Furthermore, PilA of ssp. novicida was recently shown to be involved in protein secretion that was coupled to Tfp [20, 25]. Interestingly,

mutation of pilA and loss of protein secretion resulted in increased virulence in a mouse infection model [25]. As the human pathogenic type A and type B strains do not secrete detectable levels of proteins in vitro, it is possible that one step in the evolution of human pathogenic variants of F. tularensis from ssp. novicida has involved loss of protein secretion Decitabine manufacturer as a consequence of changes in PilA structure and function. In this work we wanted to address the question if PilA is involved in virulence of the highly pathogenic type A strain SCHU S4, similarly to

what we have previously shown for type B strains, and if Tfp secretion and assembly genes are required for virulence. Results Construction of non-polar pilin gene mutants In a recent study, we were able to demonstrate that the pilA gene can be lost by a deletion event mediated by direct repeats flanking the gene [22]. Type B strains lacking pilA were found to be attenuated for virulence in a mouse infection model. In this study we wanted to extend this work to the highly pathogenic type A strain SCHU S4, and therefore we constructed a specific pilA deletion mutant using our previously described allelic exchange technique [7]. In addition, to address the significance of secretion and Rapamycin assembly of PilA, we also engineered in-frame deletions in pilC and pilQ, encoding a transmembrane protein and a secretin, respectively. For some pathogens, Tfp expression is associated with a unique ability to retract the pili, a phenotype depending on the ATPase PilT. Interestingly, pilT appears to be functional in type A

strains, while it is a pseudogene in the less pathogenic type B strains. In order to elucidate if the expression of PilT could be correlated to the higher virulence of type A strains, we also constructed an in-frame deletion in the pilT 3-mercaptopyruvate sulfurtransferase gene. In order to verify that the mutations did not have a major impact on neighboring gene transcription, each region was analysed by RT-PCR on mRNA extracted from the mutant strains and compared to the isogenic wild-type strain (Fig. 1). Thereby we could confirm that none of the deletion events caused any polar effects on transcription. Both pilC and pilT are flanked by pseudogenes situated directly downstream of each gene that were found not to be transcribed neither in the wild-type nor in the pilC or pilT mutant strains. The upstream genes of pilC and pilT were readily transcribed at similar levels in the wild-type and mutant strains. In the case of the pilQ mutant, we could verify non-polarity on the downstream aroK gene. Figure 1 A-D. Analysis of gene transcription in wild-type and mutant strains of F. tularensis using RT-PCR of mRNA.