Project description:Strains of R. rickettsii, the agent of Rocky Mountain spotted fever, differ greatly in the severity of the disease caused. The genetic differences responsible for this disparity are only now being uncovered. An avirulent, laboratory adapted strain of R. rickettsii fails to proteolytically process several large surface protein antigens. We have identified a protease that cleaves the protein precursors to their mature form. The gene encoding this protease is mutated in the avirulent strain. Complementation of the active form of the gene identifies proteolytic processing of surface antigens as important to virulence.
Project description:Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in salivary glands of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.
Project description:Boreal toads (Anaxyrus boreas boreas) of the Southern Rocky Mountain population are declining due to the introduction of the chytrid fungus Batrachochytrium dendrobatidis (Bd). Boreal toads in Colorado are generally susceptible to Bd infection, but some Bd-tolerant populations persist in parts of the Southern Rocky Mountain and broader Eastern boreal toad population. We conducted a Bd challenge with lab-reared sibling toads from Bd-susceptible Colorado and purportedly Bd-tolerant Utah populations and report on transcriptomic responses to Bd during late infection in skin and liver tissue. Fewer immune genes were expressed in response to Bd in Colorado toads, but with greater upregulation compared to Utah toads, indicating a dysregulated immune response. Signatures of Bd-tolerance in Utah toads included more moderate upregulation in immune gene expression and a significantly enriched suite of gene functions related to innate and adaptive immune responses. Our transcriptomic results support the notion that Utah toads are tolerant to Bd, rather than resistant, carrying Bd loads similar to Colorado yet having a unique transcriptomic profile and presenting minimal clinical signs of chytridiomycosis. We conclude that closely related populations have divergent transcriptomic responses to Bd with a dysregulated immune response in Bd-susceptible toads.