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

Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2009; 106(11):4430-4. DOI: 10.1073/pnas.0812074106
Source: PubMed

ABSTRACT 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 that 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, and metabolite typing. This led to development of a complex nutrient medium that supported substantial growth (approximately 3 log(10)) of C. burnetii 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 intracellular pathogens.

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Available from: Kimmo Virtaneva, Sep 29, 2015
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    • "(Kikuchi, 2009) motivate future efforts to develop suitable growing conditions and techniques to access the uncultivable majority of bacteria. Genome analysis in particular has proven a powerful method to pinpoint nutrient and oxygen requirements for microbial growth (Kikuchi, 2009; Omsland et al., 2009). The loss of key primary metabolic pathways in Ca. "
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    ABSTRACT: Ecteinascidin 743 (ET-743, Yondelis) is a clinically approved chemotherapeutic natural product isolated from the Caribbean mangrove tunicate Ecteinascidia turbinata. Researchers have long suspected that a microorganism may be the true producer of the anti-cancer drug, but its genome has remained elusive due to our inability to culture the bacterium in the laboratory using standard techniques. Here, we sequenced and assembled the complete genome of the ET-743 producer, Candidatus Endoecteinascidia frumentensis, directly from metagenomic DNA isolated from the tunicate. Analysis of the ∼631 kb microbial genome revealed strong evidence of an endosymbiotic lifestyle and extreme genome reduction. Phylogenetic analysis suggested that the producer of the anti-cancer drug is taxonomically distinct from other sequenced microorganisms and could represent a new family of Gammaproteobacteria. The complete genome has also greatly expanded our understanding of ET-743 production and revealed new biosynthetic genes dispersed across more than 173 kb of the small genome. The gene cluster's architecture and its preservation demonstrate that the drug is likely essential to the interactions of the microorganism with its mangrove tunicate host. Taken together, these studies elucidate the lifestyle of a unique, and pharmaceutically-important microorganism and highlight the wide diversity of bacteria capable of making potent natural products. This article is protected by copyright. All rights reserved.
    Environmental Microbiology 05/2015; DOI:10.1111/1462-2920.12908 · 6.20 Impact Factor
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    • "bacteria that are classified into the gamma subgroup of proteobac - teria ( Roux et al. , 1997 ) . Later on , the study by Omsland et al . ( 2009 ) reported that C . burnetti can be cultured on axenic ( host cell - free ) media . The sequence identity of the 16S rRNA gene among the Coxiella - like bacteria varies from species to species . When the 16S rRNA nucleotide sequences are compared with each other using C . burnetii as a reference genome , a maximal and a minimal nucleoti"
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    ABSTRACT: In this study, we focused on the molecular detection of Coxiella-like bacteria using a PCR technique to identify Coxiella 16S rRNA sequences in Haemaphysalis tick samples (105 adults, 8 nymph pools and 19 larval pools). Seven Haemaphysalis species obtained from 5 locations in Thailand were evaluated in this work. Coxiella endosymbionts could be detected in samples representing all 3 growth stages examined. The results also revealed that only 4 of 7 tick species were positive for Coxiella-like endosymbiont: Haemaphysalis hystricis, Haemaphysalis lagrangei, Haemaphysalis obesa, and Haemaphysalis shimoga. Haemaphysalis shimoga demonstrated the highest percentage of Coxiella-like positive samples (58.33% with n = 24), while Haemaphysalis hystricis had the lowest percentage; only 1 female tick was positive for Coxiella-like bacteria (n = 6). Interestingly, the results indicated that female Haemaphysalis ticks tended to harbour Coxiella symbionts more frequently than male ticks (59.32% of females and 21.27% of males of all species studied). Phylogenetic analyses based on 16S rRNA sequences illustrated that Coxiella-like spp. from the same tick species always grouped in same clade, regardless of the location from which they were isolated. Moreover, a phylogenetic tree also showed that Coxiella-like endosymbionts from other genera (for example, the tick genus Rhipicephalus) formed a separate group compared to Coxiella-like symbionts in the genus Haemaphysalis. This suggests that a high amount of DNA sequence variation is present in Coxiella-like bacteria harboured by ticks.
    Ticks and Tick-borne Diseases 10/2014; 6(1). DOI:10.1016/j.ttbdis.2014.09.005 · 2.72 Impact Factor
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    • "The discovery of host cell-free growth of C. burnetii in acidified citrate cysteine medium (ACCM) [37,38] allowed us to probe culture media for the presence of secreted proteins. Mass spectrometry generated a list of 105 C. burnetii proteins in ACCM culture supernatants. "
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    ABSTRACT: Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication. To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.
    BMC Microbiology 10/2013; 13(1):222. DOI:10.1186/1471-2180-13-222 · 2.73 Impact Factor
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