Characterization of Tetratricopeptide Repeat-Like Proteins in Francisella tularensis and Identification of a Novel Locus Required for Virulence

Daňková, Věra; Balonova, Lucie; Strašková, Adéla; Špidlová, Petra; Putzová, Daniela; Kijek, Todd M.; Bozue, Joel A.; Cote, Christopher K.; Mou, Sherry; Worsham, Patricia L.; Szotáková, Barbora; Červený, Lukáš; Stulı́k, Jiřı́

doi:10.1128/iai.01620-14

Cited by 3 publications

(2 citation statements)

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“…Although lacking functional annotation, FTT1597 contains tetratricopeptide repeat motifs recently shown to be present in other proteins implicated in F. tularensis virulence [ 65 ]. The similarity in interactions with AP3M1 and WDR48 highlight a possible functional similarity to FTT1538c.…”

Section: Discussionmentioning

confidence: 99%

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors

et al. 2015

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BackgroundFrancisella 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.ResultsWe 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.ConclusionsDirect 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2351-1) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors

et al. 2015

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“…These have been reported to be directly related to virulence-associated functions in bacterial pathogens [109][110][111] . In Francisella tularensis, a bacterial pathogen, a TPRcontaining protein is a membrane-associated protein that is required for intracellular replication of the microbe, in vivo virulence, and heat stress tolerance 112 . In P. infestans, TPR was predicted as one of the most common domains within proteins 113 .…”

Section: P Viticola Proteome At 6hpi Reflects Actively Regulated Processes Leading To Infection Developmentmentioning

confidence: 99%

Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes

Figueiredo

Santos

Guerra‐Guimarães

et al. 2021

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Plant apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors and several signal transduction pathways are activated. As a result of this first contact, the host triggers a defence response that involves the modulation of several extra and intracellular proteins. In grapevine-pathogen interactions, little is known about the communication between cells and apoplast. Also, the role of apoplastic proteins in response to pathogens still remains a blackbox. In this study we focused on the first 6 hours after Plasmopara viticola inoculation to evaluate grapevine proteome modulation in the apoplastic fluid (APF) and whole leaf tissue. Plasmopara viticola proteome was also assessed enabling a deeper understanding of plant and pathogen communication. Our results showed that oomycete recognition, plant cell wall modifications, ROS signalling and disruption of oomycete structures are triggered in Regent after P. viticola inoculation. Our results highlight a strict relation between the apoplastic pathways modulated and the proteins identified in the whole leaf proteome. On the other hand, P. viticola proteins related to growth/morphogenesis and virulence mechanisms were the most predominant. This pioneer study highlights the early dynamics of extra and intracellular communication in grapevine defence activation that leads to the successful establishment of an incompatible interaction.

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Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability

et al. 2015

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BackgroundChlamydia spp. are obligate, intracellular bacteria that infect humans and animals. Research on these important pathogens has been hindered due to a paucity of genetic tools. We recently adapted a group II intron (GII) mutagenesis platform for creation of ampicillin-selectable gene insertions in C. trachomatis L2. The aims of this study were: (1) to assess the stability of the intron-insertion in an in vivo infection model to gauge the efficacy of this genetic tool for long term animal studies and (2) to expand upon the utility of the method by validating a second selection marker (aadA, conferring spectinomycin resistance) for mutant construction.ResultsIntron stability was assessed using a mouse vaginal tract infection model with a C. trachomatis L2 434/Bu incA::GII(bla) mutant. Infections were performed in the absence of selection and isolates shed into the vaginal tract were isolated and expanded in cell culture (also without selection). PCR and inclusion phenotype analysis indicated that the intron was stable for at least 27 days post-infection (at which point bacteria were no longer recovered from the mouse). The aminoglycoside 3′ adenyltransferase (aadA) gene was used to create a spectinomycin-selectable GII intron, facilitating the construction of an incA::GII[aadA] C. trachomatis L2 insertion mutant. Both the GII(aadA) intron and our previously reported GII(bla) intron were then used to create an incA::GII(aadA), rsbV1::GII(bla) double mutant. Mutants were confirmed via PCR, sequencing, inclusion morphology (incA only), and western blot.ConclusionsThe stability of the intron-insertion during in vivo growth indicates that the GII-insertion mutants can be used to study pathogenesis using the well-established mouse infection model. In addition, the validation of an additional marker for mutagenesis in Chlamydia allows for gene complementation approaches and construction of targeted, double mutants in Chlamydia. The aadA marker also could be useful for other genetic methods. Collectively, our results expand upon the rapidly growing chlamydial genetic toolkit and will aid in the implementation of studies dissecting the contribution of individual genes to infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1542-9) contains supplementary material, which is available to authorized users.

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Characterization of Tetratricopeptide Repeat-Like Proteins in Francisella tularensis and Identification of a Novel Locus Required for Virulence

Cited by 3 publications

References 48 publications

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors

Revealing the secrets beneath grapevine and Plasmopara viticola early communication: a picture of host and pathogen proteomes

Use of aminoglycoside 3′ adenyltransferase as a selection marker for Chlamydia trachomatis intron-mutagenesis and in vivo intron stability

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