Characterization of the pathogenicity island protein PdpA and its role in the virulence of Francisella novicida

Abstract

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. A research surrogate for F. tularensis is Francisella novicida, which causes a tularaemia-like disease in mice, grows similarly in macrophages, and yet is unable to cause disease in humans. Both Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. In this work we examined the properties of PdpA protein expression and localization as well as the phenotype of a F. novicida pdpA deletion mutant. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice.

Fig. 1.

Deletion of pdpA. (a) The pdpA gene is the first cistron in a low-G+C operon of the Francisella pathogenicity island (FPI). A newly revised nomenclature is used to show the two convergent operons in the FPI. (b) Primers that amplify regions within the pdpA gene were used to screen wild-type (WT) F. novicida, the ΔpdpA mutant and the in cis complement ΔpdpA/SKX : : pdpA. (c) The exact nucleotides removed to create the ΔpdpA mutant are shown. The in-frame deletion preserves the last 21 nucleotides of pdpA, which contain the ribosome-binding site (shown in italics) of the downstream gene pdpB.

Fig. 2.

Detection and subcellular localization of PdpA in Francisella. (a) Reactivity of anti-PdpA monoclonal antibody with cell extracts of F. novicida and two F. tularensis subspecies. The O-antigen in F. novicida is thought to cause aberrant migration of PdpA. The migration of an 83 kDa molecular mass marker is shown on the right. (b) Effect of deletion of pdpA and mutation of O-antigen gene. An O-antigen mutation from the previously described mutant SC92 (‘WT-LPS’) was transferred to the ΔpdpA mutant and the complemented strain ΔpdpA/SKX : : pdpA, and Western blots with anti-PdpA and anti-PdpB monoclonal antibodies are shown. For the top part of (b) the number at the right indicates actual migration of a molecular mass marker, and for the bottom part, the number indicates a calculated relative molecular mass. (c) A Western blot using anti-PdpB antibody shows that the pdpA : : Em^R gene replacement mutant NZ9 expresses greatly reduced levels of PdpB compared to wild-type (WT). (d) Bacteria isolated from a J774A.1 macrophage infection were fractionated to separate soluble and membrane-associated proteins. PdpA was found to localize with the soluble proteins, as was the transcriptional regulator MglB. PdpB localized to the Sarkosyl-soluble protein fraction, indicating its association with the inner membrane of F. novicida. NADH oxidase activity was determined in each fraction as a measure of the relative amount of inner-membrane protein. The number to the right of the top panel of (d) represents the actual migration of the molecular mass marker, whereas the numbers in the two bottom panels represent calculated relative molecular masses. The amount of sample loaded in each lane was normalized by protein content. Results are representative of duplicate experiments.

Fig. 3.

Regulation of PdpA expression by iron and transcriptional regulators. (a) Levels of PdpA, PdpB and IglB protein expression were determined in mglA and mglB mutant backgrounds. (b) Expression of these proteins, as well as IglC, was also determined in varying iron conditions. The relative level of fluorescence signal generated by reactivity of anti-PdpA, anti-PdpB, anti-IglB, or anti-IglC monoclonal antibodies, calculated as described in Methods, is indicated. The amount of sample loaded to the wells of each polyacrylamide gel was normalized by protein content. The reactivity of a non-specific, cross-reactive protein band is shown in the bottom panel. Results are representative of experiments performed in triplicate.

Fig. 4.

Attenuation of the ΔpdpA mutant in chicken embryos. Seven-day-old chicken embryos were infected with 10³ c.f.u. of wild-type (WT) (a), ΔpdpA (b) and ΔpdpA/SKX : : pdpA (c) strains of F. novicida. Time to death of the embryos was monitored over a period of 6 days. All experiments were done at three separate inoculating doses, and the experiments included at least seven embryos per infective dose per strain; the survival curves presented are representative of three separate trials. The statistical difference between the WT and ΔpdpA strain survival curve was measured by the log-rank test and yielded a P-value of <0.001. The P-value for a comparison of ΔpdpA vs ΔpdpA/SKX : : pdpA was <0.001.