Progress 07/01/02 to 06/30/06
Outputs
We have determined that the capsule of Francisella tularensis is atypical in that, unlike most bacterial capsules, it cannot be isolated from culture supernatant by precipitation with 75% ethanol or cetavlon. Scanning electron microscopy revealed that cells of F. tularensis type A and the LVS (live vaccine strain), both of which are reported to be encapsulated, appeared to have a small electron-dense surface material around them. For convenience, most work was therefore done with LVS, which can be worked with in a BSL-2 laboratory. The bacteria were passed in chemically defined broth medium for 17 passages, followed by culture on the same agar medium and incubated at 32 degrees C, rather than 37 degrees C. The electron dense material was greatly enlarged. However, it remained difficult to differentiate and separate the capsule from lipopolysaccharide (LPS). In other experiments, we generated a mutant with a base substitution in the gene wbtI, which is involved in LPS
O-antigen biosynthesis. This mutant made a complete core carbohydrate, but no O-antigen. Complementation of this mutant with a normal copy of wbtI generated a recombinant strain with a normal LPS. The mutant was also passed in defined medium, and also made a large amount of electron-dense material. These passed mutant cells were gently extracted with 0.5% phenol, the cells removed, and the high molecular weight material precipitated by addition of excess cold ethanol. The precipitate was suspended in water, and subjected to ultracentrifugation at 41,000 rpm overnight. The supernatant was precipitated with cold ethanol, and the precipitate dialyzed, and fractionated through a Sephacryl-300 column. The carbohydrate-positive void volume was lyophilized. The pellet from the ultracentrifugation step was extracted with warm (50 degrees C) 1% sodium deoxycholate, the ultracentrifugation repeated, and the supernatant treated as above. The purified material was submitted to the Complex
Carbohydrate Research Center at the University of Georgia for chemical analysis, and it was determined that the putative capsule contained glucose, galactose, mannose, and octadecanoic acid. The material was coupled to an amino-affinity column support by cyanogen bromide activation, and antibodies to the putative capsule from the immunoglobulin fraction of rabbit antiserum to LVS whole cells were affinity purified. The antibodies reacted with the antigen by ELISA, and Protein A-gold-immuno-transmission electron microscopy demonstrated that the antibodies bound to material on and just off the cell surface of LVS. This antigen had a ladder-like appearance (similar to LPS) on Western blot analysis when reacted with antiserum to whole cells, but not with antibodies to LPS.
Impacts
At this time a vaccine is not licensed for the prevention of tularemia, and a rapid diagnostic test is not available. We expect that the capsule of F. tularensis is required for virulence, and that conjugation of the capsule to key antigens that induce the proper cellular immune response may be used as a protective vaccine. In addition, antibodies to the capsule may be used in various types of diagnostic tests for rapid diagnosis or for detection of the agent. We have isolated a novel glycolipid from F. tularensis that may prove to be an important factor in the virulence of this bacterium, and which can be used to design improved diagnostic tests and vaccines against tularemia.
Publications
- Li, J., F. Ahmed, M. Mandal, C. Ryder, P. Azadi, D.S. Snyder, R. Pechous, T. Zahrt, R. Gogal, and T.J. Inzana. 2006. Identification of Francisella tularensis LVS-specific genes by suppression subtractive hybridization and characterization of an O-antigen deficient mutant. In press.
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Progress 10/01/04 to 09/30/05
Outputs
Our long term objective is to identify and characterize the putative capsule of F. tularensis and to develop rapid diagnostic tests for use under field conditions. The procedure for isolation of the NPS has been refined and is as follows: Cells were grown on Chamberlain's defined medium, scraped off plates, and suspended in 0.5 percent phenol. The cells are harvested and the supernatant containing a slime layer (capsule) is purified by ultracentrifugation. The cells are extracted with phenol/water and the phenol removed by dialysis. Insoluble material is removed and the aqueous phase (AP) was digested with DNase, RNase, and proteinase K. The material is precipitated with sodium acetate and 5 volumes of ethanol, and eluted through a Sepharcryl-400 column. The capsule was coupled to a Sepharose matrix and antibodies to the capsule (from antiserum to whole cells) were isolated by affinity chromatography. Immuno-electron microscopy demonstrated that the antibodies bound
to material on and around the cells, indicating the material was on the cell surface. The carbohydrate composition of the capsule was determined and was found to consist of galactose, mannose, and glucose. Polyacrylamide gel electrophoresis and fluorescent staining demonstrated that the capsule was of high molecular weight and heterogeneous. The antibodies to the capsule were coupled to carboxylate latex particles and these sensitized latex particles were shown to agglutinate in the presence of whole bacteria or the capsule. The antibodies are currently being incorporated into photonic biosensor chips. Antibodies to whole cells have already been shown to be capable of detecting extracts of whole F. tularensis cells in these photosensors, and IgG to the capsule and lipopolysaccharide are now being incorporated and the tests further optimized.
Impacts
F. tularensis is the etiologic agent of tularemia and is classified as a category A bioterrorism agent. Tularemia is a widespread disease in wildlife and occasionally in domestic animals. As few as 10 cells are capable of causing disease, and most hosts are susceptible. A licensed vaccine is not available to prevent tularemia, and rapid diagnostic tests are not available that can be used under field conditions. The results of this work will identify the capsule of F. tularensis, which may prove to be an important antigen in diagnostic test development and later vaccine development. The tests we are currently developing show promise as rapid, field-compatible tests, and further optimization of these tests may advance diagnostic capabilities in this field for all clinical laboratories.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
In order to understand the precise function of the F. tularensis capsule, putative capsule genes were sought for by Suppression Subtractive Hybridization (SSH), which can identify genomic differences between encapsulated F. tularensis LVS and nonencapsulated F. novicida. We identified genes encoding putative proteins responsible for capsule export, polysaccharide biosynthesis, glycosyl transferases, and cell wall biosynthesis in the subtractive library of LVS in a 21.5kb region. A portion of this region lacking 1500-bp was cloned into suicide vector pPV containing a sacB cartridge and a chloramphenicol resistance (CmR) gene, and conjugated into LVS. Possible conjugants were selected for by subculture to chlroamphenicol (Cm) and then sucrose. Six recombinants were red on Congo red agar and lacked iridescence. On Congo red agar, F. novicida colonies, which lack capsule, are red and the LVS and type A colonies are pale pink. PCR was used to verify the nature of the
mutation. Virulence studies of this mutant are in progress. LVS was also treated with nitrosoguanidine, and the colonies screened for lack of iridescence. Twelve mutants were sub-cultured to Congo red agar, and one colony was as red as F. novicida and designated LMu1. Western blots of the LMu1 LPS showed that it was less reactive or non-reactive with LVS antiserum. Mice challenged with the LVS by intraperitoneal (IP) inoculation were uniformly susceptible to lethal infection with less than 200 colony forming units (CFU). However, mice challenged with over 400 CFU of LMu1 all remained healthy. Two weeks after the last inoculation the mice were injected IP with 417 CFU of LVS, and all mice remained healthy. Recent experiments indicated that LMu1 is susceptible to complement mediated killing by normal serum from rabbits and bovines, but not mice or dogs, whereas F. novicida and LVS are resistant. Using dog serum as a complement source, LMu1 was killed by canine PMNs, but not LVS. A
phenotypically similar chemical mutant has been made of a virulent type A clinical isolate. This mutant is also red on Congo red agar. Further investigation is in progress. Attempts to purify an extracellular caspsular polysaccharide by conventional procedures have not been productive. Therefore, a whole-cell extraction approach was taken. Colonies scraped off large agar plates were extracted with 68 deg. C 50 percent phenol, the entire extract dialyzed to remove the phenol, treated with DNase, RNase, and Proteinase K, and subjected to ultracentrifugation to remove LPS. The supernatant was concentrated several times and the retentate sent to The University of Georgia Complex Carbohydrate Research Center for analysis. A polysaccharide (PS) was identified that lacked KDO and quinovosamine, indicating there is little of no LPS in this material. A difference between the LPS of LMu1 and the published composition of LVS was also noted. The LMu1 LPS lacks quninovosamine and
N-acetylgalactosamine, which are present in the O antigen of LVS. These results suggest that the LMu1 LPS is lacking O antigen.
Impacts
At this time a vaccine is not licensed for the prevention of tularemia, and a rapid diagnostic test is not available. We expect that the capsule of F. tularensis is required for virulence, and that conjugation of the capsule to key antigens that induce the proper cellular immune response may be used as a protective vaccine. In addition, antibodies to the capsule may be used in various types of diagnostic tests for rapid diagnosis or identification of the presence of the agent. The isolation and characterization of the capsule and its genes are the first step in this process.
Publications
- Inzana, T.J., G. Glindemann, and G. Snider. 2004. Characterization of a wildtype strain of Francisella tularensis isolated from a cat and review of the literature. J. Vet. Diagn. Invest. 16:374-381.
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Progress 10/01/02 to 09/30/03
Outputs
We have determined that the capsule of Francisella tularensis is atypical in that, unlike most bacterial capsules, it cannot be isolated from culture supernatant by precipitation with 75% ethanol or cetavlon. Scanning electron microscopy revealed the cells of F. tularensis type A and the LVS (live vaccine strain), both of which are reported to be encapsulated, appeared very large with a coating of extracellular material that often appeared folded. However, cells of F. tularensis subspecies novicida, which is reported to be non-encapsulated, lacked any of this material. Following vigorous stirring in an Omni Mixer, the cells of F. novicida appeared unchanged, but the LVS and type A cells now appeared identical in form to that of F. novicida, suggesting that the extracellular material had been sheared off the cells. However, we have also determined that much of the lipopolysaccharide (LPS) is removed from the cells using this procedure, although the LPS appeared to be
removed by ultracentrifugation. The addition of cetavlon to the supernatant of cells stirred in an Omni Mixer now formed a precipitate that was much larger from the LVS and type A strains than from F. novicida. The semi-purified extracellular material has been submitted to the Complex Carbohydrate Research Center at the University of Georgia for chemical analysis. We have also identified a DNA region of the Schu4 genome that contains putative genes for polysaccharide biosynthesis and transport. We are in the process of mutating this region by allelic exchange in the LVS, and using chemical mutagenesis to isolate non-encapsulated mutants. In addition, we are using subtractive hybridization to identify genes in the LVS and Schu4 strain that are absent in F. novicida, in an attempt to further identify capsule biosynthesis genes and other genes required for virulence in the more pathogenic strains.
Impacts
At this time a vaccine is not licensed for the prevention of tularemia, and a rapid diagnostic test is not available. We expect that the capsule of F. tularensis is required for virulence, and that conjugation of the capsule to key antigens that induce the proper cellular immune response may be used as a protective vaccine. In addition, antibodies to the capsule may be used in various types of diagnostic tests for rapid diagnosis or identification of the presence of the agent.
Publications
- Inzana, T.J., G. Glindemann, and G. Snider. 2003. Characterization of a wildtype strain of Francisella tularensis isolated from a cat and review of the literature. J. Vet. Diagn. Invest. In press.
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Progress 10/01/01 to 09/30/02
Outputs
Francisella tularensis is the etiologic agent of tularemia, an important zoonotic pathogen that is prevalent in the wildlife population of the United States. Tularemia can be transmitted by arthropod vectors as well as water, and therefore infections occasionally occur in food animals, and feral animals. As few as 10 organisms can cause disease in humans and only 1 cell can cause death in mice. As a result, F. tularensis is categorized as a Class A bioterrorism agent by the Centers for Disease Control, and could also be used as an agent in agroterrorism. F. tularensis is reported to be encapsulated, however, the capsule has not been purified or characterized, although non-encapsulated mutants have been generated. Our goal is to purify the capsule and determine if antibodies to the capsule can provide any protection against infection or can be used in diagnostic tests. Although funding was just obtained, we have determined that the capsule is not precipitated by
cationic detergent, which does precipitate most negatively charged bacterial polysaccharide capsules. A gel-like material consistent with glycolipid was obtained from the supernatant of F. tularensis cells killed by formalin in the presence of 0.4 M NaCl and excess, cold ethanol. Further purification and characterization of this material is in progress.
Impacts
Francisella tularensis is classified as a Class A bioterrorism agent by the Centers for Disease Control, and The National Institutes of Health. There is an immediate need for improved rapid diagnostic tests and vaccines for this agent for both animals and humans. In addition, there is a need for basic information regarding the biology of the organism, which will be crucial to the development of products to diagnose, treat, and control this disease.
Publications
- No publications reported this period
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