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. 2015 Dec 29;16:1106.
doi: 10.1186/s12864-015-2351-1.

Using Host-Pathogen Protein Interactions to Identify and Characterize Francisella Tularensis Virulence Factors

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Free PMC article

Using Host-Pathogen Protein Interactions to Identify and Characterize Francisella Tularensis Virulence Factors

Anders Wallqvist et al. BMC Genomics. .
Free PMC article

Abstract

Background: Francisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms.

Results: We initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response.

Conclusions: Direct interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.

Figures

Fig. 1
Fig. 1
Yeast two-hybrid (Y2H) host-pathogen protein-protein interactions (PPIs). Using Y2H screens against whole human and murine proteome libraries, we identified 222 unique PPIs between 18 Francisella tularensis proteins and 183 human proteins (a) and 118 unique PPIs between 13 F. tularensis proteins and 113 murine proteins (b). Green nodes represent F. tularensis proteins, whereas pink and red nodes represent host proteins. Eleven F. tularensis proteins interacted with both hosts, and six of them participated in 21 conserved interactions (red edges), i.e., six F. tularensis proteins that interacted with both human proteins (red nodes in a) and their murine orthologs (red nodes in b)
Fig. 2
Fig. 2
Fifty percent lethal dose (LD50) estimation. Using the dose-response experiments, we estimated LD50 values and 95 % confidence intervals for the wild-type strain and each of the mutant strains. The estimated wild-type LD50 of 2.62 CFU, where CFU denote colony-forming units, agreed with previously estimated values for the wild-type F. tularensis subsp. tularensis Schu S4 strain. The estimated LD50 values for ΔFTT0482c and ΔFTT1597 were higher than the wild-type value and outside its 95 % confidence intervals, whereas the LD50 value estimated for ΔFTT1538c was above the wild-type value, although with overlapping confidence intervals. These results support the notion that ΔFTT0482c, ΔFTT1538c, and ΔFTT1597 attenuate F. tularensis virulence in this animal model
Fig. 3
Fig. 3
Mouse intranasal challenge model results. We compared the survival rates of 30 (3 × 10) mice exposed to the ΔFTT0482c, ΔFTT1538c, and ΔFTT1597 mutants for intranasal doses of ≥78 CFUs, with 10 mice exposed to wild-type strain doses of 320 CFU. Animals were monitored for 21 days. All mice exposed to the high-dose of the wild-type strain died by the end of the fifth day (blue line), whereas mice exposed to mutant strains had a slower time to death and survived in larger numbers 21 days post-exposure (purple, red, and orange lines). There was a statistically significant difference in the survival rate of mice exposed to mutant strains and mice exposed to the wild-type strain (p-value ≤10-3). These results support the notion that each of the three mutants attenuates virulence when infected in mice via the intranasal route of infection
Fig. 4
Fig. 4
Host signaling and the stress response as a F. tularensis virulence factor target. F. tularensis proteins target a large number of host proteins involved in signaling (red and blue octagons) and the stress response (yellow diamond). Using the connectivity and Gene Ontology (GO) annotation, we identified two statistically significant signaling interaction modules, one containing eight host proteins and one containing five host proteins (shaded area). The larger one contains proteins related to the immune system response, whereas the smaller one contains proteins related to intracellular signaling. Further annotation assessment of proteins from the largest connected component (LCC) revealed additional proteins associated with signaling (blue octagons) and existing interaction modules. These results suggest that F. tularensis targets host proteins involved in signaling to interfere with cell-to-cell signaling and the immune response but also points toward PPIs among these host proteins as equally important targets

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