That cute bunny on Skinner Green? You might want to avoid Peter Rabbit the next time you see him. Rabbits and hares are notorious vectors of the extremely infectious and fatal human and zoonotic disease tularemia caused by the Francisella tularensis bacterium. F. tularensis is a gram negative, nonmotile, non-sporulating rod-shaped intracellular bacteria that thrives in contaminated bodies of water, soil or animal carcasses. Primary modes of infection are insect vectors (ticks, deer flies or mosquitos) and airborne particles that invade mucous membranes, skin, lungs or GI tract through insect bites, consumption of contaminated water or flesh, skin contact with infected animals or inhalation of contaminated aerosols. Tularemia type often depends on infection route, but ulceroglandular tularemia resulting from animal handling or infected bites is most common; manifesting as ulcer formation and lymph gland swelling that if left untreated can spread to other organ systems and worsen patient condition.
Due to the possibility of infection by inhaling contaminated dust or aerosols and contracting the most fatal type of tularemia, pneumonic tularemia, the extremely virulent F. tularensis is classified as a Tier 1 Category A select agent and potential bioterror agent by the Center for Disease Control (CDC). Tier 1 select agents are biological agents and toxins that are considered to have the potential to pose a severe threat to public health and safety. During World War II, the potential of using F. tularensis as a bioweapon was studied by Japan as well as the U.S. and the Soviet Union. It was one of the bioweapons stockpiled by the U.S in the late 1960s and destroyed by 1973. The Soviet Union on the other hand continued to produce antibiotic- and vaccine-resistant strains of the bacterium until the early 1990s.
Based on a model built by a World Health Organization expert committee, the CDC estimates a cost of $5.4 billion for every 100,000 people exposed, in the event of a F. tularensis aerosol attack. A live vaccine strain (LVS) has been derived, using pathogen modification to prevent disease onset but still produce a strong immune response, and is used as a vaccine against tularemia in several countries. However the adverse effects in immunized people and the insufficient protection against pneumonic tularemia have prevented the licensure of the LVS in the U.S. The LVS is used as a surrogate to study the pathogenesis of tularemia, as it has an attenuated virulence in humans which decreases the likelihood of fatal infection.
Based on a model built by a World Health Organization expert committee, the CDC estimates a cost of $5.4 billion for every 100,000 people exposed, in the event of a F. tularensis aerosol attack. A live vaccine strain (LVS) has been derived, using pathogen modification to prevent disease onset but still produce a strong immune response, and is used as a vaccine against tularemia in several countries. However the adverse effects in immunized people and the insufficient protection against pneumonic tularemia have prevented the licensure of the LVS in the U.S. The LVS is used as a surrogate to study the pathogenesis of tularemia, as it has an attenuated virulence in humans which decreases the likelihood of fatal infection.
Figure 2. F. tularensis disease vectors and two possible pathological outcomes, credit: Monique Barel and Alain Charbit, Francisella tularensis: Causative Agent of Tularemia and Biothreat Agent Volume II Springer
F. tularensis bacteria enter the body, multiply around the infection site and then trigger ulceration and necrosis locally before spreading to major target areas such as liver, lungs, kidneys, spleen, lymph nodes or serosal membranes. Their primary cell targets are growing macrophages, though they will attack other cell types, whom they will infect after being phagocytized. The most challenging part of combating F. tularensis is that it lacks the normal toxins and bacterial virulence factors that epidemiologists and macrophages typically use to disable it. The outer membrane (OM) component is one section of the bacterial multidrug efflux pump system. This system is responsible for contiguous secretion of bacterial products and plays a role in multidrug resistance. One of the three OM proteins, SilC, is similar to the silver cation efflux protein in other pathogenic bacteria and is thought to play a role in resisting oxidative stress. Oxidative stress is an immune response to a foreign body where phagocytes release reactive oxygen species, triggering respiratory burst which causes cell implosion and negative effects on protein structure/activity. Oxidative stress from the host can lead to cell death and in order for F. tularensis to be most effective, it must resist oxidative stress for as long as possible using its OM proteins like SilC. In “Characterization of a Unique Outer Membrane Protein Required for Oxidative Stress Resistance and Virulence of Francisella tularensis'' by Alqahtani et al., they examined the role of SilC in the virulence of the bacterium through the use of a ΔsilC deletion mutant and transcomplemented strain partner.
Since the other two OM components, TolC and FtlC have been shown to be necessary for resistance against detergents, dyes and antibiotics, Alqahtani et al. first tested the wild type F. tularensis LVS, the ΔsilC deletion mutant and the transcomplemented strain against different dyes, detergents, and antibiotics using disc diffusion assays and bacterial killing assays. But although the ΔsilC mutant showed slightly increased sensitivity to some antibiotics like streptomycin (used to treat many bacterial infections like tuberculosis), it showed no increased sensitivity to any detergents or dyes. This means that SilC is required for the efflux of some antibiotics but not the efflux of detergents and dyes.
Next they investigated whether the ΔsilC mutant exhibited an oxidant-sensitive phenotype by measuring the sensitivity of the ΔsilC mutant against superoxide-generating compounds (paraquat and pyrogallol) and peroxides (cumene hydroperoxide, hydrogen peroxide and tert-butyl hydroperoxide) through the same disc diffusion (Figure 3A and 3B) and bacterial killing assays (Figure 3C and 3D) as previously described. The ΔsilC mutant had a significant zone of inhibition compared to the LVS strain and transcomplement strain seen in sections A and B of Figure 3 for both compounds. The larger the zone of inhibition, the greater the effect the inhibitor is exerting on the strain present. This demonstrates the role of SilC in conferring resistance to such oxidants since their zones are significantly smaller/less restricted compared to the mutant. Greater decline in bacterial killing assays, sections C and D of Figure 3, in response to increasing concentrations of both compounds shows that SilC is able to resist such oxidants under normal conditions, but less so in the ΔsilC mutant.
The ΔsilC deletion caused an increased susceptibility to silver compounds, specifically silver nitrate, which supported the idea that the multidrug efflux system is required for silver removal in F. tularensis. SilC, the homolog to a typical silver cation efflux protein, confers resistance to silver nanoparticles which are becoming more oftenly used in virucides and bactericides. Being able to effectively remove silver from the bacterium would allow it to sidestep these potential threats.
Since the other two OM components, TolC and FtlC have been shown to be necessary for resistance against detergents, dyes and antibiotics, Alqahtani et al. first tested the wild type F. tularensis LVS, the ΔsilC deletion mutant and the transcomplemented strain against different dyes, detergents, and antibiotics using disc diffusion assays and bacterial killing assays. But although the ΔsilC mutant showed slightly increased sensitivity to some antibiotics like streptomycin (used to treat many bacterial infections like tuberculosis), it showed no increased sensitivity to any detergents or dyes. This means that SilC is required for the efflux of some antibiotics but not the efflux of detergents and dyes.
Next they investigated whether the ΔsilC mutant exhibited an oxidant-sensitive phenotype by measuring the sensitivity of the ΔsilC mutant against superoxide-generating compounds (paraquat and pyrogallol) and peroxides (cumene hydroperoxide, hydrogen peroxide and tert-butyl hydroperoxide) through the same disc diffusion (Figure 3A and 3B) and bacterial killing assays (Figure 3C and 3D) as previously described. The ΔsilC mutant had a significant zone of inhibition compared to the LVS strain and transcomplement strain seen in sections A and B of Figure 3 for both compounds. The larger the zone of inhibition, the greater the effect the inhibitor is exerting on the strain present. This demonstrates the role of SilC in conferring resistance to such oxidants since their zones are significantly smaller/less restricted compared to the mutant. Greater decline in bacterial killing assays, sections C and D of Figure 3, in response to increasing concentrations of both compounds shows that SilC is able to resist such oxidants under normal conditions, but less so in the ΔsilC mutant.
The ΔsilC deletion caused an increased susceptibility to silver compounds, specifically silver nitrate, which supported the idea that the multidrug efflux system is required for silver removal in F. tularensis. SilC, the homolog to a typical silver cation efflux protein, confers resistance to silver nanoparticles which are becoming more oftenly used in virucides and bactericides. Being able to effectively remove silver from the bacterium would allow it to sidestep these potential threats.
Figure 3. The ΔsilC mutant of F. tularensis LVS exhibits enhanced sensitivity to superoxide-generating compounds. The sensitivities of the wild-type F. tularensis (Ft) LVS, the ΔsilC mutant, and the transcomplemented strain ΔsilCpsilC were determined by disc diffusion and bacterial killing assays against superoxide-generating compounds paraquat (A and C) and pyrogallol (B and D) using the protocols and concentrations described in Materials and Methods. For disc diffusion assays, the results are expressed as the zone of inhibition diameters in millimeters in means standard deviations (SD) for triplicate samples. In the bacterial killing assay, the Francisella strains were exposed to serially diluted paraquat and pyrogallol for 1 h and spotted onto MH chocolate agar plates to determine the bacterial killing. The arrows in panels C and D indicate the concentrations of paraquat and pyrogallol that resulted in enhanced killing of the ΔsilC mutant. The results shown are representative of 3 independent experiments, which yielded identical results. The P values were determined by one-way ANOVA, and a P value of 0.05 is considered statistically significant. *, P 0.05; **, P 0.01.
In order to investigate how SilC facilitates F. tularensis intramacrophage growth and survival, Alqahtani et al. infected RAW 264.7 macrophages with wild-type F. tularensis LVS, ΔsilC deletion mutant and its transcomplement at a 100 multiplicity of infection. Equal numbers of all three strains were found in the murine macrophages at 4h but the ΔsilC deletion mutant did not replicate within the macrophage and remained about the same at 24h compared to the 10-15x increase at 24h post infection for the wild-type LVS strain and transcomplement. Without SilC, the bacterium is unable to rapidly multiply at its normal pathogenic rate within macrophages.
SilC may also be able to contribute to extreme virulence by beating oxidative stress. Wild-type C57BL/6 and NADPH-oxidase deficient phox-/- mice were infected with ranging amounts of 100% lethal doses (number of bacteria that results in lethality in 100% of hosts) of the F. tularensis ΔsilC mutant via an intranasal pathway. All mice became infected within 11 days of introduction and all mice receiving the smallest portion of the dosage, 1 lethal dose, were able to survive infection with other doses producing varying lethality. Wild-type C57BL/6 mice more readily survived than NADPH-oxidase deficient phox-/- mice since the presence of NADPH oxidase is related to oxidative stress that was able to defeat the F. tularensis bacteria before it could inflict any real damage. In comparison, the NADPH-oxidase deficient phox-/- mice died at an increased rate because there was nothing (like oxidative stress) inhibiting the free range effects of F. tularensis and it was more readily able to infect and kill the host.
While individually these features of SilC may seem random and unimportant, they amount to a great deal for the survival and virulence of F. tularensis. Alqahtani et al. found that SilC isn’t required for detergent or dye removal but is required for a small handful of antibiotics like streptomycin or nalidixic acid. SilC confers resistance to superoxide-generating compounds like paraquat and pyrogallol, organic peroxides and silver that are most commonly used as bactericidal products. SilC contributes to oxidative stress resistance through its role in the Emr multidrug efflux pump system. SilC also is involved in intramacrophage growth and the bacteria’s extremely virulent nature. Learning more about tularemia pathogenesis and identifying specific required/ integral components of F. tularensis allows scientists to expose potential targets for therapeutic treatments, improved vaccine strains and bioterrorism protection. Oh and one step closer to befriending Mopsy, Flopsy and Cottontail.
SilC may also be able to contribute to extreme virulence by beating oxidative stress. Wild-type C57BL/6 and NADPH-oxidase deficient phox-/- mice were infected with ranging amounts of 100% lethal doses (number of bacteria that results in lethality in 100% of hosts) of the F. tularensis ΔsilC mutant via an intranasal pathway. All mice became infected within 11 days of introduction and all mice receiving the smallest portion of the dosage, 1 lethal dose, were able to survive infection with other doses producing varying lethality. Wild-type C57BL/6 mice more readily survived than NADPH-oxidase deficient phox-/- mice since the presence of NADPH oxidase is related to oxidative stress that was able to defeat the F. tularensis bacteria before it could inflict any real damage. In comparison, the NADPH-oxidase deficient phox-/- mice died at an increased rate because there was nothing (like oxidative stress) inhibiting the free range effects of F. tularensis and it was more readily able to infect and kill the host.
While individually these features of SilC may seem random and unimportant, they amount to a great deal for the survival and virulence of F. tularensis. Alqahtani et al. found that SilC isn’t required for detergent or dye removal but is required for a small handful of antibiotics like streptomycin or nalidixic acid. SilC confers resistance to superoxide-generating compounds like paraquat and pyrogallol, organic peroxides and silver that are most commonly used as bactericidal products. SilC contributes to oxidative stress resistance through its role in the Emr multidrug efflux pump system. SilC also is involved in intramacrophage growth and the bacteria’s extremely virulent nature. Learning more about tularemia pathogenesis and identifying specific required/ integral components of F. tularensis allows scientists to expose potential targets for therapeutic treatments, improved vaccine strains and bioterrorism protection. Oh and one step closer to befriending Mopsy, Flopsy and Cottontail.
About the Authors:
Peyton Kim-LaTona ‘21 is a Biological Sciences and Architectural Studies double major from Los Angeles, California. She works in the Fimbel Maker and Innovation Lab as a Makerspace Consultant and in Woodard Lab studying glial cells in Drosophila melanogaster during metamorphosis. After Mount Holyoke, she hopes to work as a biomedical research tech before pursuing an MD/PhD looking at zoonotic origins of infectious disease. She enjoys riding competitively for the Intercollegiate Horse Show Association, baking pastries, ocean kayaking and reading Stephen King novels.
Laraiba Seibou’21 is a Biochemistry major with a Five College African Studies certificate from Niamey, Niger. Outside of academics, she swims for the Mount Holyoke varsity swim team and enjoys cooking, knitting and sleeping. After graduation she plans on taking a gap year to gain some research experience. In the future, she hopes to work in biomedical research specializing in infectious disease in West Africa.
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