Francisella tularensis

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A Microbial Biorealm page on the genus Francisella tularensis

Contents

Classification

Higher order taxa

Domain: Bacteria

Phylum: Proteobacteria

Class: Gamma Proteo Bacteria

Order: Thiotrichales

Family: Francisellaceae

Genus

Genus species: Francisella tularensis


NCBI: Taxonomy

Description and significance

Francisella tularensis is a gram-negative bacteria (Gram-negative bacteria contain an outer membrane outside the peptidoglycan cell wall, unlike Gram-positive bacteria that have a thicker layer of cell wall and no outer membrane. Many of the Gram-negative bacteria are pathogenic), with pili on the surface. It is nonmotile, aerobic, and non-spore forming bacteria. In nature, it can survive up to weeks at low temperatures in water, soil, and animal carcasses. In laboratory settings, Francisella tularensis appears as small rods (0.2 by 0.2 µm), and is grown best at 35-37 degrees Celcius. (2)

Francisella tularensis is a highly contagious bacteria that causes tularemia, or "rabbit fever" (It is called rabbit fever because rabbits are vectors for the disease) that is contagious to humans. There are four known subspecies of Francisella tularensis . There are two strains of Francisella tularensis that are studied the most: the more virulent Type A strain (found in North America), and the less virulent Type B (subspecies holarctica , also referred to as palearctica) strain (found in Europe). Two other subspecies are the non-virulent mediasiatica, found in central Asia, and novicida, which not much is known about.(2)

The bacteria was used in development of biological weapons in the World War II and post WWII years, and is considered a very dangerous biological terror threat today.(5) Francisella tularensis is listed as a "Category A select agent" by the United States government due to its high virulence and ease of spread(14), and if the disease is left untreated, the mortality rate can be as high as 30 to 60% of the cases.(5) Most research today regarding the bacteria is for the creation of a vaccine for tularemia.

Genome structure

Francisella tularensis has a circular chromosome, and its entire 1,898,476 nt long genome is sequenced. It has 52 RNA genes. It has a G+C content of 32% (G and C being guanine and cytosine), 79% of the genes are functional(3)

F. tularensis Type A's complete genome has been sequenced, and it was discovered that mutations disrupt several metabolic and synthetic pathways required for survival, indicating that F. tularensis has evolved to depend on host organisms for certain nutrients. Several virulence-associated genes were located in a pathogenicity island, and more than 10% of those contain deteriorating mutations, which explains why metabolic and synthetic pathways are being disrupted and why F. tularensis is host dependent.(7)

Type B of F. tularensis has also been sequenced, and it was discovered that the difference between the two types is the amount of genomic rearrangement. Most of the rearrangements are due to homologous recombination between ISFtu1 and ISFtu2, 2 insertion elements. In type A, many pseudogenes have been found in the genome while no rearrangements have been found in type B, making it a likely cause to the difference in virulence between the two strains.(12) Discovering the cause of the virulence of the different strains can lead to discoveries of vaccines that can effectively combat the bacteria F. tularensis

Other parts of the bacterial genome consists of fslA,B,C,and D in the F. tularensis genome which code for siderophores, which are important for pathogen survival within the host (see Cell structure and metabolism section for more information). F. tularensis also has a transcription factor, MglA, which helps express several genes that allow for replication in macrophages, aiding in its virulence.(14)

Cell structure and metabolism

Most of the Francisella bacteria are homogeneous in shape and size. They are covered by a capsule-like coat with well-defined borders. The virulent strains, like Francisella tularensis, have thick capsules while avirulent strains have thinner capsules. Some Francisella tularensis bacteria are able to produce protrustions on the outer membrane. The bacteria contains "Type 4 pili" on their surface, which is a type of pili used by gram-negative pathogenic bacteria to adhere to the host tissue, biofilm formation, DNA uptake and motility.(4,9) Francisella tularensis also contains siderophores which grow under iron-limiting conditions. Siderophores are small molecules that can bind to iron from inorganic and host sources and then siderophore-iron complex are bound by receptors on the bacterial membrane and taken in by the bacteria. This feature is important to the bacteria because intracellular replication of F. tularensis is iron-dependent, as shown with deferroxamine having inhibitory effects in a tissue during infection(10), and even virulence of the bacteria is iron dependent.(11)

High virulent strains of the bacteria contain AcpA, which is a repiratory, burst-inhibiting acid phosphatase inhibited by metal oxyanions orthovanadate, molybdate, and tungstate. AcpA inhibts the respiratory burst (which is release of chemicals by immune cells such as neutrophils and macrophages when they encounter bacteria)of encountered neutrophils, which suggests that AcpA is an important enzyme that helps F. tularensis avoid the host's immune system during infection.(8)

Ecology

In nature, Francisella tularensis are suspected to reside in protozoan cells like amoebas.(13), and Francisella tularensis affects its environment by infecting small mammals such as rabbit and rodents with the disease tularemia. The animals can acquire the disease through contact with fleas, flies, or contaminated soil, water, and vegetation (5) Francisella tularensis can infect humans through contact with infected animals or vectors such as fleas and mosquitoes. The disease can also be spread by human handling of animal or flesh infected with the disease. It can also be spread to humans by being in their water or food supply (It can survive for along time in animal carcasses). (5) However, the disease is not known to spread through human-human contact.

While more uncommon, it is also possible for airborne infection of tularemia to occur in nature. In 1966-67, an outbreak of Type B F. tularensis occurred in Sweden in a large farming area. The outbreak affected more than 600 patients, most of whom "acquired infection while doing farm work that created contaminated aerosols." Rodent-infested hay was also suspected as a source of the outbreak.(5)

Pathology

The F. tularensis is a highly contagious bacteria that can be spread from animals to humans, through vectors such as mosquitos and fleas, or from being breathed in from the air. The bacteria infects humans through skin, mucous membranes, lungs, and the gastrointestinal tract. It primarily infects macrophages of the host organism after it is ingested by phagocytosis. F. tularensis multiply inside the macrophage and later break out the macrophage and invades other cells. The major target organs are lymph nodes, lungs, liver, and kidneys. People infected with tularemia through inhalation also develop hemorrhagic inflammation of the airways early in the disease, and it might develop into bronchopneumonia. No proven vaccine has been created for tularemia, and the general treatment for the disease is antibiotics.(1,5,6)

F. tularensis is very infectious because it is capable of causing a "debilitating or fatal disease with doses as low as 10 colony-forming units".(6) Some symptoms of tularemia are fever, ulcers, dyspnea, and others (depending on what part of the body is affected). Here are some different types of tularemia:

Ulceroglandular-the most common type of tularemia, usually caused by a insect bite. At the site there is a skin sore, which becomes an ulcer. Glands in the area of the ulcer will swell. The ulcer is accompanied by fever, chills, headaches, and fatigue.

Glandular- There is no obvious ulcer, and symptoms are fever and swollen glands.

Typhoidal- Usually caused by breathing in the bacteria, but it can also be caused by insect bite and contaminated water and food. Symptoms are fever, weight loss, fatigue, and usually pneumonia. (2)

Application to Biotechnology

Francisella tularensis was researched and used as a biological weapon during World War II and two decades afterwards. During World War II, Japan conducted research on the bacteria for its potential as a biological weapon. Francisella tularensis has the potential to be a biological weapon because it is very infectious (a small number, about 10-50 organisms, can cause disease)(7). As a biological weapon it is most effective when spread airborne, and the United States developed weapons that can deliver aerosol Francisella tularensis in the 1950s-60s. The Soviet Union, along with using Francisella tularensis as a weapon, also developed antibiotic and vaccine resistant strains against the bacteria. (1,5,6)

The effects of the biological weapon are severe respiratory illness including pneumonia and systemic infection that if not treated, can result in death. To put into perspective how powerful a biological weapon F. tularensis can be, the WHO in 1969 estimated that "an aerosol dispersal of 50 kg of virulent F. tularensis over a metropolitan area with 5 million inhabitants in a developed country would result in 250,000 illnesses, including 19,000 deaths."(2)

Current Research

1. This research deals with the genetic manipulation of Francisella tularensis . This bacteria is a "category A bioterror pathogen" that can cause fatal human infection, however, very few virulence factors are known in this species due to lack of tools for genetic manipulation and difficulty working with it. In this study, a vector that can replicate in E. coli and F. tularensis is created that allows the research of the bacteria by identifying the promoters that are activated during intracellular growth and survival of the bacteria. (Current Research 1)

2. This research investigated the virulence of different strains of F. tularensis on chicken embryos. The experiments are conducted by infecting 7 day old chicken embryos with different strains (a wild strain and a live vaccine strain). The wild strain is determined to be more virulent. Later, more mutant strains of the bacteria are tested, and it is determined that they all have a wide range of virulence in chicken embryos. (Current Research 2)

3. This research attempts to identify F. tularensis mutants that can be useful in the developmental of a vaccine for tularemia in humans and other animals. F novicida transposon mutants are screened to identify mutants that showed reduced growth in mouse microphages. Results yielded 16 F. novicida mutants that were "100-fold more attenuated for virulence in a mouse model than the wild-type parental strain". The mutants are then tested for their ability to protect the mice against high doses of wild type strains. 5 of the 16 mutants exhibit protection for the mice. This is believed to be valuable in designing a vaccine against tularemia. (Current Research 3)

4. Outer membrane proteins of Type A(Schu S4) and Type B(LVB) are separated from the cell by spheroplasting (using antibiotics to remove the cell wall). Sucrose gradients used to separate the outer membrane shows that F. tularensis's outer membrane usually migrates in sucrose gradients between "densities of 1.17 and 1.20 g/ml", differing from typical gram-negative bacteria that migrate at 1.21 to 1.24 g/ml. Identities of immunogenic proteins on the outer membrane are determined by separation using gel electrophoresis and mass spectrometry analysis. This study is to gain a deeper understanding of F. tularensis structure and more information to develop useful vaccine and antibiotics against the bacteria. (Current Research 4)

References

1.National Center for Disease Control and Prevention: Tularemia http://www.cdc.gov/ncidod/dvbid/tularemia.htm

2.Tularemia: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, treatment, and prophylaxis http://www.cidrap.umn.edu/cidrap/content/bt/tularemia/biofacts/tularemiafactsheet.html

3. Francisella tularensis subsp. tularensis WY96-3418, complete genome http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome&cmd=Retrieve&dopt=Overview&list_uids=20753

4. Gil, H., J. L. Benach, and D. G. Thanassi. 2004. Presence of pili on the surface of Francisella tularensis. Infect. Immun. 72:3042-3047. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&rendertype=abstract&artid=387894

5. Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. JAMA 285:2763-2773 http://jama.ama-assn.org/cgi/content/full/285/21/2763

6. Oyston P, Sjostedt A, Titball R (2004). "Tularaemia: bioterrorism defence renews interest in Francisella tularensis.". Nat Rev Microbiol 2 (12): 967-78. PMID 15550942

7. Larsson P, Oyston PC, Chain P, Chu MC, Duffield M, Fuxelius HH, Garcia E, Halltorp G, Johansson D, Isherwood KE, Karp PD, Larsson E, Liu Y, Michell S, Prior J, Prior R, Malfatti S, Sjostedt A, Svensson K, Thompson N, Vergez L, Wagg JK, Wren BW, Lindler LE, Andersson SG, Forsman M, Titball RW. The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet. 2005 Feb;37(2):153-9. Epub 2005 Jan 9. PMID: 15640799 http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15640799

8. Felts RL, Reilly TJ, Tanner JJ. Structure of Francisella tularensis AcpA: PROTOTYPE OF A UNIQUE SUPERFAMILY OF ACID PHOSPHATASES AND PHOSPHOLIPASES C J. Biol. Chem. 2006;281:30289-30298 http://www.jbc.org/cgi/content/full/281/40/30289

9. Gerasimov,Dolotov, Stepanov, Urakov, Morphology, ultrastructure and populational features of bacteria francisella, Vestn Ross Akad Med Nauk. 1997;(6):24-30 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=9289274&dopt=Abstract

10. Fortier, A. H., D. A. Leiby, R. B. Narayanan, E. Asafoadjei, R. M. Crawford, C. A. Nacy, and M. S. Meltzer. 1995. Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth. Infect. Immun. 63:1478-1483 http://iai.asm.org/cgi/content/abstract/63/4/1478?ijkey=47812778fa42e9fbc61673435f5994fc98fa1c3e&keytype2=tf_ipsecsha

11. Sullivan,Jeffery,Shannon,Ramakrishnan. Characterization of the Siderophore of Francisella tularensis and Role of fslA in Siderophore Production, Journal of Bacteriology, June 2006, p. 3785-3795, Vol. 188, No. 11 http://jb.asm.org/cgi/content/full/188/11/3785

12. J. Bacteriol, Chromosome Rearrangement and Diversification of Francisella tularensis Revealed by the Type B (OSU18) Genome Sequence. Petrosino et al.2006;188:6977-6985. http://jb.asm.org/cgi/content/full/188/19/6977

13. Abd H, Johansson T, Golovliov I, et al. Survival and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol 2003 Jan;69(1):600-6 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12514047

14. Brotcke A, Weiss DS, Kim CC, et al. Identification of MglA-regulated genes reveals novel virulence factors in F tularensis. Infect Immun 2006 Dec;74(12):6642-55 http://iai.asm.org/cgi/content/abstract/IAI.01250-06v1

Current Researches

1. Rasko DA, Esteban CD, Sperandio V, Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids. 2007 http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17459476&itool=iconabstr&query_hl=1&itool=pubmed_docsum

2. Eli B. Nix,1 Karen K. M. Cheung,1 Diana Wang,2 Na Zhang,1 Robert D. Burke,1,2 and Francis E. Nano Virulence of Francisella spp. in Chicken Embryos, 2006 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1539577

3. Rebecca Tempel,†* Xin-He Lai,† Lidia Crosa, Briana Kozlowicz, and Fred Heffron. Attenuated Francisella novicida Transposon Mutants Protect Mice against Wild-Type Challenge, 2006 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&rendertype=abstract&artid=1594869

4. Jason F. Huntley, Patrick G. Conley, Kayla E. Hagman, and Michael V. Norgard. Characterization of Francisella tularensis Outer Membrane Proteins, 2006 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1797401

Edited by Dan Su of Rachel Larsen and Kit Pogliano

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