Regulated, sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin

Abstract

Summary Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C‐terminal ~1,200 amino acid ‘prodomain’ is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C‐terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein.

DegP initiates regulated processing of filamentous hemagglutinin in Bordetella bronchiseptica

“…FhaB CP is stabilized in whole-cell lysates (WCLs) harvested from B. bronchiseptica ctpA cultures, consistent with the discovery that the native FhaB C terminus is a poor substrate for CtpA (22) and the hypothesis that FhaB CP is the preferred substrate for CtpA. The presence of FhaB CP in ctpA WCLs indicates that yet another protease, that we are calling the initiating protease, is responsible for converting FhaB into FhaB CP through removal of the ECT.…”

“…We hypothesized that the initiating protease is responsible for removing the ECT to convert full-length FhaB into the CtpA substrate FhaB CP . To test this hypothesis, we first showed (as we have done previously (22)) that addition of an HA epitope Nterminal to the PRR (Fig. 3A, strain FhaB HA-PRR ) has no effect on FhaB processing (Fig.…”

“…In fact, our data suggest that SphB1-dependent processing only occurs after extended time in culture and may have no functional relevance under normal (in vivo) circumstances. We have also shown previously that the ECT inhibits CtpA from degrading the PD, as deletion of the ECT (ECT) causes rapid and thorough conversion of FhaB to FHA (which we call hyperprocessing) in a CtpA-dependent manner (11,22). We hypothesized that the initiating protease is responsible for removing the ECT to convert full-length FhaB into the CtpA substrate FhaB CP .…”

“…We also created a derivative of degP in which a wild-type copy, driven by the constitutive S12 promoter, was inserted at the attTn7 site (degP +degP). We grew the bacteria overnight in media containing 20mM MgSO 4 (which inactivates the BvgAS regulatory system that activates expression of fhaB and other virulence factor-encoding genes) and then outgrew the bacteria in media without MgSO 4 (BvgAS-activating conditions) for four hours to induce production of FhaB before collecting WCLs as previously described (22). As with the degP strain, nearly all of the FhaB protein in the ∆degP strain was the full-length ~371 kDa form, and normal processing was restored in the complemented strain (Fig.…”

“…Previous work from our laboratory determined that the last 98 amino acids of FhaB, called the 'extreme C-terminus' (ECT), negatively regulate CtpA-dependent FhaB processing (11). While characterizing CtpA, we identified a novel polypeptide intermediate called the "FhaB clipped product" (FhaB CP ), which lacks ~90 amino acids (i.e., most of the ECT) from the C terminus (22).…”