340 burnetii, probably because the iron-protoporphyrin molecule is critical to the pathogen's ability to 341 grow within human macrophages(Moses et al. 2017). ...
Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the reproductive success of ticks, a major disease vector, while Coxiella burnetii is the etiological agent of human Q fever, and uncharacterized coxiellae cause infections in both animals and humans. To better understand the evolution of pathogenesis and symbiosis in this group of intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales. Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen, than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts and pathogens, indicating that several CLE lineages have evolved independently from pathogenic Coxiella. We also determined that the last common ancestor of CLEOA and C. burnetii was equipped to infect macrophages, and that even though HGT contributed significantly to the evolution of C. burnetii, most acquisition events occurred primarily in ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution of C. burnetii, which previously was assumed to have emerged when an avirulent tick endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the human pathogen but not the tick symbiont, and show that the use of heme analogs is a promising approach to controlling C. burnetii infections.
Bacterial obligate intracellular parasites (BOIPs) represent an exclusive group of bacterial pathogens that all depend on invasion of a eukaryotic host cell to reproduce. BOIPs are characterized by extensive adaptation to their respective replication niches, regardless of whether they replicate within the host cell cytoplasm or within specialized replication vacuoles. Genome reduction is also a hallmark of BOIPs that likely reflects streamlining of metabolic processes to reduce the need for de novo biosynthesis of energetically costly metabolic intermediates. Despite shared characteristics in lifestyle, BOIPs show considerable diversity in nutrient requirements, metabolic capabilities, and general physiology. In this review, we compare metabolic and physiological processes of prominent pathogenic BOIPs with special emphasis on carbon, energy, and amino acid metabolism. Recent advances are discussed in the context of historical views and opportunities for discovery.
The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.
Coxiella burnetii is a bacterial obligate intracellular parasite and the etiological agent of Query (Q) fever. While the C. burnetii genome has been reduced to approximately 2 Mb as a likely consequence of genome streamlining in response to parasitism, components for a nearly complete central metabolic machinery are encoded by the genome. However, lack of a canonical hexokinase for phosphorylation of glucose and an apparent absence of the oxidative branch of the pentose phosphate pathway, a major mechanism for regeneration of the reducing equivalent NADPH, have been noted as potential metabolic limitations of C. burnetii. By complementing C. burnetii with the zwf gene encoding the glucose-6-phosphate (G6P)-consuming and NADPH-producing enzyme glucose 6-phosphate dehydrogenase (G6PD), we demonstrate a severe metabolic fitness defect for C. burnetii under conditions of glucose limitation. Supplementation of the medium with the gluconeogenic carbon source glutamate did not rescue the growth defect of bacteria complemented with zwf. Absence of G6PD in C. burnetii therefore likely relates to the negative effect of its activity under conditions of glucose limitation. C. burnetii central metabolism with emphasis on glucose, NAD+, NADP+ and NADPH is discussed in a broader perspective, including comparisons to other bacterial obligate intracellular parasites.
Both symbiotic and pathogenic bacteria in the family Coxiellaceae cause morbidity and mortality in humans and animals. For instance, Coxiella-like endosymbionts (CLEs) improve the reproductive success of ticks - a major disease vector, while Coxiella burnetii causes human Q fever, and uncharacterized coxiellae infect both animals and humans. To better understand the evolution of pathogenesis and symbiosis in this group of intracellular bacteria, we sequenced the genome of a CLE present in the soft tick Ornithodoros amblus (CLEOA) and compared it to the genomes of other bacteria in the order Legionellales. Our analyses confirmed that CLEOA is more closely related to C. burnetii, the human pathogen, than to CLEs in hard ticks, and showed that most clades of CLEs contain both endosymbionts and pathogens, indicating that several CLE lineages have evolved independently from pathogenic Coxiella. We also determined that the last common ancestor of CLEOA and C. burnetii was equipped to infect macrophages, and that even though horizontal gene transfer (HGT) contributed significantly to the evolution of C. burnetii, most acquisition events occurred primarily in ancestors predating the CLEOA-C. burnetii divergence. These discoveries clarify the evolution of C. burnetii, which previously was assumed to have emerged when an avirulent tick endosymbiont recently gained virulence factors via HGT. Finally, we identified several metabolic pathways, including heme biosynthesis, that are likely critical to the intracellular growth of the human pathogen but not the tick symbiont, and show that the use of heme analog is a promising approach to controlling C. burnetii infections.
