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Identification of trkH, encoding a potassium uptake protein required for Francisella tularensis systemic dissemination in mice.

ABSTRACT: Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the potassium uptake permease TrkH. Inactivation of trkH impaired bacterial growth in CDM. Normal growth of the mutant was only restored when CDM was supplemented with potassium at high concentration. Strikingly, although not required for intracellular survival in cell culture models, TrkH appeared to be essential for bacterial virulence in the mouse. In vivo kinetics of bacterial dissemination revealed a severe defect of multiplication of the trkH mutant in the blood of infected animals. The trkH mutant also showed impaired growth in blood ex vivo. Genome sequence analyses suggest that the Trk system constitutes the unique functional active potassium transporter in both tularensis and holarctica subspecies. Hence, the impaired survival of the trkH mutant in vivo is likely to be due to its inability to survive in the low potassium environment (1-5 mM range) of the blood. This work unravels thus the importance of potassium acquisition in the extracellular phase of the F. tularensis infectious cycle. More generally, potassium could constitute an important mineral nutrient involved in other diseases linked to systemic dissemination of bacterial pathogens.

SUBMITTER: Alkhuder K

PROVIDER: S-EPMC2813290 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Identification of trkH, encoding a potassium uptake protein required for Francisella tularensis systemic dissemination in mice.

Alkhuder Khaled K Meibom Karin L KL Dubail Iharilalao I Dupuis Marion M Charbit Alain A

PloS one 20100129 1

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularaemia. During its infectious cycle, F. tularensis is not only exposed to the intracellular environment of macrophages but also resides transiently in extracellular compartments, in particular during its systemic dissemination. The screening of a bank of F. tularensis LVS transposon insertion mutants on chemically defined medium (CDM) led us to identify a gene, designated trkH, encoding a homolog of the pota ...[more]

PMID: 20126460

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Project description:pool 16 and pool 29 arrays represent the Francisella transposon insertion mutants isolated from the spleens of two separate groups of 5 intraperitoneally infected mice. skin arrays represent the Francisella transposon insertion mutants isolated from the skin of 5 individual, subcutaneously infected mice. input arrays represent Francisella transposon insertion mutants surviving after growth in TSB broth. Detailed Materials and Methods from Weiss et al, PNAS, 2007, In vivo negative selection screen identifies genes required for Francisella virulence: Mice and Francisella infections Female C57BL/6 mice (Jackson) between 7 and 10 wk of age were kept under specific pathogen-free conditions in filter-top cages at Stanford University and provided with sterile water and food ad libitum. Experimental studies were in accordance with IACUC Guidelines. Mice were inoculated with 2x105 cfu subcutaneously in 50 ul sterile PBS or intraperitoneally (IP) in 500 ul. Spleens were harvested 2 days post-infection, homogenized, and dilutions were plated on supplemented MH agar plates containing kanamycin (infections with library) or MH plates with and without the appropriate antibiotic (competition experiments). Plates were grown overnight at 37oC, 5% CO2 and colonies were enumerated. After infections with the library, approximately 10^5 cfu were collected in sterile PBS for isolation of DNA. Competitive Index (CI) values were calculated using the formula CI = (mutant cfu in output/wild-type cfu in output)/(mutant cfu in input/wild-type cfu in input). Microarrays Genomic DNA was purified from bacterial pellets by phenol-chloroform extraction. Each DNA sample was divided in two and digested separately with BfaI or RsaI (NEB). The digested DNA was used as the template for in vitro transcription with the AmpliScribe T7-Flash transcription kit (Epicentre) following the manufacturer's protocol, except that 2 ?g of digested DNA was used, and the reaction was allowed to proceed for 12 to 16 h. Purified RNA was used in a RT reaction using Superscript II(?) (Invitrogen) and random hexamers as primers. cDNA was labeled with amino-allyl dUTP using the Klenow (exo-) enzyme. The ssDNA containing amino-allyl dUTP from the mouse output or the library input pools were labeled with Cy3 and Cy5, respectively, prior to hybridization to our Francisella microarray as described previously. Data analysis Normalized data were downloaded from the Stanford Microarray Database according to the median log2 Cy5/Cy3 (logRAT2N). Filters for feature quality, including a Cy3 net median intensity of 150 and regression correlation of >0.6, were applied. To compare data from separate IP infection experiments, each experimental sample was zero-transformed to the input/input control. Features (spots) missing values for 40% of the arrays were removed from the data set. The data sets were analyzed with the SAM program, using the two-class analysis option to identify features that consistently deviated from the input and samples across all of the arrays with a false discovery rate of 1%.

Dataset Information

Identification of trkH, encoding a potassium uptake protein required for Francisella tularensis systemic dissemination in mice.

Publications

Identification of trkH, encoding a potassium uptake protein required for Francisella tularensis systemic dissemination in mice.

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