Neurotransmitter System-Targeting Drugs Antagonize Growth of the Q Fever Agent, Coxiella burnetii, in Human Cells.

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. IMPORTANCE Coxiella 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.

Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens.

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking-Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM-/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol.

Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

Hijacking Host Cell Highways: Manipulation of the Host Actin Cytoskeleton by Obligate Intracellular Bacterial Pathogens.

Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.

Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived ß1,3-glucans contribute to the EPS matrix structure, while fungal mannan and ß-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

Suppressor of cytokine signalling protein SOCS1 and UBP43 regulate the expression of type I interferon-stimulated genes in human microvascular endothelial cells infected with Rickettsia conorii.

Rickettsia conorii, the causative agent of Mediterranean spotted fever, preferentially infects human microvascular endothelium and activates pro-inflammatory innate immune responses as evidenced by enhanced expression and secretion of cytokines and chemokines. Our recent studies reveal that human microvascular endothelial cells (HMECs) infected with R. conorii also launch 'antiviral' host defence mechanisms typically governed by type I interferons. To summarize, infected HMECs secrete IFN-ß to activate STAT1 in an autocrine/paracrine manner and display increased expression of IFN-stimulated genes, for example ISG15, which in turn activate innate responses to interfere with intracellular replication of rickettsiae. We now present evidence that UBP43 and SOCS1, known negative regulators of JAK/STAT signalling, are also induced in R. conorii-infected HMECs, of which UBP43 but not SOCS1 functions to negatively regulate STAT1 activation. Interestingly, UBP43 induction is almost completely abolished in the presence of IFN-ß-neutralizing antibody, implicating an important role for UBP43 as a feedback inhibitor for IFN-ß-mediated STAT1 activation. In contrast, SOCS1 expression is only partially affected by IFN-ß neutralization, implicating potential involvement of as-yet-unidentified IFN-independent mechanism(s) in SOCS1 induction during R. conorii infection. A number of IFN-stimulated genes, including ISG15, OAS1, MX1, IRF1, IRF9 and TAP1 are also induced in an IFN-ß-dependent manner, whereas GBP1 remains unaffected by IFN-ß neutralization. Increased STAT1 phosphorylation in HMECs subjected to UBP43 knockdown led to transcriptional activation of OAS1, MX1 and GBP1, confirming the negative regulatory role of UBP43. Although IRF1, IRF9 and TAP1 were induced by IFN-ß, siRNA-mediated silencing of UBP43 or SOCS1 did not significantly affect their transcriptional activation. Expression of ISG15 was, however, increased in HMECs transfected with siRNA for UBP43 and SOCS1. Thus, unique regulatory patterns of induced expression of UBP43, SOCS1 and IFN-stimulated genes represent pathogen-specific responses underlying IFN-ß-mediated host endothelial signalling during the pathogenesis of spotted fever group rickettsiosis.

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells.

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-ß, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-ß, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-ß activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-ß-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.

Beta interferon-mediated activation of signal transducer and activator of transcription protein 1 interferes with Rickettsia conorii replication in human endothelial cells.

Infection of the endothelial cell lining of blood vessels with Rickettsia conorii, the causative agent of Mediterranean spotted fever, results in endothelial activation. We investigated the effects of R. conorii infection on the status of the Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling pathway in human microvascular endothelial cells (HMECs), the most relevant host cell type, in light of rickettsial tropism for microvascular endothelium in vivo. R. conorii infection induced phosphorylation of STAT1 on tyrosine 701 and serine 727 at 24, 48, and 72 h postinfection in HMECs. Employing transcription profile analysis and neutralizing antibodies, we further determined that beta interferon (IFN-ß) production and secretion are critical for STAT1 activation. Secreted IFN-ß further amplified its own expression via a positive-feedback mechanism, while expression of transcription factors interferon regulatory factor 7 (IRF7) and IRF9, implicated in the IFN-ß-STAT1 feedback loop, was also induced. Metabolic activity of rickettsiae was essential for the IFN-ß-mediated response(s) because tetracycline treatment inhibited R. conorii replication, IFN-ß expression, and STAT1 phosphorylation. Inclusion of IFN-ß-neutralizing antibody during infection resulted in significantly enhanced R. conorii replication, whereas addition of exogenous IFN-ß had the opposite inhibitory effect. Finally, small interfering RNA-mediated knockdown further confirmed a protective role for STAT1 against intracellular R. conorii replication. In concert, these findings implicate an important role for IFN-ß-mediated STAT1 activation in innate immune responses of vascular endothelium to R. conorii infection.

Rickettsia rickettsii infection protects human microvascular endothelial cells against staurosporine-induced apoptosis by a cIAP(2)-independent mechanism.

BACKGROUND: Manipulation of host cell death is an important determinant of the outcome of an infection. Here, we investigate whether Rickettsia rickettsii-infected host endothelial cells resist the effects of staurosporine, a potent inducer of apoptosis, and we explore the mechanisms underlying the anti-apoptotic effect of infection. METHODS: Human microvascular endothelial cells infected with R. rickettsii for 24 or 48 h were challenged with staurosporine. The extent of apoptosis was evaluated with flow cytometry. mRNA and protein expression levels were determined by use of microarray or polymerase chain reaction and immunoblotting, respectively. RESULTS: Staurosporine-induced apoptosis in endothelial cells infected for 24 and 48 h was significantly reduced, compared with simultaneously treated uninfected cells. A microarray of human genes involved in apoptosis and polymerase chain reaction analyses revealed increased steady-state mRNA expression of cIAP2 (a member of the inhibitor-of-apoptosis family of proteins) at 24 h after infection. The levels of cIAP2 protein (+/-SD) in infected cells were 3.5 +/- 1.7 -fold and 2.3 +/- 1.2 -fold higher than that in uninfected control cells at 24 and 48 h after infection. Nucleofection of human-specific cIAP2-targeted siRNA resulted in inhibition of protein expression by > or = 50% but had no effect on infection-induced protection against apoptosis. CONCLUSIONS: R. rickettsii-induced expression of cIAP2 in host endothelial cells is likely not a major contributor to protection against staurosporine-induced cell death.