Genetic Manipulation of Coxiella burnetii

Beare, Paul A.

doi:10.1007/978-94-007-4315-1_13

Cited by 31 publications

(30 citation statements)

References 67 publications

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“…A second-generation medium, acidified citrate cysteine medium-2 (ACCM-2), supported increased replication and viability (9,10). Propagation of C. burnetii in ACCM-2 also accelerated the development of genetic tools and allowed cloning of antibioticresistant transformants by colony formation (11)(12)(13).…”

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confidence: 99%

“…The use of antibiotic resistance markers in the genetic transformation of C. burnetii, a category B select agent, is restricted to those that confer resistance to antibiotics without clinical relevance, such as chloramphenicol, kanamycin, and ampicillin (13). Acidic conditions similar to those of ACCM-2 and C. burnetii's lysosome-like intracellular niche inhibit the activity of aminoglycoside antibiotics (18).…”

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confidence: 99%

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Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Sandoz

Beare

Cockrell

et al. 2016

Appl Environ Microbiol

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101

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Host cell-free (axenic) culture of Coxiella burnetii in acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required for C. burnetii growth. Metabolic reconstruction of C. burnetii predicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examine C. burnetii amino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5-to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealed C. burnetii auxotrophy for 11 amino acids, including arginine. Heterologous expression of Legionella pneumophila argGH in C. burnetii permitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection of C. burnetii genetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impair C. burnetii replication. Together, these results highlight the advantages of a nutritionally defined medium in investigations of C. burnetii metabolism and the development of genetic tools. IMPORTANCEHost cell-free growth and genetic manipulation of Coxiella burnetii have revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies of C. burnetii physiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability of C. burnetii. Growth studies confirmed that C. burnetii cannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in the L. pneumophila arginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies of C. burnetii metabolism and allowing non-antibiotic-based selection of C. burnetii genetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.C oxiella burnetii is a wide-ranging bacterial pathogen that causes the zoonosis Q fever (1, 2). Humans are generally infected by inhalation of contaminated aerosols generated by domestic livestock, with sheep, goats, and dairy cattle being the prim...

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confidence: 99%

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Sandoz

Beare

Cockrell

et al. 2016

Appl Environ Microbiol

Self Cite

101

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“…As mutants must be selected and propagated in host cells, mutating genes that are essential for cell invasion and survival remains problematic. Last, complementation of a mutated gene with the wild-type gene under the control of its own promoter remains a major bottleneck that has only been overcome for three obligate intracellular bacteria: C. burnetii 7 , Chlamydia trachomatis serovar L2 (REFS 8,9) and Rickettsia parkeri 10 . Despite these difficulties, there has been substantial progress in the development of genetic tools for obligate intracellular bacteria (BOX 2).…”

Section: Genetic Tools: Methods and Limitationsmentioning

confidence: 99%

“…C. burnetii uses the Dot/Icm type IV secretion system to remodel its intracellular niche (Supplementary information S1 (figure)), and detailed knowledge of substrates was gained through TEM β-lactamase reporter assays 38 . The pJB-CAT plasmid backbone was used to generate other shuttle vectors that enabled the ectopic expression of tagged proteins in C. burnetii and complementation of mutated genes 7 . Although the copy numbers of shuttle vectors are difficult to control and may cause polar effects, they provide a convenient method to examine gene function directly in obligate intracellular bacteria.…”

Section: Genetic Tools: Methods and Limitationsmentioning

confidence: 99%

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Engineering of obligate intracellular bacteria: progress, challenges and paradigms

et al. 2017

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It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host–pathogen interactions.

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Use of Axenic Culture Tools to Study Coxiella burnetii

2018

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Coxiella burnetii is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C. burnetii. In this unit, the media currently utilized for axenic culture of C. burnetii are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for C. burnetii axenic growth under control conditions are provided as a reference. © 2018 by John Wiley & Sons, Inc.

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Genetic Manipulation of Coxiella burnetii

Cited by 31 publications

References 67 publications

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Engineering of obligate intracellular bacteria: progress, challenges and paradigms

Use of Axenic Culture Tools to Study Coxiella burnetii

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