Coxiella Burnetii: Lessons from Genomics»

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May 8, 2011 Library « Coxiella burnetii: lessons from genomics»

ÖQuestions to genomicsLecture ÖGenomics based ressourcesauthor Onlineby ÖAnswers © Didier Raoult Marseille –France [email protected]

ESCMID U R Questions to genomics

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ÖSource Lecture ÖEasy culture author ÖClinical findingsOnlineby © ÖTreatment ESCMID Coxiella burnetii : Bacteriology

Library z Pleomorphic Lecture z Coccobacillus z Gram‐negative author z 0.2 ‐ 0.7 mm Onlineby z Gimenez staining © z Agent of Q fever

ESCMID U R Coxiella burnetii : Bacteriology z Obligate intracellular bacterium Library z Any animals z Gene exchanges in Amoeba with Legionella Lecture z Multiplication within the phagolysosome of author macrophages Onlineby z Survives in acidic vacuole© (low pH activates metabolism) z Growing in axenic medium ESCMID U R Q fever, free amoeba, and air conditioning.Raoult D. Clin Infect Dis. 2010 Oct 1;51(7):869-70.Library To the Editor—I congratulate the team of Amitai et al [1] on their investigation of an epidemic of Q fever in a school in Israel. This work is remarkable because, for the first time, to the best of my knowledge, it evokes the role of the air conditioning as a potential source of the Q fever. This assumption deserves to be considered in the context of a reflection on diseases transmitted by air conditioning and, more generally, on diseases caused bacteria hosted by the free amoebas of water present in air conditioning devices and cooling towers. Legionnaires disease was the first disease recognized to have free amoebas as reservoirs, which explained persistence of Legionella pneumophila in water supply networks, in the circuits of air conditioning, and in hospitals and hotels. The resistance of the amoebas to the process of sterilization explains the difficulty in eliminating L. pneumophila from water circuits [2]. In recent years, there has been a considerable increaseLecture of the number of bacteria identified in amoebas, which used free amoeba as reservoir and Trojan horse to infect human beings [2, 3]. In an interesting way, certain bacterial pathogens transmissible by aerosols can be found in the free amoebas of water: Legionella species, but also Fransicella tularensis, Chlamydia-related organisms, Mycobacterium species other than Mycobacterium tuberculosis [2, 3], and Coxiella burnetii. C. burnetii is mainly transmitted by aerosols [1] and has a capacity to survive in the free amoeba [4], which led researchers to suspect that intra-amoebal survival had played a role in the selection of the pathogenicity ofauthor these bacteria for humans. Moreover, C. burnetii and L. pneumophila obviously had exchanged genes in the amoebas [5]. On the basis of these data, one may suspect that C. burnetii was a candidate to be transmitted by air conditioning. Finally, amoebas of water are also used as vehicles and reservoirs by many mycobacteria that are at the origin of postoperativeOnline nosocomialby infections of the skin.

In conclusion, the association with Q fever and air conditioning© is not really a surprise and makes sense if it is acknowledged that C. burnetii can survive in amoebas and, thus, be conveyed by water pipelines. The capacity of C. burnetii to be transmitted by aerosol in air conditioning mean that, like other bacteria resisting the phagocytic capabilities of the free-living amoebas, it has the potential to determine infections by way of aerosols. Moreover, free-living protists (including amoebas), by organizing gene exchanges of intracellular organisms, are participating in the creation of new genomic repertoires and may help in the creation of new respiratory pathogens [6]. I suggest that the amoeba-resisting organisms, including C. burnetii [7], should be tested if unexplained cases of pneumonia are observedESCMID in patients exposed to air conditioning, including patients with nosocomial pneumonia. Questions to genomics

ÖLife style Library

ÖSource Lecture ÖEasy culture author ÖClinical findingsOnlineby © ÖTreatment ESCMID Epidemiology

Library z Primary reservoirs z Sources of transmission – Sheep – Uterus – Goats – Placenta – Cattle Lecture– Feces – Cats – Urine – Dogs author– Milk – Pigeons – Straw, manure – Humans Onlineby – Ticks © – Sperm – Blood – Intentional

ESCMID U R Epidemiology Survival in domestic animals and in the environment Library

z Cows: shedding in milk up to 32 month

z Sheep: 109 microorganisms per gram of Lecture placental tissue author z Skim milk at room temperature: > 40 month Onlineby z Refrigerated meat:© > 1 month z Dust on walls at 15-200 C: 7-10 months

z Water: amoebae

ESCMID U R Epidemiology Sheep and goats are the major source of infection Library z Sheep/goats – Abattoirs (Briançon) – Wind (Martigues) Lecture – Milk and cheese (Banon) z Birds (Luberon) author Onlineby ©

ESCMID Epidemiology Q fever: Modes of transmission to man Library

z Inhalation of contaminated aerosols +++

z Oral route (contaminated milk and cheese) Lecture z Percutaneous route (intradermal inoculation) author z Vertical transmission Onlineby z Person-to-person ©transmission (autopsies, deliveries, blood transfusion)

z Sexual transmission

ESCMID U R Questions to genomics

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ÖSource Lecture ÖEasy culture author ÖClinical findingsOnlineby © ÖTreatment ESCMID Diagnostic Library z Serology z Isolation (Shell vial technique): blood and heart valves z Immunofluorescence or Lecture Immunohistochemistry: heart valves author z PCR Onlineby ©

ESCMID U R Diagnostic Cutoff proposal for Q fever diagnosis using the microimmunofluorescence and interpretation of serological results obtained with a single serum sample Library Phase II Phase I antibodies Interpretation antibodies IgG IgM IgG IgA Lecture < 100 Active Q fever authorimprobable > 200 > 50 Acute Q fever Onlineby (100 % © predictive) > 1600 > 100 Chronic Q fever (94 % predictive)

ESCMID U R Diagnostic : Isolation Library z Hazardous – P3-laboratories – Laminar-flow hoods Lecture z 3 systems – Laboratory animals author (guinea-pig) – Embryonated Onlineeggs by – Continuous cell lines© (HEL; Vero) z < 100 isolates worldwide available ESCMID U R PCR z Discrepancies among laboratories – Some team report positive PCR in asymptomaticLibrary patients or years after infection – Our team find positive PCR only during the early phase of acute infection and during chronic infection when antibodies anti phase I IgG are betweenLecture 1/800 and 1/6400 z Indications author – Acute cases negative for IgM – Suspicion of chronicOnline infectionby with Ig anti phase I: IgG ≥ 800 © – Use IS IIII (7 to 20 copies) as a target – Increased used of threat samples

Rolain JM, Raoult D. Molecular detection of Coxiella burnetii in blood and sera during Q fever. ESCMID U QJM. 2005 ;98:615-7. R Questions to genomics

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ÖSource Lecture ÖEasy culture author ÖClinical findingsOnlineby © ÖTreatment ESCMID Natural history and pathophysiology of Q fever D Raoult, T J Marrie, J L Mege Lancet Infect Dis 2005; 5:219–26

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Lecture author Onlineby ©

ESCMID Acute Q fever : Variations from country to country Library

Hepatitis or pneumonia ? Lecture Hepatitis Pneumonia Febrile illness

Basque county + +++author+ AndalusiaOnline +++ by ++ + France© +++ ++

Canada + +++ +

Australia +++ ++

ESCMID U R Maurin M, Raoult D. Q fever. Clin Microbiol Rev. 1999 ;12:518-53. : R U :

Host factors -Sex -Age -Immune status -Pregnancy Bacterial factors genotype -Strain -Inoculum of infection -Way Library Library

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Coxiella burnetii Questions to genomics

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ÖSource Lecture ÖEasy culture author ÖClinical findingsOnlineby © ÖTreatment ESCMID Treatment : acute Q fever

z Usually acute Q fever resolves without treatmentLibrarywithin 15 days z Various antibiotics have been reported to be effective : – Ofloxacin, Pefloxacin – Erythromycin – Chloramphenicol Lecture – Cotrimoxazole – Ceftriaxone author z Doxycycline (200 mg/day) for 2-3 weeks remains the antibiotic-regimenOnline of choiceby z Quinolones should be ©considered in Q fever meningoencephalitis z In cases of hepatitis with autoimmune manifestations short term corticosteroid-therapy should be associated to antibiotics ESCMID U Raoult D. Antimicrob Agents Chemother 1993; 37:1733-6. R Treatment Library z In vitro studies : – Phagolysosomes of C. burnetii -infected cell lines maintain an acidic pH Lecture during persistent infection – Phagolysosomal author alkalinization is critical for the bactericidalOnline effectsby of antibiotics © z Therapeutic implications

ESCMID U R Treatment Library z Coxiella burnetii + P388 D1 – Doxycycline pH 4.8 Bacteriostatic Rifampin – Pefloxacine Lecture + Chloroquine (1 mg/ml) pH 5.7 Pefloxacin – Doxycycline Bacteriocidal author – Pefloxacine Bacteriostatic Doxycycline + NH4Cl (1 mg/ml)Online pHby 7 – Doxycycline © Bacteriocidal – Pefloxacin

Maurin M, et al. Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm.

J. Infect. Dis. 1992;ESCMID 166:1097-1102 Treatment Library z Doxycycline should be monitored and ≥ 5mcg/ml serum z OH-Chloroquine shouldLecture be 1 ± 0.2 mcg/ml z When possible, the strainsauthor susceptibility should be testedOnlineby © z Resistance is increasing more strains than half have a MIC > 2 mcg/ml

ESCMID U R Genomic based ressources Library ÖGenome

ÖGenotyping Lecture author ÖProteomics and Immunoproteomics Onlineby ©

ESCMID Development of sensitive PCR assays Mobilome Resistome

Repeated Mobile genetic Resistance Conserved Development of sequences elements markers sequences diagnostic tests Library Comparative Strain-specific genomics sequences

Reverse Surfaceome Putative vaccinology Total proteic extract antigens NewLecture bacterium Metabolic Proteome Genome pathways (bidimentional sequence Metabolome gels, DIGE) Mass spectrometry author Immunoproteomics SNPs, VNTR, Micro (Maldi‐TOF MS, MS/MS) MST array Virulence Onlineby markers Candidate Development Genotyping DNA RNA Pathophysiology proteins © of culture media Pathophysiology Identification Spectrum (proteins involved in of drug targets, pathological processes, Development of new Protein either by their presence identification or altered expression) antimicrobials

Development of Cloning + ESCMID diagnostic tests Vaccine development Expression (serology, monoclonal antibodies) Library

Proc Natl Acad Sci U S A. 2003Lecture Apr 29;100(9):5455-60 The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellularauthor acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion,Online intracelby lular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis© of this organism. Although the lifestyle and parasitic strategies of C. burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in theESCMIDC. burnetii genome. These analyses suggest that the obligate intracellular lifestyle of C. burnetii may be a relatively recent innovation. Coxiella burnetii : Genome

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Lecture author Onlineby ©

Seshadri R. et al. Complete genome sequence of the Q-fever pathogen ESCMID U Coxiella burnetii. PNAS, 2003;100:5455-60 R Genome repertoire Exchanging gene in amoeba Library Lateral gene transfer between Rickettsia bellii and Amoeba resistant microorganisms Bacteria LectureNumber of genes • Legionella • 49 • Burkholderiaceae •author27 • Parachlamydia Onlineby • 20 • Coxiella © • 15 • Francisella • 6 • Mimivirus • 3

ESCMID U R HGT and genome size

¾Whole genome phylogenetic analysis gave evidence for Lateral acquisition of genetic material facilitated by Libraryhost’s coinfection

Common Intracellular Intracellular bacterial Lecturebacteria bacteria ancestor 1.17 Mb

author Intracellular bacteria Onlineby © Intracellular bacteria Free-living Intracellular Intracellular bacteria bacteria bacteria 3.82 Mb R. felis ESCMID 1.48 Mb Genomic based ressources Library ÖGenome

ÖGenotyping Lecture author ÖProteomics and Immunoproteomics Onlineby ©

ESCMID Emerg Infect Dis, 2005; 11: 1211-1217.Library

Lecture author Onlineby ©

ESCMID U R Genomotyping of Coxiella burnetii using microarrays reveals a conserved genomotype for hard tick isolates

Quentin Leroy1, Fabrice Armougom1, Pascal Barbry2 and Didier Raoult1*

Unité de Recherche en maladies Infectieuses et Tropicales Emergentes (URMITE), CNRS-IRD, UMR 6236-198, Faculté de Médecine, Université de la Méditerranée, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, FranceLibrary1 Abstract C. burnetii is a Gram-negative intracellular γ-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method basedLecture on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographicauthor areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organizedby into 3 groups, with a topology congruent with that obtainedOnline through multi-spacer typing. We also found that only 4 genomotypes were specifically© associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype. ESCMID PlosOne Submitted Chapter III: Comparison of typing methods

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Group A3 LibraryGT : Genomotyping

MST : Multi Spacer Typing

A A2 Group

A1 Lecture

B5 author

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B4 ESCMIDB6 GT MST Genomic based ressources Library ÖGenome

ÖGenotyping Lecture author ÖProteomics and Immunoproteomics Onlineby ©

ESCMID Figure 5. Fondamental research Applied research

BIOTYPING by MS Library PROTEOMICS : fondamental, IMMUNOPROTEOMICS clinique & GLYCOMICS (LPS) (sera, Mabs) SERODIAGNOSTIC RECOMBINANT PROTEINS: ELISA& protein array VACCINE Lecture CANDIDATES

THERAPEUTIC author STRATEGIES C.burnetii strains characterization by MS LIBRARY of C. burnetii approaches Onlineby BIOMARKERS

Cell biology of C. burnetii ©

DEVELOPMENT of NEW COMBINED APPROACHES (Immuno‐PCR, Immuno‐MALDI ESCMID TOF, others..) Identification of protein candidates for the serodiagnosis of Q fever endocarditis by an immunoproteomic approach

Z. Sekeyová & M. Kowalczewska & P. Decloquement &N. Pelletier & E. Špitalská & D. Raoult

Eur J Clin Microbiol Infect Dis (2009) 28:287–295Library

Lecture author Onlineby ©

ESCMID Answers from genomics Library ÖC.burnetii life style

ÖEpidemiology strain andLecture source

ÖAxenic culture and diagnosticauthor Onlineby Ö Link between strains© and clinical findings

Ö Better treatment? ESCMID Library

Lecture author Onlineby ©

ESCMID Library

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ESCMID Library Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18997-9001

Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts,whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encodedLecture by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This differenceauthor suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in supportOnline of this hypothesis.by We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host© protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directedESCMID against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts. Library

Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21755-60

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ESCMID Coxiella burnetii Transcriptional Analysis Reveals Serendipity Clusters of Regulation in Intracellular Bacteria Library

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ESCMID PLoS ONE 5(12): e15321. doi:10.1371/journal.pone.0015321 Transcriptional profiles of the early responses to temperature stress Library

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ESCMID Library

ESCMID Answers from genomics Library ÖC.burnetii life style

ÖEpidemiology strain andLecture source

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ESCMID 1 animal Emerg Infect Dis. 2011 Apr;17(4):668-675.

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ESCMID Answers from genomics Library ÖC.burnetii life style

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Ö Better treatment? ESCMID Library

The inability to propagate obligate intracellular pathogens under axenic (host cell- free) culture conditions imposes severe experimental constraints that have negatively impacted progress in understanding pathogen virulence and disease mechanisms. Coxiella burnetii, the causative agent of human Q (Query) fever, is an obligate intracellular bacterial pathogen thatLecture replicates exclusively in an acidified, lysosome-like vacuole. To define conditions that support C. burnetii growth, we systematically evaluated the organism’s metabolic requirements using expression microarrays, genomic reconstruction, andauthor metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log10) of C.Online burnetiiby in a 2.5% oxygen environment. Importantly, axenically grown C. burnetii were highly infectious for Vero cells and exhibited developmental forms characteristic© of in vivo grown organisms. Axenic cultivation of C.burnetii will facilitate studies of the organism’s pathogenesis and genetics and aid development of Q fever preventatives such as an effective subunit vaccine. Furthermore, the systematic approach used here may be broadly applicable to development of axenic media that support growth of other medically important obligate intracellularESCMID pathogens. Supplemented CCM supports enhanced C. burnetii metabolic activity Library

ESCMID Library

ESCMID Answers from genomics Library ÖC.burnetii life style

ÖEpidemiology strain andLecture source

Ö Better treatment? ESCMID Role of strains in acute Q fever z Acute infection : Library – Nine Mile lower inoculum for lung lesion than Priscilla Lecture

Stein A, et al. Q fever pneumonia: virulence of Coxiella burnetii pathovars in a murine model of aerosol infection. Infect Immun. 2005;73:2469-77. author Onlineby – Priscilla group: no© isolate from acute infection Glazunova, et al. Coxiella burnetii genotyping. Emerg Infect Dis. 2005;11:1211-7. ESCMID U R Association with gene repertoires and information Association of the source of isolation and the number of deleted genes

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ESCMID Chapter III: Conclusion and outlook

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ESCMID Answers from genomics Library ÖC.burnetii life style

ÖEpidemiology strain andLecture source

Ö Better treatment? ESCMID Library

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ESCMID CONCLUSION Library

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