Article

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

Center for Immunology and Microbial Disease, MC 151, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Journal of bacteriology (Impact Factor: 2.81). 09/2009; 191(20):6447-56. DOI: 10.1128/JB.00534-09
Source: PubMed

ABSTRACT

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated
reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide
dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide
radicals (O2−). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated
microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a
mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O2− or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type
F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ−/−, inos−/−, and phox−/− mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and
SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.

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Available from: Juan Andre Melendez
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    • "In IFN-γ-activated macrophages, sodC mutants were attenuated for macrophage growth and were found to be partially attenuated in a mouse i.n. infection model (Melillo et al., 2009). RipA (FTL1914), an IM protein, first was identified by screening a LVS transposon library for mutants that failed to replicate in alveolar epithelial cells (Fuller et al., 2008). "
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    ABSTRACT: Francisella tularensis is a highly-infectious bacterium that causes the rapid, and often lethal disease, tularemia. Many studies have been performed to identify and characterize the virulence factors that F. tularensis uses to infect a wide variety of hosts and host cell types, evade immune defenses, and induce severe disease and death. This review focuses on the virulence factors that are present in the F. tularensis envelope, including capsule, LPS, outer membrane, periplasm, inner membrane, secretion systems, and various molecules in each of aforementioned sub-compartments. Whereas no single bacterial molecule or molecular complex single-handedly controls F. tularensis virulence, we review here how diverse bacterial systems work in conjunction to subvert the immune system, attach to and invade host cells, alter phagosome/lysosome maturation pathways, replicate in host cells without being detected, inhibit apoptosis, and induce host cell death for bacterial release and infection of adjacent cells. Given that the F. tularensis envelope is the outermost layer of the bacterium, we highlight herein how many of these molecules directly interact with the host to promote infection and disease. These and future envelope studies are important to advance our collective understanding of F. tularensis virulence mechanisms and offer targets for future vaccine development efforts.
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    • "In contrast, katG mutants of Schu S4 were sensitive to hydrogen peroxide and reactive nitrogen species in vitro but retained lethality in mice, indicating that KatG is a sufficient but not necessary virulence factor.29 Similarly, the superoxide dismutases SodB and SodC are essential for bacterial resistance to superoxide radicals, as F. tularensis LVS mutants of either enzyme are highly attenuated in mice challenged intranasally.30,31 "
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    ABSTRACT: Francisella tularensis is an intracellular Gram-negative bacterium that causes life-threatening tularemia. Although the prevalence of natural infection is low, F. tularensis remains a tier I priority pathogen due to its extreme virulence and ease of aerosol dissemination. F. tularensis can infect a host through multiple routes, including the intradermal and respiratory routes. Respiratory infection can result from a very small inoculum (ten organisms or fewer) and is the most lethal form of infection. Following infection, F. tularensis employs strategies for immune evasion that delay the immune response, permitting systemic distribution and induction of sepsis. In this review we summarize the current knowledge of F. tularensis in an immunological context, with emphasis on the host response and bacterial evasion of that response.
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    • "This mechanism occurs over and above the activity of the superoxide dismutase that neutralises both reactive oxygen and nitrogen spp. [31] and is just one of many mechanisms employed by F. tularensis to manipulate and evade the host response [32]. "
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    ABSTRACT: Reactive nitrogen is critical for the clearance of Francisella tularensis infections. Here we assess the role of nitric oxide in control of intracellular infections in two murine macrophage cell lines of different provenance: the alveolar macrophage cell line, MH-S, and the widely used peritoneal macrophage cell line, J774A.1. Cells were infected with the highly virulent Schu S4 strain or with the avirulent live vaccine strain (LVS) with and without stimuli. Compared to MH-S cells, J774A.1 cells were unresponsive to stimulation and were able to control the intracellular replication of LVS bacteria, but not of Schu S4. In MH-S cells, Schu S4 demonstrated control over cellular NO production. Despite this, MH-S cells stimulated with LPS or LPS and IFN- γ were able to control intracellular Schu S4 numbers. However, only stimulation with LPS induced significant cellular NO production. Combined stimulation with LPS and IFN- γ produced a significant reduction in intracellular bacteria that occurred whether high levels of NO were produced or not, indicating that NO secretion is not the only defensive cellular mechanism operating in virulent Francisella infections. Understanding how F. tularensis interacts with host macrophages will help in the rational design of new and effective therapies.
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