Proteolytic processing of the Yersinia pestis YapG autotransporter by the omptin protease Pla and the contribution of YapG to murine plague pathogenesis

Abstract

Autotransporter protein secretion represents one of the simplest forms of secretion across Gram-negative bacterial membranes. Once secreted, autotransporter proteins either remain tethered to the bacterial surface or are released following proteolytic cleavage. Autotransporters possess a diverse array of virulence-associated functions such as motility, cytotoxicity, adherence and autoaggregation. To better understand the role of autotransporters in disease, our research focused on the autotransporters of Yersinia pestis, the aetiological agent of plague. Y. pestis strain CO92 has nine functional conventional autotransporters, referred to as Yaps for Yersinia autotransporter proteins. Three Yaps have been directly implicated in virulence using established mouse models of plague infection (YapE, YapJ and YapK). Whilst previous studies from our laboratory have shown that most of the CO92 Yaps are cell associated, YapE and YapG are processed and released by the omptin protease Pla. In this study, we identified the Pla cleavage sites in YapG that result in many released forms of YapG in Y. pestis, but not in the evolutionarily related gastrointestinal pathogen, Yersinia pseudotuberculosis, which lacks Pla. Furthermore, we showed that YapG does not contribute to Y. pestis virulence in established mouse models of bubonic and pneumonic infection. As Y. pestis has a complex life cycle involving a wide range of mammalian hosts and a flea vector for transmission, it remains to be elucidated whether YapG has a measurable role in any other stage of plague disease.

Fig. 1.

Localization of YapG in Y. pestis and Y. pseudotuberculosis and requirement of Pla for proteolytic cleavage. Western blot demonstrating the localization of YapG in the WT, pla mutant and pla-2 mutant strains of Y. pestis CO92 (pCD1-), and two WT strains, YPIII and IP32953, of Y. pseudotuberculosis (Y. ptb). Protein samples from cell pellets (a) and culture supernatants (b) were probed with anti-YapG serum. −, Uninduced culture; +, culture induced with aTc. Molecular masses (kDa) are indicated on the left. The solid arrowhead indicates mature-length YapG and the open arrowheads denote the Pla-processed forms of YapG.

Fig. 2.

Amino acid residues of YapG important for processing by the omptin protease Pla. (a) Schematic of the domain organization of YapG. The signal peptide (SP) is shown in white, the PD in grey and the β-domain in black. The basic Lys and Arg residues within the PD that are demonstrated to be important for Pla cleavage are located within two direct repeats (R1 and R2) and in a third partial direct repeat (R3′). Cleavage of sites 1–5 (K₅₁₂, KR_548–549, K₅₅₈, KR_594–595 and K₆₀₄) by Pla (illustrated with scissors) results in released fragments A–E. (b) Western blot demonstrating the secretion of WT and mutant YapG in the avirulent (pCD1⁻) Y. pestis yapG mutant. Bacteria were treated with aTC to induce yapG expression, and culture supernatants were probed with anti-YapG serum. The site-directed mutations (individual or multiple) examined for cleavage are indicated above the blot. The arrows to the right correspond to the predicted fragment sizes illustrated in (a). Sites 1 (K₅₁₂), 2 (KR_548–549) and 4 (KR_594–595) represent the major cleavage sites of YapG resulting in fragments A, B and C, respectively, whereas, sites 3 (K^*₅₅₈) and 5 (K^*₆₀₄) represent the secondary alternative cleavage sites of YapG resulting in fragments D and E, respectively. Molecular mass is indicated to the left of the blot in kDa.

Fig. 3.

Comparison of WT and yapG mutant colonization and dissemination during pneumonic (a) and bubonic (b) plague. (a) Mice were i.n. inoculated with ~1×10⁴ c.f.u. WT (▪) or yapG mutant (□). (b) Mice were s.c. injected in the neck with ~1×10² c.f.u. WT (▪) or yapG mutant (□). For (a) and (b), data are representative of two independent experiments. Mice were sacrificed at various times p.i. (as indicated below the graphs), and organs were homogenized and plated to determine c.f.u. (g tissue)⁻¹. Each symbol represents bacteria recovered from an individual mouse. Horizontal bars denote the median of each population. Numbers with an ‘×’ below each population indicate the number of mice that were euthanized or found dead at that time point. The dashed lines indicate the limits of detection for each organ.