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1.  Toxic Introns and Parasitic Intein in Coxiella burnetii: Legacies of a Promiscuous Past▿  
Journal of Bacteriology  2008;190(17):5934-5943.
The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.
PMCID: PMC2519523  PMID: 18606739
2.  The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG▿  
Journal of Bacteriology  2007;189(18):6572-6579.
We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.
PMCID: PMC2045182  PMID: 17644584
3.  Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii▿ 
Journal of Bacteriology  2011;193(19):5292-5299.
The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.
PMCID: PMC3187410  PMID: 21803999
4.  A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant▿  
Journal of Bacteriology  2009;191(12):4044-4046.
Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.
PMCID: PMC2698393  PMID: 19376857
5.  Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening 
PLoS Pathogens  2014;10(3):e1004013.
Coxiella burnetii is the agent of the emerging zoonosis Q fever. This pathogen invades phagocytic and non-phagocytic cells and uses a Dot/Icm secretion system to co-opt the endocytic pathway for the biogenesis of an acidic parasitophorous vacuole where Coxiella replicates in large numbers. The study of the cell biology of Coxiella infections has been severely hampered by the obligate intracellular nature of this microbe, and Coxiella factors involved in host/pathogen interactions remain to date largely uncharacterized. Here we focus on the large-scale identification of Coxiella virulence determinants using transposon mutagenesis coupled to high-content multi-phenotypic screening. We have isolated over 3000 Coxiella mutants, 1082 of which have been sequenced, annotated and screened. We have identified bacterial factors that regulate key steps of Coxiella infections: 1) internalization within host cells, 2) vacuole biogenesis/intracellular replication, and 3) protection of infected cells from apoptosis. Among these, we have investigated the role of Dot/Icm core proteins, determined the role of candidate Coxiella Dot/Icm substrates previously identified in silico and identified additional factors that play a relevant role in Coxiella pathogenesis. Importantly, we have identified CBU_1260 (OmpA) as the first Coxiella invasin. Mutations in ompA strongly decreased Coxiella internalization and replication within host cells; OmpA-coated beads adhered to and were internalized by non-phagocytic cells and the ectopic expression of OmpA in E. coli triggered its internalization within cells. Importantly, Coxiella internalization was efficiently inhibited by pretreating host cells with purified OmpA or by incubating Coxiella with a specific anti-OmpA antibody prior to host cell infection, suggesting the presence of a cognate receptor at the surface of host cells. In summary, we have developed multi-phenotypic assays for the study of host/pathogen interactions. By applying our methods to Coxiella burnetii, we have identified the first Coxiella protein involved in host cell invasion.
Author Summary
Infectious diseases are among the major causes of mortality worldwide. Pathogens‚ invasion, colonization and persistence within their hosts depend on a tightly orchestrated cascade of events that are commonly referred to as host/pathogen interactions. These interactions are extremely diversified and every pathogen is characterized by its unique way of co-opting and manipulating host functions to its advantage. Understanding host/pathogen interactions is the key to face the threats imposed by infectious diseases and find alternative strategies to fight the emergence of multi-drug resistant pathogens. In this study, we have setup and validated a protocol for the rapid and unbiased identification of bacterial factors that regulate host/pathogen interactions. We have applied this method to the study of Coxiella burnetii, the etiological agent of the emerging zoonosis Q fever. We have isolated, sequenced and screened over 1000 bacterial mutations and identified genes important for Coxiella invasion and replication within host cells. Ultimately, we have characterized the first Coxiella invasin, which mediates bacterial internalization within non-phagocytic cells. Most importantly, our finding may lead to the development of a synthetic vaccine against Q fever.
PMCID: PMC3961360  PMID: 24651569
6.  Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway 
Infection and Immunity  2014;82(9):3740-3752.
Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.
PMCID: PMC4187803  PMID: 24958706
7.  Pentamidine Inhibition of Group I Intron Splicing in Candida albicans Correlates with Growth Inhibition 
We previously demonstrated that pentamidine, which has been clinically used against Pneumocystis carinii, inhibits in vitro a group I intron ribozyme from that organism. Another fungal pathogen, Candida albicans, also harbors a group I intron ribozyme (Ca.LSU) in the essential rRNA genes in almost half of the clinical isolates analyzed. To determine whether pentamidine inhibits Ca.LSU in vitro and in cells, phylogenetically closely related intron-containing (4-1) and intronless (62-1) strains were studied. Splicing in vitro of the Ca.LSU group I intron ribozyme was completely inhibited by pentamidine at 200 μM. On rich glucose medium, the intron-containing strain was more sensitive to growth inhibition by pentamidine than was the intronless strain, as measured by disk or broth microdilution assays. On rich glycerol medium, they were equally susceptible to pentamidine. At pentamidine levels selectively inhibiting the intron-containing strain (1 μM) in glucose liquid cultures, inhibition of splicing and rRNA maturation was detected by quantitative reverse transcription-PCR within 1 min with a 10- to 15-fold accumulation of precursor rRNA. No comparable effect was seen in the intronless strain. These results correlate the cellular splicing inhibition of Ca.LSU with the growth inhibition of strain 4-1 harboring Ca.LSU. Broth microdilution assays of 13 Candida strains showed that intron-containing strains were generally more susceptible to pentamidine than the intronless strains. Our data suggest that ribozymes found in pathogenic microorganisms but absent in mammals may be targets for antimicrobial therapy.
PMCID: PMC89798  PMID: 10722497
8.  Interleukin-10 Stimulates Coxiella burnetii Replication in Human Monocytes through Tumor Necrosis Factor Down-Modulation: Role in Microbicidal Defect of Q Fever 
Infection and Immunity  2001;69(4):2345-2352.
Coxiella burnetii, an obligate intracellular bacterium, is the agent of Q fever. The chronic form of the disease is associated with the overproduction of interleukin-10 and deficient C. burnetii killing by monocytes. We hypothesized that the replication of C. burnetii inside monocytes requires a macrophage-deactivating cytokine such as interleukin-10. In the absence of interleukin-10, C. burnetii survived but did not replicate in monocytes. C. burnetii replication (measured 15 days) was induced in interleukin-10-treated monocytes. This effect of interleukin-10 is specific since transforming growth factor β1 had no effect on bacterial replication. C. burnetii replication involves the down-modulation of tumor necrosis factor (TNF) release. First, interleukin-10 suppressed C. burnetii-stimulated production of TNF. Second, the addition of recombinant TNF to interleukin-10-treated monocytes inhibited bacterial replication. Third, the incubation of infected monocytes with neutralizing anti-TNF antibodies favored C. burnetii replication. On the other hand, deficient C. burnetii killing by monocytes from patients with chronic Q fever involves interleukin-10. Indeed, C. burnetii replication was observed in monocytes from patients with Q fever endocarditis, but not in those from patients with acute Q fever. Bacterial replication was inhibited by neutralizing anti-interleukin-10 antibodies. As monocytes from patients with endocarditis overproduced interleukin-10, the defective bacterial killing is likely related to endogenous interleukin-10. These results suggest that interleukin-10 enables monocytes to support C. burnetii replication and to favor the development of chronic Q fever.
PMCID: PMC98164  PMID: 11254592
9.  A unique Coxiella burnetii lipoprotein involved in metal binding (LimB) 
Microbiology  2011;157(Pt 4):966-976.
Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. The limB gene was maximally expressed at 3–7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
PMCID: PMC3139440  PMID: 21212117
10.  Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages 
Infection and Immunity  2012;80(6):1980-1986.
Coxiella burnetii is the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission, C. burnetii replicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms of C. burnetii intracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling during C. burnetii infection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV and C. burnetii replication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulent C. burnetii isolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated during C. burnetii infection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA in C. burnetii infection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.
PMCID: PMC3370589  PMID: 22473604
11.  A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis 
PLoS Pathogens  2014;10(7):e1004286.
Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.
Author Summary
Coxiella burnetii is the causative agent of the human disease Q fever. This bacterium uses the Dot/Icm type IV secretion system to deliver effectors into the cytosol of host cells. The Dot/Icm system is required for intracellular replication of C. burnetii. To determine the contribution of individual proteins to the establishment of a vacuole that supports C. burnetii replication, we conducted a visual screen on a library of C. burnetii transposon insertion mutants and identified genes required for distinct stages of intracellular replication. This approach was validated through the identification of intracellular replication mutants that included insertions in most of the dot and icm genes, and through the identification of individual effector proteins delivered into host cell by the Dot/Icm system that participate in creating a vacuole that supports intracellular replication of C. burnetii. Complementation studies showed convincingly that the effector Cig57 was critical for intracellular replication. The effector protein Cig2 was found to play a unique role in promoting homotypic fusion of C. burnetii vacuoles. Disrupting host autophagy phenocopied the defect displayed by the cig2 mutant. Thus, our visual screen has successfully identified effectors required for intracellular replication of C. burnetii and indicates that Dot/Icm-dependent subversion of host autophagy promotes homotypic fusion of CCVs.
PMCID: PMC4117601  PMID: 25080348
12.  Virulent Coxiella burnetii Pathotypes Productively Infect Primary Human Alveolar Macrophages 
Cellular microbiology  2013;15(6):1012-1025.
The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen’s infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii-host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harbored single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short term phosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C. burnetii.
PMCID: PMC3655087  PMID: 23279051
13.  Coxiella burnetii Expresses a Functional Δ24 Sterol Reductase▿  
Journal of Bacteriology  2010;192(23):6154-6159.
Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.
PMCID: PMC2981196  PMID: 20870767
14.  Coxiella burnetii Acid Phosphatase Inhibits the Release of Reactive Oxygen Intermediates in Polymorphonuclear Leukocytes ▿  
Infection and Immunity  2010;79(1):414-420.
Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Replication of C. burnetii during infection has been shown to be increased by decreasing oxidative stress using p47phox −/− and iNOS−/− mice in vivo and by pharmacologic inhibitors in vitro. Building upon this model, we investigated the role polymorphonuclear leukocytes (PMN) play in the control of infection, since NADPH oxidase-mediated release of reactive oxygen intermediates (ROI) is a primary bactericidal mechanism for these cells that is critical for early innate clearance. Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN through expression of an unidentified acid phosphatase (ACP). Recent genomic annotations identified one open reading frame (CBU0335) which may encode a Sec- and type II-dependent secreted ACP. To test this model, viable C. burnetii propagated in tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP) was combined with human PMN induced with 4-phorbol 12-myristate 13-acetate (PMA). The release of ROI was inhibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were challenged with electron beam-inactivated C. burnetii. C. burnetii extracts and rACP were also able to inhibit PMA-induced formation of NADPH oxidase complex on PMN membranes, suggesting a molecular mechanism responsible for this inhibition. These data support a model in which C. burnetii eludes the primary ROI killing mechanism of activated PMN by secreting at least one acid phosphatase.
PMCID: PMC3019863  PMID: 21078859
15.  Persistent Coxiella burnetii Infection in Mice Overexpressing IL-10: An Efficient Model for Chronic Q Fever Pathogenesis 
PLoS Pathogens  2008;4(2):e23.
Interleukin (IL)-10 increases host susceptibility to microorganisms and is involved in intracellular persistence of bacterial pathogens. IL-10 is associated with chronic Q fever, an infectious disease due to the intracellular bacterium Coxiella burnetii. Nevertheless, accurate animal models of chronic C. burnetii infection are lacking. Transgenic mice constitutively expressing IL-10 in macrophages were infected with C. burnetti by intraperitoneal and intratracheal routes and infection was analyzed through real-time PCR and antibody production. Transgenic mice exhibited sustained tissue infection and strong antibody response in contrast to wild-type mice; thus, bacterial persistence was IL-10-dependent as in chronic Q fever. The number of granulomas was low in spleen and liver of transgenic mice infected through the intraperitoneal route, as in patients with chronic Q fever. Macrophages from transgenic mice were unable to kill C. burnetii. C. burnetii–stimulated macrophages were characterized by non-microbicidal transcriptional program consisting of increased expression of arginase-1, mannose receptor, and Ym1/2, in contrast to wild-type macrophages in which expression of inducible NO synthase and inflammatory cytokines was increased. In vivo results emphasized macrophage data. In spleen and liver of transgenic mice infected with C. burnetii by the intraperitoneal route, the expression of arginase-1 was increased while microbicidal pathway consisting of IL-12p40, IL-23p19, and inducible NO synthase was depressed. The overexpression of IL-10 in macrophages prevents anti-infectious competence of host, including the ability to mount granulomatous response and microbicidal pathway in tissues. To our knowledge, this is the first efficient model for chronic Q fever pathogenesis.
Author Summary
The interaction between immune system and invading bacteria is sufficient to eradicate microorganisms in the majority of bacterial infections, but the suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. Coxiella burnetii, an obligate intracellular bacterium, is responsible for Q fever. This infectious disease is characterized by a primary infection that may become chronic as endocarditis in patients with valvular damage and immunocompromised patients. Clinical and in vitro studies have suggested a role for interleukin-10 in the chronic evolution of Q fever. However, an efficient mouse model for chronic Q fever pathogenesis, which could serve as a platform for anti–C. burnetii drug or immunotherapy development, is lacking. Here we use transgenic mice with constitutive overexpression of interleukin-10 in the macrophage lineage to study C. burnetii infection. We report an efficient mouse model for chronic Q fever pathogenesis, which associates high levels of specific antibodies, sustained tissue infection, and reduced granuloma formation, as in human Q fever. We also find an anti-inflammatory transcriptional program and altered expression of chemokines in infected tissues.
PMCID: PMC2222951  PMID: 18248094
16.  Bactericidal effect of doxycycline associated with lysosomotropic agents on Coxiella burnetii in P388D1 cells. 
There is no consistently reliable treatment for endocarditis resulting from chronic Coxiella burnetii infection, the causative agent of Q fever. Although certain antibiotics are recommended on the basis of their in vitro bactericidal activities, results of therapy with these antibiotics are often disappointing. To evaluate whether the currently recommended antibiotic susceptibility tests for C. burnetii give misleading results because of continued division of uninfected cells, thereby resulting in the dilution of infected cells and, hence, a false picture of antibiotic efficacy, we blocked cell division during antibiotic susceptibility testing with cycloheximide. Using this new method, we found that the currently recommended antibiotics for the treatment of Q fever, doxycycline, pefloxacin, and rifampin, did not reduce the ratio of infected to noninfected cells (either L929 or P388D1) by 9 days postinfection. To test the hypothesis that this lack of antibacterial activity is due to antibiotic inactivation by the low pH of the phagolysosomes in which C. burnetii is found, we used alkalinizing lysosomotropic agents (chloroquine or amantadine) concurrently with doxycycline. This resulted in the sterilization of C. burnetii infection in P388D1 cells. This finding seems to confirm our suspicion that the acidic conditions of the phagolysosomes in which C. burnetii is located inhibit antibiotic activity. This inhibition can be reversed in vitro when lysosomotropic alkalinizing agents are used.
PMCID: PMC171863  PMID: 2221859
17.  Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding 
Nucleic Acids Research  2002;30(13):2961-2971.
The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 µM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.
PMCID: PMC117049  PMID: 12087182
18.  The Uptake of Apoptotic Cells Drives Coxiella burnetii Replication and Macrophage Polarization: A Model for Q Fever Endocarditis 
PLoS Pathogens  2008;4(5):e1000066.
Patients with valvulopathy have the highest risk to develop infective endocarditis (IE), although the relationship between valvulopathy and IE is not clearly understood. Q fever endocarditis, an IE due to Coxiella burnetii, is accompanied by immune impairment. Patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes, as determined by the measurement of active caspases and nucleosome determination. The binding of apoptotic cells to monocytes and macrophages, the hosts of C. burnetii, may be responsible for the immune impairment observed in Q fever endocarditis. Apoptotic lymphocytes (AL) increased C. burnetii replication in monocytes and monocyte-derived macrophages in a cell-contact dependent manner, as determined by quantitative PCR and immunofluorescence. AL binding induced a M2 program in monocytes and macrophages stimulated with C. burnetii as determined by a cDNA chip containing 440 arrayed sequences and functional tests, but this program was in part different in monocytes and macrophages. While monocytes that had bound AL released high levels of IL-10 and IL-6, low levels of TNF and increased CD14 expression, macrophages that had bound AL released high levels of TGF-β1 and expressed mannose receptor. The neutralization of IL-10 and TGF-β1 prevented the replication of C. burnetii due to the binding of AL, suggesting that they were critically involved in bacterial replication. In contrast, the binding of necrotic cells to monocytes and macrophages led to C. burnetii killing and typical M1 polarization. Finally, interferon-γ corrected the immune deactivation induced by apoptotic cells: it prevented the replication of C. burnetii and re-directed monocytes and macrophages toward a M1 program, which was deleterious for C. burnetii. We suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for bacterial persistence.
Author Summary
Infective endocarditis (IE) is a problem of public health that still causes high mortality despite antibiotic treatments. Most of the patients who develop an IE have pre-existing cardiac lesions, although the relationship between IE and valvulopathy is not clearly understood. We showed here that patients with valvulopathy exhibited increased levels of circulating apoptotic leukocytes. As the binding of apoptotic cells to monocytes and macrophages is known to inhibit their inflammatory activity, we hypothesized that the high levels of circulating apoptotic leukocytes may be responsible for the immune impairment observed in Q fever endocarditis, an IE due to Coxiella burnetii, a bacterium that survives in monocytes and macrophages. The binding of apoptotic lymphocytes to monocytes and macrophages increased the replication of C. burnetii by stimulating their anti-inflammatory response. In contrast, the binding of necrotic lymphocytes to monocytes and macrophages induced C. burnetii killing and stimulated an inflammatory response. Interferon-γ, which is associated with the control of C. burnetii infection, prevented the replication of C. burnetii in monocytes and macrophages that have bound apoptotic lymphocytes by stimulating their inflammatory response. In conclusion, we suggest that leukocyte apoptosis associated with valvulopathy may be critical for the pathogenesis of Q fever endocarditis by deactivating immune cells and creating a favorable environment for pathogen persistence.
PMCID: PMC2361190  PMID: 18483547
19.  Coxiella burnetii Survives in Monocytes from Patients with Q Fever Endocarditis: Involvement of Tumor Necrosis Factor 
Infection and Immunity  2000;68(1):160-164.
Endocarditis is the most frequent form of chronic Q fever, an infectious disease caused by Coxiella burnetii. As this obligate intracellular bacterium inhabits monocytes and macrophages, we wondered if pathogenesis of Q fever endocarditis is related to defective intracellular killing of C. burnetii by monocytes. Monocytes from healthy controls eliminated virulent C. burnetii within 3 days. In contrast, monocytes from patients with ongoing Q fever endocarditis were unable to eliminate bacteria even after 6 days. In patients who were cured of endocarditis, the monocyte infection was close to that of control monocytes. This killing deficiency was not the consequence of generalized functional impairment, since patient monocytes eliminated avirulent C. burnetii as did control cells. The addition of supernatants of C. burnetii-stimulated monocytes from patients with ongoing endocarditis to control monocytes enabled them to support C. burnetii survival, suggesting that some soluble factor is responsible for bacterial survival. This factor was related to tumor necrosis factor (TNF): expression of TNF mRNA and TNF release were increased in response to C. burnetii in patients with ongoing endocarditis compared to cured patients and healthy controls. In addition, neutralizing anti-TNF antibodies decreased C. burnetii internalization, an early step of bacterial killing, in monocytes from patients with ongoing endocarditis but did not affect delayed steps of intracellular killing. We suggest that Q fever-associated activation of monocytes allows the survival of C. burnetii by modulating early phases of microbial killing.
PMCID: PMC97115  PMID: 10603382
20.  Sustained Axenic Metabolic Activity by the Obligate Intracellular Bacterium Coxiella burnetii▿ †  
Journal of Bacteriology  2008;190(9):3203-3212.
Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [35S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell.
PMCID: PMC2347409  PMID: 18310349
21.  Characterization of the GDP-D-Mannose Biosynthesis Pathway in Coxiella burnetii: The Initial Steps for GDP-β-D-Virenose Biosynthesis 
PLoS ONE  2011;6(10):e25514.
Coxiella burnetii, the etiologic agent of human Q fever, is a Gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.
PMCID: PMC3204966  PMID: 22065988
22.  A DNA-Binding Peroxiredoxin of Coxiella burnetii Is Involved in Countering Oxidative Stress during Exponential-Phase Growth▿  
Journal of Bacteriology  2010;192(8):2077-2084.
Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100°C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.
PMCID: PMC2849434  PMID: 20173000
23.  The Effect of C. burnetii Infection on the Cytokine Response of PBMCs from Pregnant Goats 
PLoS ONE  2014;9(10):e109283.
In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.
PMCID: PMC4184872  PMID: 25279829
24.  Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening 
mBio  2013;4(1):e00606-12.
Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.
Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.
PMCID: PMC3560531  PMID: 23362322
25.  αvβ3 Integrin and Bacterial Lipopolysaccharide Are Involved in Coxiella burnetii-Stimulated Production of Tumor Necrosis Factor by Human Monocytes 
Infection and Immunity  2000;68(10):5673-5678.
Coxiella burnetii, the agent of Q fever, enters human monocytes through αvβ3 integrin and survives inside host cells. In addition, C. burnetii stimulates the synthesis of inflammatory cytokines including tumor necrosis factor (TNF) by monocytes. We studied the role of the interaction of C. burnetii with THP-1 monocytes in TNF production. TNF transcripts and TNF release reached maximum values within 4 h. Almost all monocytes bound C. burnetii after 4 h, while the percentage of phagocytosing monocytes did not exceed 20%. Cytochalasin D, which prevented the uptake of C. burnetii without interfering with its binding, did not affect the expression of TNF mRNA. Thus, bacterial adherence, but not phagocytosis, is necessary for TNF production by monocytes. The monocyte αvβ3 integrin was involved in TNF synthesis since peptides containing RGD sequences and blocking antibodies against αvβ3 integrin inhibited TNF transcripts induced by C. burnetii. Nevertheless, the cross-linking of αvβ3 integrin by specific antibodies was not sufficient to induce TNF synthesis. The signal delivered by C. burnetii was triggered by bacterial lipopolysaccharide (LPS). Polymyxin B inhibited the TNF production stimulated by C. burnetii, and soluble LPS isolated from C. burnetii largely mimicked viable bacteria. On the other hand, avirulent variants of C. burnetii induced TNF production through an increased binding to monocytes rather than through the potency of their LPS. We suggest that the adherence of C. burnetii to monocytes via αvβ3 integrin enables surface LPS to stimulate TNF production in THP-1 monocytes.
PMCID: PMC101522  PMID: 10992470

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