key: cord-0020624-41673wlu
authors: Fullerton, Marissa S.; Colonne, Punsiri M.; Dragan, Amanda L.; Brann, Katelynn R.; Kurten, Richard C.; Voth, Daniel E.
title: Neurotransmitter System-Targeting Drugs Antagonize Growth of the Q Fever Agent, Coxiella burnetii, in Human Cells
date: 2021-07-07
journal: nan
DOI: 10.1128/msphere.00442-21
sha: 41339298f5a37ed8b394561d114fb42d8aebb6af
doc_id: 20624
cord_uid: 41673wlu

Coxiella burnetii is a highly infectious, intracellular, Gram-negative bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis. C. burnetii is transmitted to humans via aerosols and has long been considered a potential biological warfare agent. Although antibiotics, such as doxycycline, effectively treat acute Q fever, a recently identified antibiotic-resistant strain demonstrates the ability of C. burnetii to resist traditional antimicrobials, and chronic disease is extremely difficult to treat with current options. These findings highlight the need for new Q fever therapeutics, and repurposed drugs that target eukaryotic functions to prevent bacterial replication are of increasing interest in infectious disease. To identify this class of anti-C. burnetii therapeutics, we screened a library of 727 FDA-approved or late-stage clinical trial compounds using a human macrophage-like cell model of infection. Eighty-eight compounds inhibited bacterial replication, including known antibiotics, antipsychotic or antidepressant treatments, antihistamines, and several additional compounds used to treat a variety of conditions. The majority of identified anti-C. burnetii compounds target host neurotransmitter system components. Serotoninergic, dopaminergic, and adrenergic components are among the most highly represented targets and potentially regulate macrophage activation, cytokine production, and autophagy. Overall, our screen identified multiple host-directed compounds that can be pursued for potential use as anti-C. burnetii drugs. IMPORTANCECoxiella burnetii causes the debilitating disease Q fever in humans. This infection is difficult to treat with current antibiotics and can progress to long-term, potentially fatal infection in immunocompromised individuals or when treatment is delayed. Here, we identified many new potential treatment options in the form of drugs that are either FDA approved or have been used in late-stage clinical trials and target human neurotransmitter systems. These compounds are poised for future characterization as nontraditional anti-C. burnetii therapies.

progression (1) (2) (3) (4) (5) . The PV fuses with numerous host compartments, including autophagosomes and lysosomes, resulting in an acidic, degradative vacuole that is not conducive to antibiotic activity (6) (7) (8) (9) . C. burnetii manipulates the host cell using a type IV secretion system (T4SS) and replicates within the PV throughout a lengthy infectious cycle (5, (10) (11) (12) (13) . Currently, doxycycline effectively treats acute Q fever, which presents with flu-like symptoms and pneumonia (14, 15) . However, chronic disease requires up to 1.5 years of combination therapy using doxycycline and hydroxychloroquine that often does not completely eradicate infectious bacteria (14) . In addition, a doxycyclineresistant isolate of C. burnetii was recently reported (16) , stressing the need for alternatives to this long-established treatment.

Host-directed compounds that prevent intracellular pathogen growth have been investigated as alternatives to traditional antibiotics (17) (18) (19) (20) (21) , and a recent study identified 75 compounds that antagonize C. burnetii intracellular growth and typical PV expansion (22) . These compounds are termed host-directed antimicrobial drugs (HDADs) because they do not directly target C. burnetii, like traditional antibiotics, but impact host processes required for intracellular growth. This study also compared the efficacy of 640 compounds against a panel of intracellular pathogens, including Brucella abortus, Rickettsia conorii, and Legionella pneumophila. The authors demonstrated multiple pathogen-specific and pan-pathogen activities of individual compounds, indicating broad applicability of HDADs in treatment of infectious diseases. In addition to drug screens, individual chemical inhibitors have been used in a similar manner to study the importance of distinct host signaling pathways during C. burnetii infection. Previous studies used chemical inhibitors to demonstrate the importance of mammalian signaling cascades, including PKA, PKC, eIF-2a, Akt, and Erk1/2, for C. burnetii replication, PV expansion, or prevention of host cell apoptosis (23) (24) (25) (26) (27) . Together, drug screens and individual chemical inhibitor studies have demonstrated the utility of HDADs in preventing C. burnetii infection events required for host cell parasitism.

In this study, we screened an NIH clinical collection (NCC) of 727 FDA-approved or late-stage clinical trial compounds for their ability to inhibit C. burnetii growth in host cells. This screen identified 88 compounds that significantly inhibited C. burnetii growth within human THP-1 macrophage-like cells. Members of the largest group of identified inhibitory compounds target diverse components of neurotransmitter systems and have been largely used to treat psychosis and mood-related disorders. Overall, we identified a new class of anti-C. burnetii drugs that can be pursued in future studies to improve Q fever therapy.

A subset of compounds from the NCC library antagonizes C. burnetii growth in human cells. To identify new anti-C. burnetii compounds, we used the established THP-1 macrophage-like cellular infection model that is widely accepted in the C. burnetii field as an effective mimic of primary human macrophages (2, 3, 7, 9, (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) . As shown in Fig. 1A , our initial screen consisted of differentiated THP-1 cells treated with individual drugs 2 h prior to infection with avirulent C. burnetii expressing red fluorescent mCherry (NMII-mCherry) to ensure compound effects existed at initiation of infection. At 72 h postinfection (hpi), cells were analyzed by bright-field microscopy to assess cytopathic effects (cell rounding) and fluorescence microscopy to observe intracellular accumulation of NMII-mCherry, indicative of bacterial growth. As shown in Fig. 1B and Table 1 , 88 compounds prevented normal C. burnetii growth within THP-1 cells, similar to chloramphenicol treatment (34) . Importantly, 52 of the 88 compounds identified have not been previously reported as anti-C. burnetii agents, indicating the discovery of novel anti-C. burnetii compounds in our screen. In contrast, 37 compounds enhanced C. burnetii replication (Fig. 1B and Table 2 ); however, these drugs were not pursued further in this study. The 88 inhibitory compounds were separated based on known clinical use, as shown in Fig. 1C . Unsurprisingly, 25 inhibitory compounds were common antibiotics, including levofloxacin, azithromycin, and doxycycline (the best current Q fever treatment). Seven compounds have been used as antihistamines, and the largest group with anti-C. burnetii activity included 30 antipsychotic and antidepressant drugs used to treat psychosis and mood-related disorders, respectively.

Antipsychotic and antidepressant drugs often target distinct neurotransmitter machinery components. Eighteen identified inhibitory compounds outside the antipsychotic and Chloramphenicol, an antibiotic that prevents C. burnetii intracellular growth, was included as a control. Eighty-eight compounds inhibit typical C. burnetii growth (paroxetine is shown as an example). Thirty-seven compounds enhance C. burnetii growth (enalapril maleate is shown as an example). (C) Reported clinical use of the identified 88 inhibitory compounds. (D) Forty-eight inhibitory compounds target neurotransmitter system machinery. These compounds are divided into known target components and accompanying mechanism of action. Trimethoprim is typically used in a 1:5 mixture with sulfamethoxazole, known as cotrimoxazole. Reports cited reflect this use. antidepressant group also target neurotransmitter machinery. These results make neurotransmitter systems the target of over half of the inhibitory compounds (48 of 88) identified in this screen. Figure 1D separates these compounds by known target and mechanism of action. The largest subset of this group antagonizes neurotransmitter receptors, and members of the smallest subset act as neurotransmitter agonists. This category of compounds has not been previously explored for anti-C. burnetii properties and represents a novel class of potential HDADs that antagonize C. burnetii replication.

Neurotransmitter system-targeting compounds prevent typical C. burnetii intracellular growth. To further investigate neurotransmitter system-targeting compounds as HDADs against C. burnetii, we assessed a representative sampling of these drugs (28 of the 48 compounds identified in Fig. 1 ). Most of these compounds are antipsychotic or antidepressant drugs and represent the largest HDAD group, targeting diverse neurotransmitter system components. In growth inhibitor screens, it is critical to differentiate compounds that specifically inhibit bacterial growth within host cells from those that are directly toxic to bacteria using a traditional antibiotic mode of action. To distinguish between these two scenarios, we compared C. burnetii treated with individual compounds during infection of THP-1 cells to treated bacteria growing in axenic media. It is also important to differentiate between compounds that prevent bacterial entry into host cells and those that prevent intracellular growth following uptake, which mimics treatment of a previously infected patient. Thus, all compounds were added at 24 hpi to ensure that growth defects resulted from inhibiting intracellular growth independent of host cell uptake.

Of the 28 inhibitory neurotransmitter system-targeting compounds identified, only aripiprazole and nefazodone were substantially cytotoxic to THP-1 cells (Table S1) , with no direct antibacterial effects on C. burnetii (Table 3) . Lofepramine was the only compound that reduced C. burnetii growth in axenic media by almost 50% compared to vehicle control-treated cultures at 7 days postinoculation, suggesting the drug acts similar to a traditional antibiotic on C. burnetii at the concentration tested. Atomoxetine was the only compound that demonstrated no detectable inhibitory effect on C. burnetii intracellular growth (Table 4 ) or in axenic media. These data, combined with the observation that pretreatment with atomoxetine inhibits C. burnetii growth (Fig. 1A and Table 1 ), suggests the drug prevents bacterial entry into host cells. Amoxapine and perospirone treatment resulted in less than 30% reduction in intracellular growth compared to vehicle control-treated cells at 5 days postinfection (dpi). The former had no direct antibacterial effect, while the latter reduced C. burnetii growth by less than 20% compared to vehicle control-treated axenic cultures. This result suggests inhibitory effects observed in our original microscopy screen (Fig. 1A ) are due to reduced bacterial entry. Of the remaining 22 identified compounds, 18 reduced intracellular growth by more than 45% and 4 reduced intracellular growth by more than 30%. While 11 of these 22 compounds had statistically significant antibacterial effects, none reduced C. burnetii growth in axenic media more than 30% at 7 days postinoculation. Collectively, we identified only one potential direct antibacterial compound and 22 neurotransmitter machinery-targeting HDADs that prevent typical intracellular C. burnetii growth.

Multiple neurotransmitter systems and components are targeted by the identified HDADs. Figure 2A shows fluorescence-based 5-day NMII-mCherry growth curves in THP-1 cells treated with representative compounds that target diverse components of monoamine neurotransmitter systems, including the serotonin (also known as 5-hydroxytryptamine or 5-HT), dopamine, or norepinephrine systems. Perphenazine and thioridazine are antipsychotic compounds that antagonize dopamine receptors or 5-HT and dopamine receptors, respectively. Nortriptyline, paroxetine, and bifemelane are antidepressant compounds. Nortriptyline and paroxetine target monoamine transporters, inhibiting reuptake of extracellular norepinephrine and/or 5-HT into host cells. Nortriptyline also antagonizes 5-HT 2 receptors. Bifemelane inhibits monoamine oxidases involved in monoamine metabolism. In contrast, cisapride is a 5-HT agonist that has been used to treat gastrointestinal ailments. All 6 example compounds inhibited typical C. burnetii intracellular replication from 3 to 5 dpi. Importantly, inhibitory effects were not due to THP-1 cell cytotoxicity (Fig. 2B ) or direct bactericidal effects (Table 3) . Overall, our results suggest diverse components of monoamine neurotransmitter systems can be targeted to inhibit C. burnetii intracellular growth.

HDADs prevent expansion of the C. burnetii replication vacuole. To replicate within eukaryotic cells, C. burnetii must form a phagolysosome-like PV to activate metabolism and allow bacterial cell division as the vacuole expands (35) (36) (37) . To determine if HDADs that target monoamine neurotransmitter system components disrupt PV expansion, we measured PV area in infected THP-1 cells treated with perphenazine, thioridazine, nortriptyline, paroxetine, bifemelane, or cisparide using fluorescence microscopy. As shown in Fig. 3 , each compound prevented typical PV expansion when added to cells at 24 hpi, indicating PVs were unable to undergo heterotypic fusion and appropriately expand. These results suggest that host 5-HT, dopamine, or norepinephrine system activity positively impacts C. burnetii PV expansion needed for bacterial replication to high numbers.

HDADs antagonize virulent C. burnetii replication in primary human alveolar macrophages. THP-1 cells have been used in numerous studies as a reliable in vitro model of C. burnetii interactions with human macrophages (7, 27, 28). However, cell line results should be confirmed in primary cells when possible to ensure disease a Student's t test was used to compare percent growth of C. burnetii in compound-treated cultures to DMSOtreated cultures. SD, standard deviation from the mean. *, P , 0.05; **, P , 0.01; ****, P , 0.0001. relevance, particularly when assessing new HDADs. We previously established primary human alveolar macrophages (hAMs) as a disease-relevant system to determine if in vitro findings extend to cells preferentially targeted by C. burnetii in the human lung (3, 29, 38) . Here, nortriptyline was used as a representative monoamine neurotransmitter system-targeting, anti-C. burnetii HDAD. hAMs were infected with avirulent NMII-mCherry and treated with nortriptyline at 24 hpi. Reduced NMII-mCherry within hAMs (Fig. 4A) confirmed that the inhibitory properties of this compound, which correlates with findings from our THP-1 cell line model, are reproducible in a disease-relevant context. Next, we treated hAMs infected with virulent NMI C. burnetii (acute disease isolate) with nortriptyline at 24 hpi and monitored PV expansion. Nortriptyline efficiently prevented PV expansion and accumulation of large numbers of virulent C. burnetii in hAMs (Fig. 4B) . Together, these results indicate targeting host monoamine neurotransmitter system machinery is a therapeutic strategy relevant in a natural disease cellular setting involving virulent C. burnetii.

In this study, we identified 88 compounds with anti-C. burnetii activity and validated 22 of these drugs as potential HDADs to treat Q fever. New therapies are desperately needed to combat C. burnetii infection, particularly for chronic disease, the most lifethreatening form of Q fever. The current regimen for treating Q fever endocarditis is up to 1.5 years of doxycycline treatment combined with a pH-elevating compound (14) . This time course is inefficient, and treatment does not always clear infectious bacteria. As an alternative to traditional antibiotics, multiple studies have repurposed a Student's t test was used to compare percent growth of C. burnetii in compound-treated infections to DMSOtreated infections. SD, standard deviation from the mean. **, P , 0.01; ***, P , 0.001; ****, P , 0.0001. host-directed compounds that target eukaryotic proteins usurped by intracellular pathogens (17) (18) (19) 22) . A key feature of these HDADs is the ability to suppress bacterial replication or degrade bacteria while maintaining host cell viability despite altering host processes. Therapeutics of this nature hold great promise in the current era of antibiotic resistance. HDADs are problematic for disease-causing bacteria because they do not target the pathogen directly, making development of classical genetics-based resistance unlikely and ultimately improving treatment. Based on our extensive drug screen, 22 identified compounds specifically inhibit C. burnetii intracellular growth in a host-directed manner. Our in vitro experimental approach mimics a natural scenario in which a patient receives treatment after infection and diagnosis. The majority of these compounds have been used extensively to treat a variety of psychoses or mood disorders, including schizophrenia and depression, respectively. These drugs target distinct components of neurotransmitter systems, most commonly the serotoninergic, dopaminergic, and adrenergic systems comprised of components that respond to 5-HT, dopamine, or epinephrine and norepinephrine. Prior to this study, Czy_ z et al. assessed the antibacterial activity of 640 compounds against L. pneumophila, B. abortus, R. conorii, and C. burnetii, identifying 75 HDADs that prevent typical C. burnetii growth (22) . Many of these compounds target G protein-coupled receptors (GPCRs), intracellular calcium signaling, or sterol homeostasis machinery. In line with these findings, we found many compounds that significantly impede C. burnetii intracellular growth and target neurotransmitter receptors that are part of the GPCR family. Although the parameters between studies were not identical, Czy_ z et al. reported 4 of the 22 HDADs identified in our study, confirming the effectiveness of our approach in replicating previously confirmed HDADs. Of these compounds, 2 also antagonized growth of L. pneumophila, suggesting broader applicability in infectious disease treatment. Compounds assessed by Czy_ z et al. and many of the compounds in the current study have already been approved by the FDA to treat disorders unrelated to infection, suggesting they can be safely administered to humans, but they have not been used to treat Q fever.

Neurotransmitter system-targeting drugs also alter the life cycle of other intracellular pathogens beyond those reported by Czy_ z et al. and our current study. For example, Mycobacterium tuberculosis is sensitive to thioridazine and nortriptyline, two compounds with anti-C. burnetii activity in our study, further supporting the potential for broad-spectrum use (39) (40) (41) . In addition, pimozide, a dopamine receptor inhibitor used to treat schizophrenia and Tourette's syndrome, reduces entry of Listeria monocytogenes into host phagocytes and host entry and intracellular replication of the eukaryotic parasite Toxoplasma gondii (17, 18) . Interestingly, the effect of pimozide on T. gondii replication is independent of neurotransmitter receptor signaling. As demonstrated for T. gondii, it is important to note the potential for off-target effects when conducting drug studies. Therefore, while neurotransmitter system-targeting compounds represent a major class of potential therapeutics to treat intracellular pathogen infections, future studies should investigate whether anti-C. burnetii activity is due to traditional or alternative activities of these compounds. At the whole-host level, neurotransmitter systems are not typically considered in mechanistic studies of respiratory infections. However, numerous reports demonstrate a role for 5-HT, dopamine, and norepinephrine in macrophage function that could impact alveolar physiology. For example, 5-HT impacts alveolar macrophage production of tumor necrosis factor alpha (TNF-a) and interleukin-10 (IL-10) (42) , which are involved in proinflammatory and anti-inflammatory responses, respectively. In addition, norepinephrine signaling through b 2 -adrenergic receptors may drive macrophage IL-10 production (43). C. burnetii triggers a robust human macrophage inflammatory response characterized by production of TNF-a, IL-6, and IL-8, and the pathogen stimulates anti-inflammatory IL-10 production (3, 29) . Thus, HDAD alteration of the innate immune response may promote C. burnetii clearance and less severe acute disease. In line with this prediction, dopamine receptor activity modulates production of IL-6 and TNF-a (44), and 5-HT signaling impacts production of the monocyte chemoattractant CCL2 (45) . Other bacterial pathogens are similarly susceptible to HDADs that alter the inflammatory response. For example, resolvin and clavanin modulate immune responses to Escherichia coli and Staphylococcus aureus, indicating that dampening the host inflammatory response prevents robust bacterial growth, effectively inhibiting disease progression (46) (47) (48) . Moreover, neurotransmitter receptor expression and signaling impacts macrophage polarization that defines the inflammatory state of the cell (43, 49) . C. burnetii promotes alveolar macrophage transition from M1 to M2 polarization to provide a more hospitable growth niche (3), and 5-HT, dopamine, or norepinephrine signaling may contribute to this event. Thus, future studies should determine if neurotransmitter signaling controls cytokine/chemokine production by, and polarization of, C. burnetii-infected macrophages.

At the cellular level, some neurotransmitter system-targeting compounds that inhibit PV expansion impact host autophagy. Autophagy is a homeostatic process that recycles damaged cytosolic components, regulates inflammation, and clears invading bacteria by delivery to degradative lysosomes (50, 51) . However, many intracellular pathogens modulate this process for their own benefit, including C. burnetii. PV expansion involves T4SS-dependent recruitment of, and fusion with, autophagosomes (1, 7, 52, 53) . Multiple antipsychotic and antidepressant drugs, including thioridazine, nortriptyline, and paroxetine, can induce autophagy by increasing processing of autophagy protein microtubule-associated light chain 3 (LC3) or modulating mammalian target of rapamycin (mTOR), a kinase component of the autophagy regulator mTOR complex 1 (mTORC1) (39, 50, 51) . During infection, LC3 is recruited to the PV, and Coxiella vacuolar protein F (CvpF) promotes LC3 processing to its lipidated form (LC3-II), demonstrating that autophagy is manipulated by the pathogen (1, 7, 52, 53) . C. burnetii also inhibits mTORC1 in a T4SS-dependent manner to promote PV expansion and bacterial replication (2) . As with any homeostatic process, activation and deactivation of autophagy is a delicate balance that can favor the pathogen or host depending on their respective needs. Thus, although C. burnetii actively recruits autophagosomes, HDADs may overactivate autophagy, or reroute autophagic machinery, to negate proper PV expansion. Together, these findings provide a basis for future mechanistic studies of HDAD prevention of C. burnetii intracellular growth.

Overall, our drug screen results present multiple new options for future anti-Q fever therapeutic investigation. These options are needed in light of the nonspecific flu-like nature of acute disease and the potential for chronic infection leading to life-threatening endocarditis. The immunomodulatory role of peripheral neurotransmitters on macrophages suggests the traditional activity of neurotransmitter system-targeting compounds should not be dismissed when investigating anti-C. burnetii activity. These potential therapeutics now await testing in animal models to assess utility as anti-Q fever treatments. This testing will ultimately provide novel therapies that suppress disease progression following C. burnetii infection and limit pathogen development of resistance due to host-directed activity. Moreover, due to the wide-ranging effect of specific antipsychotic or antidepressant drugs on multiple intracellular pathogens, these therapies may serve broad-spectrum purposes in infectious disease treatment. For example, a study by Cao et al. used the compound library in the current study to identify drugs that prevent mouse hepatitis virus infection (54) . Although addressing potential side effects of neurotransmitter system-targeting drugs would be critical prior to human administration, these HDADs hold immense promise as antimicrobial agents that potentially can be used to combat multiple, disparate infections.

Bacterial and eukaryotic cell culture. Avirulent (Nine Mile Phase II [NMII]; RSA 439) Coxiella burnetii expressing fluorescent mCherry (NMII-mCherry) was cultured in acidified citrate cysteine medium 1 (ACCM-1) containing chloramphenicol (3 mg/ml) at 378C, 5% CO 2 , and 2.5% O 2 . After 7 days, bacterial cultures were pelleted by centrifugation and washed with 250 mM sucrose phosphate (SP) buffer. Bacterial stocks were stored in SP buffer at 2808C. Wild-type NMII or virulent C. burnetii (Nine Mile I [NMI]; RSA 493) isolates were cultured, harvested, and stored as described above without antibiotic. A multiplicity of infection of 10 to 30 was used for each experiment. Experiments using virulent C. burnetii were conducted in the UAMS biosafety level-3 laboratory approved by the Centers for Disease Control and Prevention.

THP-1 cells (TIB-202; American Type Culture Collection) were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Bio-techne) at 378C and 5% CO 2 . Before infection, THP-1 cells were differentiated into macrophage-like cells by incubating with medium containing phorbol 12-myristate 13acetate (PMA; 200 nM; Calbiochem) overnight. PMA-containing medium was removed prior to infection.

Primary human alveolar macrophages (hAMs) were isolated from human lungs postmortem (National Disease Research Interchange) by bronchoalveolar lavage (BAL) as previously described (29) . BAL fluid was centrifuged and 0.86% ammonium chloride added to lyse red blood cells. Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12; Gibco) containing 10% FBS, 1% antibiotic-antimycotic (10,000 U/ml penicillin, 10,000mg/ml streptomycin, and 25mg/ml amphotericin B; Gibco), and gentamicin (10mg/ml; Gibco) was used to neutralize the lysis reaction. Cells were allowed to adhere to tissue culture dishes for 1.5 to 2 h at 378C and 5% CO 2 . Culture medium was then replaced with fresh medium to remove nonadherent cells. Medium was replaced every other day for 1 week, and at least 24 h prior to infection, medium was replaced with antibiotic-antimycotic-free media.

Small-molecule screen. THP-1 cells cultured on glass coverslips in 24-well plates were treated with individual compounds (10 mM) or dimethyl sulfoxide (DMSO) 2 h prior to infection with NMII-mCherry. Compounds were obtained from the NIH Clinical Compound Library (NIH Clinical Collection 1 and 2). At 24 h postinfection (hpi), medium was replaced with fresh media containing fresh compounds. Cells were fixed with ice-cold methanol and blocked with PBS containing 0.5% bovine serum albumin (BSA; Cell Signaling) at 72 hpi. Coverslips were mounted onto slides with MOWIOL (Sigma-Aldrich). Bright-field microscopy was used to visualize infected cells, and mCherry expression allowed visualization of C. burnetii (Nikon Ti-U microscope).

Cytotoxicity assay. THP-1 cells cultured in 96-well clear, flat-bottom plates were infected with NMII-mCherry, and the inoculum was removed and replaced with fresh medium containing DMSO or individual drugs (10mM) at 24 hpi. Medium in cell death control wells was replaced with medium containing DMSO (10%) 24 h prior to the endpoint. A Cell Counting Kit-8 (Dojindo Laboratories) was used according to the manufacturer's instructions at 5 days postinfection (dpi) to detect viable cells and calculate percent survival.

Intracellular bacterial growth assay. THP-1 cells were cultured in RPMI 1640 phenol red-free medium (Gibco) supplemented with 10% FBS in 96-well glass, flat-bottom black plates. Cells were infected with NMII-mCherry and then treated with medium containing DMSO or individual drugs (10 mM) following removal of the inoculum at 24 hpi. mCherry fluorescence was measured for 5 days, starting at day 0, using a Biotek Synergy H1 microplate reader (excitation at 585 nm and emission at 620 nm [Ex 585 / Em 620 ]). Bacterial growth was calculated using the formula percent growth = [(sample 2 average uninfected)/(average DMSO 2 average uninfected)] Â 100.

Axenic bacterial growth assay. NMII-mCherry was grown in 96-well glass, flat-bottom, black plates in ACCM-1 treated with DMSO or individual drugs (10 mM). Starting at day 0, mCherry fluorescence was measured for 7 days with a Biotek Synergy H1 microplate reader (Ex 585 /Em 620 ). Cultures were mixed every other day. Bacterial growth was calculated using the formula percent growth = [(sample 2 average ACCM-1)/ (average DMSO 2 average ACCM-1)] Â 100.

Immunofluorescence microscopy. THP-1 cells or primary hAMs, plated on glass coverslips in a 24well plate, were infected with NMII-mCherry, wild-type NMII, or virulent NMI C. burnetii. The inoculum was removed and replaced with fresh medium containing DMSO or individual drugs (10 mM) at 24 hpi. At 96 hpi, cells were washed with cold PBS three times and fixed with PBS containing 4% formaldehyde for 15 min or ice cold methanol for 3 to 5 min. Cells were washed with cold PBS and blocked overnight at 4°C in PBS containing 0.5% BSA (methanol-fixed) or this solution supplemented with 0.3% Triton X-100 (formaldehyde-fixed cells). Cells were then incubated with the appropriate block solution containing primary antibodies for 1 h with rocking at room temperature (RT). Cells were then washed in cold PBS and placed in the appropriate block solution containing secondary antibodies for 1 h with rocking at RT. Cells were washed with cold PBS and then incubated with 49,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 5 min. Coverslips were mounted onto slides with MOWIOL. Images were acquired using a 40Â objective or under oil immersion using a 60Â objective with either a Nikon Ti-U Eclipse One microscope or Nikon Ti2 Eclipse microscope. A D5-QilMc digital camera was used to obtain images shown in Fig. 3 (perphenazine, thioridazine, and nortriptyline) and Fig. 4A , and a DS-Qi2 digital camera was used to acquire images shown in Fig. 3 (paroxetine, bifemelane, and cisapride) and Fig. 4B . NIS elements software (Nikon) was used to measure the areas (NMII-infected THP-1 cells) or diameter (NMI-infected hAMs) of 50 PV, which were then averaged. Primary antibodies were used to detect CD63 (BD Biosciences) or C. burnetii. Secondary antibodies used were mouse antibody conjugated to Alexa Fluor 488 and rabbit or guinea pig antibodies conjugated to Alexa Fluor 594.

Statistical analysis. All statistical analyses were performed using Student's t test and Prism software (GraphPad 8 or 9). A P value of ,0.05 was considered significant in all experiments.

Supplemental material is available online only. TABLE S1, DOCX file, 0.01 MB.

Coxiella burnetii: international pathogen of mystery

Noncanonical inhibition of mTORC1 by Coxiella burnetii promotes replication within a phagolysosome-like vacuole

Characterization of early stages of human alveolar infection by the Q fever agent Coxiella burnetii

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth

Dot/ Icm-translocated proteins important for biogenesis of the Coxiella burnetii-containing vacuole identified by screening of an effector mutant sublibrary

Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii type IV secretion-dependent recruitment of macrophage autophagosomes

The early secretory pathway contributes to the growth of the Coxiella-replicative niche

Coxiella burnetii phase I and II variants replicate with similar kinetics in degradative phagolysosome-like compartments of human macrophages

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions

Coxiella burnetii employs the Dot/Icm type IV secretion system to modulate host NF-kappaB/RelA activation

Coxiella burnetii effector proteins that localize to the parasitophorous vacuole membrane promote intracellular replication

Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole

Antimicrobial therapies for Q fever

Diagnosis and management of Q fever-United States, 2013: recommendations from CDC and the Q Fever Working Group

Genome sequence of Coxiella burnetii 109, a doxycycline-resistant clinical isolate

A small-molecule screen identifies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection

Drug repurposing screening identifies novel compounds that effectively inhibit Toxoplasma gondii growth

A dual-targeting approach to inhibit Brucella abortus replication in human cells

Mitigating the impact of antibacterial drug resistance through host-directed therapies: current progress, outlook, and challenges

Combating intracellular pathogens with repurposed host-targeted drugs

Host-directed antimicrobial drugs with broad-spectrum efficacy against intracellular bacterial pathogens

Coxiella burnetii exploits host cAMP-dependent protein kinase signalling to promote macrophage survival

Coxiella burnetii alters cyclic AMPdependent protein kinase signaling during growth in macrophages

Host kinase activity is required for Coxiella burnetii parasitophorous vacuole formation

Sustained activation of Akt and Erk1/2 is required for Coxiella burnetii antiapoptotic activity

Coxiella burnetii requires host eukaryotic initiation factor 2a activity for efficient intracellular replication

Coxiella burnetii subverts p62/sequestosome 1 and activates Nrf2 signaling in human macrophages

Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages

Coxiella burnetii inhibits apoptosis in human THP-1 cells and monkey primary alveolar macrophages

Coxiella burnetii RpoS regulates genes involved in morphological differentiation and intracellular growth

A Coxiella burnetii phospholipase A homolog PldA is required for optimal growth in macrophages and developmental form lipid remodeling

Coxiella burnetii Nine Mile II proteins modulate gene expression of monocytic host cells during infection

Maturation of the Coxiella burnetii parasitophorous vacuole requires bacterial protein synthesis but not replication

Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii

Coxiella burnetii exhibits morphological change and delays phagolysosomal fusion after internalization by J774A.1 cells

Host cell-free growth of the Q fever bacterium Coxiella burnetii

Development of an ex vivo tissue platform to study the human lung response to Coxiella burnetii

Integration of chemical and RNAi multiparametric profiles identifies triggers of intracellular mycobacterial killing

Thioridazine alters the cell envelope permeability of Mycobacterium tuberculosis

Clinical concentrations of thioridazine kill intracellular multidrug-resistant Mycobacterium tuberculosis

Serotonin modulates the cytokine network in the lung: involvement of prostaglandin E2

Adrenergic regulation of immune cell function and inflammation

Dopamine uses the DRD5-ARRB2-PP2A signaling axis to block the TRAF6-mediated NF-kappaB pathway and suppress systemic inflammation

Serotonin activates murine alveolar macrophages through 5-HT2C receptors

Resolvin D4 stereoassignment and its novel actions in host protection and bacterial clearance

Identification of resolvin D2 receptor mediating resolution of infections and organ protection

Clavanin A improves outcome of complications from different bacterial infections

Serotonin skews human macrophage polarization through HTR2B and HTR7

Is there a role of autophagy in depression and antidepressant action? Front Psychiatry

Mechanisms and therapeutic significance of autophagy modulation by antipsychotic drugs

The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell

Autophagy induction favours the generation and maturation of the Coxiella-replicative vacuoles

A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs

Treatment of chronic fatigue syndrome with antibiotics: pilot study assessing the involvement of Coxiella burnetii infection

Use of axenic media to determine antibiotic efficacy against Coxiella burnetii

Bacteriostatic and bactericidal activity of levofloxacin against Rickettsia rickettsii, Rickettsia conorii

Fulminant hepatic failure and acute renal failure as manifestations of concurrent Q fever and cytomegalovirus infection: a case report

Q fever community-acquired pneumonia in a patient with Crohn's disease on immunosuppressive therapy

Diffuse abdominal uptake mimicking peritonitis in gallium inflammatory scan: an unusual feature of acute Q fever

The effect of pH on antibiotic efficacy against Coxiella burnetii in axenic media

In vitro susceptibility of Coxiella burnetii to trovafloxacin in comparison with susceptibilities to pefloxacin, ciprofloxacin, ofloxacin, doxycycline, and clarithromycin

In vitro susceptibility of Coxiella burnetii to linezolid in comparison with its susceptibilities to quinolones, doxycycline, and clarithromycin

Q fever during pregnancy: an emerging cause of prematurity and abortion

Acute myocardial failure in a young man: Q fever myocarditis

In vitro susceptibilities of spotted fever group rickettsiae and Coxiella burnetii to clarithromycin

Bacteriostatic and bactericidal activities of tigecycline against Coxiella burnetii and comparison with those of six other antibiotics

Q fever pneumonia: are clarithromycin and moxifloxacin alternative treatments only?

The in vitro antirickettsial activity of macrolides

Unusual manifestations of acute Q fever: autoimmune hemolytic anemia and tubulointerstitial nephritis

Bacteriostatic and bactericidal activities of moxifloxacin against Coxiella burnetii

Cure of Q fever pneumonia with moxifloxacin: case report

Susceptibility of Coxiella burnetii to pefloxacin and ofloxacin in ovo and in persistently infected L929 cells

Antibiotic susceptibilities of two Coxiella burnetii isolates implicated in distinct clinical syndromes

Bactericidal effect of doxycycline associated with lysosomotropic agents on Coxiella burnetii in P388D1 cells

Antibiotic treatment of rickettsiosis, recent advances and current concepts

Acute Q fever pneumonia: a review of 80 hospitalized patients

Doxycycline desensitization in chronic Q fever-a critical tool for the clinician

Acute and probable chronic Q fever during anti-TNFa and anti B-cell immunotherapy: a case report

Abdominal aortic aneurysm and Coxiella burnetii infection: report of three cases and review of the literature

Imported brucellosis and Q fever coinfection in Croatia: a case report

Q fever (Coxiella burnetii) causing an infected thoracoabdominal aortic aneurysm

An unusual manifestation of Q Fever: peritonitis

Q fever endocarditis on porcine bioprosthetic valves. Clinicopathologic features and microbiologic findings in three patients treated with doxycycline, cotrimoxazole, and valve replacement

Q fever in the Southern California desert: epidemiology, clinical presentation and treatment

Q fever cases in the Northern Territory of Australia from 1991 to

Treatment of Q fever endocarditis: comparison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine

Azithromycin for acute Q fever in pregnancy

Azithromycin: single 1.5 g dose in the treatment of patients with atypical pneumonia syndrome-a randomized study

Epidemiological survey on the route of Coxiella burnetii infection in an animal hospital

Acute cerebellitis caused by Coxiella burnetii

Q fever with clinical features resembling systemic lupus erythematosus

Antibiotic susceptibility of rickettsia and treatment of rickettsioses

Unexpected antibiotic susceptibility of a chronic isolate of Coxiella burnetii

Evaluation of Coxiella burnetii antibiotic susceptibilities by real-time PCR assay

Q fever during pregnancy: a cause of poor fetal and maternal outcome

In vitro susceptibility of Coxiella burnetii to antibiotics, including several quinolones

Contemporary unconventional clinical use of co-trimoxazole

Managing Q fever during pregnancy: the benefits of long-term cotrimoxazole therapy

Coxiella burnetii inhabits a cholesterolrich vacuole and influences cellular cholesterol metabolism

This research was supported by funding to D.E.V. from the Arkansas Biosciences Institute and the Center for Microbial Pathogenesis and Host Inflammatory Responses (NIH/NIGMS P20GM103625).