1. Host and aquatic environment dependent cost and benefits of the Shigella flexneri virulence plasmid and Shiga toxin production.
Dr. Thomas Platt, College of Arts & Sciences
Many emerging infectious diseases maintain significant populations outside of host environments. The consequences of dynamics occurring in these environmental reservoirs on the pathogenesis of these pathogens is seldom investigated and poorly characterized. This project examines how the molecular systems that facultative pathogens use to exploit hosts are shaped by the consequences of dynamics occurring within both host and environmental reservoir environments. Specifically, the researchers aim to test the hypothesis that context dependent costs and benefits result in selective dynamics within aquatic environments that lead to loss of Shigella flexneri virulence plasmid encoded virulence factors while promoting the maintenance of chromosomally encoded Shiga toxin production. The researchers will use a combination of competition experiments to measure the costs and benefits of pINV virulence plasmid carriage and virulence regulon expression in both host and aquatic environments. Further the researchers will determine how Shiga toxin expression in host and aquatic environments influences the fitness of pathogenic Shigella. Finally, the researchers will test predictions with respect to the evolutionary dynamics of both pINV encoded and Shiga toxin virulence factors using an experimental evolution approach. The researchers predict that increased duration of bouts of selection within aquatic reservoirs will result in the spread of non-invasive Shigella while the presence of Tetrahymena thermophila predation in these reservoirs will result in increased Shiga toxin production. The proposed work will determine the context dependent fitness consequences of Shigella virulence factors and determine whether selection in environmental reservoirs may contribute to the recent emergence of novel Shiga toxin producing Shigella pathogens.
2. Mechanisms of effector-mediated host defense using Legionella.
Dr. Stephanie Shames, College of Arts & Sciences
Emerging and zoonotic infectious diseases (EZIDs) cause global health devastation and are increasing in prevalence due to human activity and other factors that facilitate disease spread. Often, the etiological agents of EZIDs are difficult to study due to lack of established tools and genetic systems to perform hypothesis-driven research. The objective of this proposal is to use bacteria of the genus Legionella as model pathogens to characterize host defense mechanisms applicable to EZIDs. Legionella species are natural pathogens of protozoa and accidental human pathogens that can cause disease upon inhalation of contaminated aerosols and subsequent bacterial replication within alveolar macrophages. As transmission between mammals is rare, Legionella has not acquired sophisticated immune evasion mechanisms, and are, therefore, excellent model pathogens to reveal host immune defense mechanisms. Legionella replication within phagocytic cells is facilitated by a Dot/Icm type IV secretion system (T4SS), which translocates a repertoire of bacterial proteins called effectors into infected host cells. Although effector translocation is required for intracellular replication, effector functions can also impair Legionella fitness in mammals. The overall objective is to elucidate mechanisms by which effector function contributes to pathogen clearance by the innate immune system and determine whether these can be used to enhance defense against other pathogens. The central hypothesis is that LegC4 interacts with host factors to enhance cytokine-mediated host defense. To test the central hypothesis, we will test the following specific aims. Aim 1 is to define the mechanism of LegC4-mediated attenuation of bacterial replication in macrophages; Aim 2 is to identify host factors modulated by LegC4; and Aim 3 is to determine if LegC4 is able to protect mice from infection with a highly virulent Legionella species that does not naturally encode LegC4. Through this work, we will elucidate the function of the effector LegC4 and further determine if LegC4 is sufficient to promote host defense against non-pneumophila pathogens. To address these questions, we will use cell biology, imaging, immunology, biochemistry, genetic techniques and animal models. The proposed project is innovative and has potential to positively impact public health. Mechanisms by which the mammalian immune system detects and eradicates pathogens can be harnessed to treat and prevent infectious diseases. The benefits of studying innate immune activation by effectors are (1) previously uncharacterized pathogen detection strategies may be identified and targeted for therapeutic intervention; and (2) the effectors can be used as therapeutics to enhance immune clearance of pathogenic microbes. Ultimately, the work will culminate in enhanced understanding of host defense strategies and provide the means to develop therapeutics that will be effective against a broad range of infectious agents. This is especially important to limit disease outbreaks and decrease the global health burden associated with EZIDs. This work will provide preliminary data required for a successful R01 application to investigate mechanisms of effector-mediated host immune restriction of bacterial pathogens.
3. Cutaneous human papillomavirus as a novel model of viral oncogenesis.
Dr. Nicholas Wallace, College of Arts & Sciences
Emerging infectious diseases account for at least 12% of all human pathogens. Increased globalization among other factors led the World Health Organization to predict that novel infectious agents will continue to appear at an unprecedented rate. To protect society against these pathogens, it is essential to know all of the potential mechanisms by which infectious agents can cause disease. While viral infections are known to cause 15-20% of cancers, persistent genus β human papillomavirus (β-HPV) infections cause non-melanoma skin cancers. β- HPV’s role in these malignancies is through a novel mechanism that could be shared with emerging pathogens. Specifically, β-HPV infections act as co-factor that along with UV, blocks DNA repair and reduces host genome fidelity. The resulting mutations can drive tumorigenesis without continued exposure to UV or β-HPV. In addition to abundant supportive epidemiological, animal model, and cell culture evidence from other labs, the PI and his group have established the ability of a β-HPV gene (β-HPV E6) to attenuate the expression of four cellular DNA repair factors. β-HPV E6’s inhibition of repair stem primarily from the viral protein’s degradation of a cellular transcription factor, p300. This proposal defines the extent that the p300 loss prevents cells from mitigating genome destabilizing events, particularly events occurring during S-phase. AIM1 interrogates β-HPV E6’s inhibition of signaling events triggered by DNA crosslinks. AIM2 defines the mechanisms of β-HPV E6’s impairment of double strand DNA break repair. AIM3 determines how β-HPV E6 attenuates the regulation of centrosome duplication. The research team uses a combination of cutting-edge techniques as well as traditional molecular biology and biochemical approaches. Virus-free systems confirm all mechanisms. The selective forces (dependence on host replication factors and a tropism for sun-exposed cells) that make it advantageous for β- HPV to disrupt cell cycle regulation and DNA repair are not unique to β-HPV. Thus, p300 inactivation by other novel cutaneous viruses would be a good marker of oncogenic potential. The overall goal of this study is to understand the mutagenic potential of p300 destabilization to improve risk assessment of emerging viruses. More specific to β-HPV, the expected results have preventative implications as the current FDA-approved HPV vaccine technology could be adapted to target β-HPV and β-HPV specific inhibitors could be developed and added to formulations used to block UV light (e.g. sunscreen).
4. Rational design of live-attenuated vaccines for flaviviruses.
Dr. Yan-Jang (Scott) Huang, College of Veterinary Medicine
The objective of the proposed study is to develop broadly effective attenuation strategies for the rational design of live-attenuated vaccines (LAVs) against flaviviruses. Pathogenic flaviviruses cause severe human diseases such as hemorrhagic fever and encephalitis. The use of two legacy vaccines, yellow fever virus (YFV) 17D and Japanese encephalitis virus SA14-14-2, has demonstrated how immunization can be an efficient strategy for disease control. However, the empirical approach used for the development of these LAVs has proven ineffective in producing candidate LAVs for other flaviviruses, thereby demanding new strategies for rational vaccine design. Previously, the rational design of flavivirus LAVs was based on the introduction of mutations that lead to attenuated phenotypes observed in the two legacy vaccines and other attenuated mutants. This failed to produce broadly effective attenuation concepts, because the mutagenesis targets lacked conserved sequences or interacted with diverse host molecules. A major challenge in designing LAVs is the field’s limited knowledge of flavivirus virulence mechanisms. Target genes that contain consensus sequences, functionally important for the virulence of different flaviviruses, are yet to be identified. In this study, the PI will develop two attenuation strategies by interfering with the interdomain movements of flavivirus envelope (E) proteins. Interdomain movements of E proteins are universally conserved mechanisms in all flaviviruses and critical for viral membrane fusion and virion assembly; they are controlled by highly conserved sequences in two sets of interdomain peptides, the envelope protein domain I (EDI) – envelope protein domain II (EDII) hinge and the EDI – envelope protein domain III (EDIII) linker. The central hypothesis is that conserved residues in the interdomain peptides are functionally important for flavivirus virulence, regardless of their tissue tropism and disease pathogenesis. To remove virulence determinants in the two interdomain peptides, mutagenesis analyses will be conducted in the following two specific aims: In Aim 1, highly conserved hydrophobic residues will be mutated to interfere with the hydrophobic interactions that contribute to EDI-EDII hinge structure and functions; in Aim 2, the structures and functions of EDI-EDIII linker will be disrupted by removing functionally important side-chains of conserved residues and inserting additional glycine/prolin residues to increase peptide flexibility. Broadly effective attenuation strategies will be developed by engineering selected mutations into the full-length complementary DNA infectious clones of two model flaviviruses, West Nile virus and YFV, and then demonstrating the loss of virulence in respective mouse models. The completion of the proposed study will lead to an advancement in knowledge regarding the functional importance of the EDI-EDII hinge and EDI-EDIII linker interdomain regions for flavivirus virulence. The results of this study are expected to provide the basis for broadly effective attenuation strategies for pathogenic flaviviruses and facilitate the rational design of future candidate flavivirus LAVs.