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Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

ABSTRACT: UNLABELLED:The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ?kdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ?kdhAB mutant strain had a 50% lethal dose (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-?) and interleukin-1? (IL-1?) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ?kdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ?kdhAB mutant induced higher levels of TNF-? and IL-1? in a TLR2-dependent manner. In addition, TLR2(-/-) mice were more susceptible than wild-type mice to ?kdhAB bacterial infection. Finally, immunization of mice with ?kdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE:The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification-Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal--has recently been reported in F. tularensis. We found that the kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

SUBMITTER: Okan NA

PROVIDER: S-EPMC3573668 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

Okan Nihal A NA Chalabaev Sabina S Kim Tae-Hyun TH Fink Avner A Ross Robin A RA Kasper Dennis L DL

mBio 20130212 1

<h4>Unlabelled</h4>The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori ...[more]

PMID: 23404403

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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

Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

Publications

Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

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