Francisella tularensis, a pathogenic gram-negative bacteria and the only bacteria recognized under the genus Francisellaceae of the gammaproteobacteria family, was isolated by George Walter McCoy from ground squirrels in 1912. There are four subspecies of F. tularensis: biovar tularensis (type A), biovar holartica (type B), subspecies novicida, and biovar mediasiatica. Type A, most commonly found within North America, is the most virulent subspecies and includes the fully sequenced laboratory strain, SCH4U. Type B is most commonly found within Europe and Asia but is rarely linked to fatal disease while subspecies novicida and biovar mediasiatica, found within North America and central Asia respectively, are non-virulent strains in human hosts, however, little is known about the latter. F. tularensis is non-motile , aerobic and rod shaped with an approximate size of 0.2 µm.
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Despite F. tularensis having been categorized as a Class A Select Agent by the U.S. government as a prospective instrument of bioterrorism, human-to-human transmission of the bacteria has not been observed. F. tularensis is most commonly spread through vectors such as ticks or deer flies, with aquatic rodents, deer and lagomorphs being common reservoir hosts. F. tularensis has the ability to infect a host via skin contact and inhalation, leading to ulceroglandular forms of tularemia and pneumonic tularemia. Without treatment, pneumonic tularemia has been shown to be fatal (mortality rate of 30-60% if left untreated), causing symptoms such as fever, anorexia, and septicemia within approximately three to four days after infection. Although human-to-human transmission has not been identified, F. tularensis’ ability to infect human hosts is not hindered. F. tularensis requires a low infectious dose (approximately 10-50 bacteria), contributing to its high virulence. Being an intracellular bacterium requiring cysteine for growth can be limiting, however F. tularensis is capable of surviving outside of a host for weeks at a time. This characteristic leads to its easy spread when one comes in contact with water and grasslands, particularly during activities like brushcutting or lawn mowing in which the carcasses of dead reservoir animals may be present within the environment, leading to what is commonly known as “lawnmower disease” or “rabbit disease”. Tularemia is endemic in North America, Europe and Asia with 5-10% of cases being waterborne infection opposed to the most common method, via contact with reservoir animals. At risk groups include those who are immunosuppressed.
The life cycle of this intracellular pathogen is important in determining which aspects are of main focus when dealing with potential virulence factors. There are approximately five steps of this pathogens life cycle: entry (associated with type IV pili), phagosomal escape, cytoplasmic multiplication (associated with biofilms and ppGpp), lysis and release (associated with MglA). The entry of this pathogen is dependent on complement factor C3, mannose receptors and cell surface-expressed nucleolin (encoded by the ncl gene). Within the cell, the bacterium colocalizes within arrested, late endosomal phagosomes. Within 15-30 minutes, the phagosomes are destroyed and the bacteria escape into the cytoplasm of the host cell. After multiplication, two distinct mechanisms for host cell lysis occur: Type 1-induced apoptosis and caspase 1-mediated pyropoptosis (in which immune cells that recognize signals of infection within themselves, go through programmed cell death via production of cytokines). The pathogens response to innate immunity within the host cell also plays a role in it’s virulence as evading innate responses (such as production of cytokines) is crucial when infecting host cells, macrophages in particular. Being able to detect environmental changes through use of two-component systems help the pathogen survive in different conditions within or outside of host cells, which can have a huge impact on its virulence as well.
Biofilms also play a significant role in the virulence of F. tularensis. Biofilms are a community of microbes attached to a surface, encased in a matrix. Biofilms help provide protection from antimicrobial agents as well as protection from the immune system of the host. Biofilms play a role in chronic infections as they protect the bacteria which are most resistant to antibiotics or antimicrobials that may be administered to the host. In the case of F. tularensis, biofilm formation is increased when the relA gene is inactivated. Production of hyperphosphorylated guanosine diphosphate and triphosphate analogs or (p)ppGpp, is used to combat limited nutrient conditions. Uncharged tRNA molecules bind the ribosome resulting in stalling of translation and activation of ribosome associated RelA. This causes the production of (p)ppGpp which is then converted to ppGpp. ppGpp molecules bind RNA polymerase to alter gene expression under certain conditions. Inactivation of the relA gene causes defective production of (p)ppGpp which leads to increased biofilm formation and increased resistance to stress. These biofilms make it hard to treat infections resulting from this pathogen as eliminating the pathogen from the body becomes more difficult with increased production of biofilms and increased resistance.
The ability for this pathogen to survive without a host is due, in part, to its ability to adapt to different environments by activation and repression of genes, some of which are found within what are known as pathogenicity islands. In the case of F. tularensis, the pathogenicity island FP1, contains 17 highly controlled genes that are crucial to its survival. In the laboratory strain of Type A alone, the expression of approximately 658 genes are up regulated and/or down regulated during infection within macrophages. The change in expression of these genes has helped researchers understand F. tularensis’ reactions to specific environmental stimuli such as temperature, limited iron source and oxidative stress. In terms of temperature as an environmental stimuli, F. tularensis is able is to alter its outer surface when growing in temperatures of 25°C as opposed to 37°C in a human host, by modifying the lipid A of lipopolysaccharide (LPS). This pathogen is capable of temperature changes through involvement of alternative sigma factors as well as heat-shock proteins. In F. tularensis, the only alternative sigma factor that is encoded is RpoH. In terms of iron acquisition, intracellular pathogens require the ability to acquire iron because of the limited availability within mammalian host cells, as intracellular replication is iron-dependent. F. tularensis contains siderophores. These small molecules grow under iron-limiting conditions and bind iron from inorganic and host sources. Studies show that iron-starved F. tularensis expresses an additional 80 genes, including some within FP1, and that F. novidica growth in broth and macrophages is inhibited as well as it virulence in regards to causing pneumonic tularemia in mouse models. In addition to temperature and iron acquisition, the detection of oxidative stress is important as oxidative stress is hugely involved in innate antimicrobial responses by macrophages in the host. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have the ability to produce superoxide and hydrogen peroxide molecules which cause damage to biological molecules within bacterial cells, such as DNA, as well as important enzymes which lead to metabolic defects. F. tularensis has the ability to inactivate these reactive species by inactivation of the phagocyte NADPH oxidase which is found within the phagolysosome and is required to reduce oxygen to superoxide anions.
Environmental stimulus is important when studying the virulence of this pathogen as it is directly related to regulation of virulence gene expression. In fact, the gene MglA (or macrophage growth locus) has been linked with responses to oxidative stress. MglA in particular, is required for replication within macrophages. MglA is highly up-regulated during infection and mutant strains are unable to escape macrophage phagosomes. MglA binds with SspA (a transcription factor that responds to nutrient limitation) as well as RNA polymerase in a heterodimer which is required for FP1 gene activation. In order for the pathogen to detect these environmental stimuli, however, two component regulatory systems composed of membrane-bound sensor kinases and cytoplasmic response regulators are essential. The F. tularensis genome codes for two of these systems, one of which is of major importance and contains a response regulator that resembles PmrA of E. coli bacteria, involved in LPS modification. Inactivation of this gene increases susceptibility to killing through antimicrobial actions, decreased growth and inability to escape macrophages. PmrA is described as a DNA binding protein that allows for binding of the MglA and SspA complex bound to RNA polymerase to initiate FP1 gene transcription.
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Aside from gene expression relating to virulence, genes within F. tularensis’ genome have also been shown to produce structures that aid with its life cycle and its virulence. One structure in particular is type IV pili. Multifunctional and flexible, these appendages are capable of adhesion, motility, biofilm formation and conjugation, all of which are important aspects of colonization of pathogens. In human strains of the virus, type IV pili have been shown to be essential for virulence, specifically in type A. In type B strains (which have relatively low virulence), however, pseudogenes of genes encoding type IV pili have been found which further support the idea that virulence is somehow connected to type IV pili. In the case of F. tularensis, type IV pili are essential for the binding of the bacterial cell to host cells to allow phagocytosis to occur. Mutant strains lacking the genes responsible for the production of pili (mainly pilA, pilB, pilC, pilD, pilT, and pilQ) are considerably attenuated in pathogenicity.
Another structural virulence determinant is the ability of Francisella tularensis to suppress and avoid early innate immune responses (which slows progression of infection and allows for adaptive immunity to develop) by modifying its LPS, as mentioned previously in regards to PmrA and temperature as an environmental stimulus. Through removal of Kdo (3-deoxy-D-manno-octulosonic acid) saccharide, F. tularensis is able to kill the host before adaptive immunity matures. Mutant strains with the inability to modify its LPS are shown to be attenuated in mice models, inducing an early innate immune response. The O-antigen present within the LPS of F. tularensis is also important for multiplication. O-antigen is a repetitive glycan polymer, composing the outermost domain of the LPS. Mutant strains lacking O-antigen show susceptibility to killing by serum. F. tularensis also produces AcpA (burst-inhibiting acid phosphatase) which inhibits respiratory bursts (release of neutrophils by macrophages when they encounter bacteria) in order to help evade host immune system responses.
Though the genome of F. tularensis has been sequenced, not many genes have been shown to be identical or even similar to those currently within bioinformatics databases. This makes it challenging to determine what genes are responsible for what functions. As described above, the major factors that determine F. tularensis’ virulence are mainly two-component systems which detect environmental stimuli, helping the bacteria adapt to environmental change (including temperature, iron limitation and oxidative stress), which leads to expression of important regulatory genes such as MgIA, PmrA, and relA, all of which are associated with specific stages of its life cycle. MglA is of major importance because of its role in initiating transcription of another virulence factor, the FP1 pathogenicity island. Biofilms, LPS modification and type IV pili also influence virulence through their ability to aid in multiplication, resistance, and evasion of innate immunity as well as entry into host cells. Even though the functions of many genes within the genome of F. tularensis are unknown, this pathogen is still very important to the world of biotechnology because of its ability to be used as a biological weapon. This is due to several characteristics of F. tularensis including being: easy to aerosolize, highly infective (requiring only a small dose of 10-50 bacteria for infection) highly incapacitating to infected hosts (with a relatively high mortality rate if its associated disease is left untreated). WHO estimated, in 1969, that 50kg of aerosolized virulent F. tularensis could result in 250,000 illnesses and 19,000 deaths if dispersed over a population of approximately 5 million people. This has led to production of a live vaccine as well as an attenuated; however the live vaccine has not yet been approved within the United States and the attenuated vaccine is only available in special cases. Disease associated with this pathogen is currently treated with antibiotics, the drug choice being streptomycin or tetracycline-class drugs. The best way to prevent an infection by F. tularensis is through proper protection when skinning wild animals, particularly lagomorphs (rabbits), avoiding ingestion of uncooked reservoir animals and untreated water sources in which these animals inhabit as well as wearing repellent to prevent tick bites.
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