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Title: ATPase-Independent Type-III Protein Secretion in Salmonella enterica}
Article:
Abstract:
Type-III protein secretion systems are utilized by gram-negative pathogens to secrete building blocks of the bacterial flagellum, virulence effectors from the cytoplasm into host cells, and structural subunits of the needle complex. The flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate unfolding and translocation. We report formation of functional flagella in the absence of type-III ATPase activity by mutations that increased the proton motive force and flagellar substrate levels. We additionally show that increased proton motive force bypassed the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion. Our data support a role for type-III ATPases in enhancing secretion efficiency under limited secretion substrate concentrations and reveal the dispensability of ATPase activity in the type-III protein export process.
Introduction:
Many bacteria move by rotating a rigid, helical organelle, the flagellum[1]. The flagellum represents one of the smallest motor complexes known and enables bacteria to move through liquids( swimming)[2] and highly viscous environments or surfaces( swarming)[3]. In addition to the chemotactic behavior, flagellar motility contributes to bacterial pathogenesis by promoting bacteria-host interactions, adherence, biofilm formation and invasion of eukaryotic cells[4]. A schematic overview of flagellar biogenesis in Salmonella enterica is shown in Figure 1. Export of the building blocks of the flagellum is mediated by a flagellar-specific type-III secretion system( fT3SS), whose core cytoplasmic and inner-membrane export apparatus components( FliHIJ FliPQR FlhAB) are evolutionarily and functionally related to the virulence-associated type-III secretion systems( vT3SS) of pathogenic Gram-negative bacteria[5]. The core export apparatus components of both fT3SS and vT3SS mediate the translocation of proteins across the inner membrane and exhibit stringent substrate recognition and high speed of protein translocation[6]. Core export apparatus assembly initiates in the cytoplasmic membrane with the stepwise addition of FliF, FliG, FliM and FliN to produce a structure resembling a cytoplasmic-facing cup[7]. The inner membrane export apparatus( export gate) of the fT3SS assembles within the cup-like structure and consists of the integral membrane proteins FlhB, FlhA, FliO, FliP, FliQ and FliR[8]. Functionally associated with the fT3SS is a cytoplasmic ATPase complex composed of the FliI, FliH and FliJ proteins[6]. The ATPase FliI forms a hexameric ring-shaped structure[9] and together with the negative regulator FliH[10] regulates the initial entry of substrates into the export gate by ensuring that FliI-dependent ATP hydrolysis occurs in concert with secretion. FliH interacts with the N-terminal region of FliI and prevents its ATPase activity until the export apparatus is competent to utilize ATP hydrolysis in the export process[10],[11]. Interestingly, FliI and FliJ show remarkable structural similarity to the α/β and γ-subunits of FOF1 ATPases, respectively[12],[13]. FliH is homologous to the stalk subunits b and δ of FOF1 ATPases[14]. Until recently, it was presumed that ATP hydrolysis was the energy source for type-III secretion. However, it was demonstrated that elimination of the proton motive force( PMF, consisting of the protein gradient ΔpH and the membrane potential Δψ) inhibited flagellar type-III secretion[15],[16]. ATP hydrolysis might energize chaperone release and secretion substrate unfolding[17]. Further functions of the FliHIJ proteins might be in localizing the ATPase/secretion substrate complexes to the vicinity of the export gate by interaction with the switch-complex protein FliN[18],[19] and the cytoplasmic domain of FlhA[12],[20]. An efficient interaction of FliJ with FlhA requires support of FliH and FliI upon which FliJ switches the conformation of the export gate from a low-efficient proton-protein antiporter to a highly efficient, Δψ-driven export system. FliJ is thus essential for the Δψ-driven type-III protein export and in the absence of FliH and FliI, the type-III export apparatus requires both Δψ and ΔpH of the PMF to couple the energy derived from the proton influx with substrate protein secretion[21]. Additional described functions of FliJ include chaperone-like activities by preventing premature aggregation of both early and late secretion substrates[22], as well as a function in substrate selectivity as a chaperone escort protein that specifically recruits unladen substrate-specific chaperones FlgN and FliT during assembly of the filament junction and cap substructures[23]. FliJ is also implicated in facilitating FliI hexamer ring formation, thereby stimulating the ATPase activity[12],[23]. Similar to the fT3SS, the vT3SS is an essential component of the virulence-associated needle complexes encoded on Salmonella Pathogenicity Islands 1 and 2( Spi1 and Spi2)[5],[24]. The ATPase associated with the Spi1 vT3SS is encoded by invC[25]. InvC functions in effector substrate recognition and induces chaperone release and subsequent unfolding of the respective secretion substrate in an ATP-dependent manner[17]. Type-III protein secretion is a process of great complexity and the importance of ATP hydrolysis for the type-III secretion process was unclear. In the present work, we demonstrate that type-III protein secretion can efficiently occur in the absence of ATPase activity in both flagellar and virulence-associated T3SS.
Discussion:
The results demonstrate that ATPase activity is universally dispensable for protein export via bacterial type-III secretion systems. We propose that the function of the T3SS-associated ATPase complex is to deliver secretion substrates to the export apparatus and ensure efficient substrate unfolding, which was first reported by Akeda and Galán[17]. Either increased substrate production or increased PMF overcomes the requirement for the ATPase complex( Fig. S7). However, increased PMF or substrates do not bypass the requirement for proton flux through FlhA in contrast to the dispensability of ATP hydrolysis by the FliHIJ complex( Fig. S6). In summary, our data demonstrate that efficient type-III protein export occurs in the absence of the ATP hydrolyzing components; therefore it is reasonable to assume that the export gate itself has a primary function in substrate unfolding and translocation. We speculate that the function of the ATPase complex in substrate unfolding and chaperone release is secondary to assure a highly efficient export process. Our data would suggest that chaperone release and unfolding occur inefficiently without ATPase activity, but is catalyzed by FliHIJ. Increasing PMF thus shifts the equilibrium of the spontaneous process. The observation that export of a FlgE-Bla reporter construct was similarly increased in both the fliHI and fliJ mutant strains indicated that the FliHI and FliJ components of the ATPase complex acted in different pathways of the type-III export process. A possible scenario would be that FliJ – which interacts with FlhA of the export gate – activates the efficient Δψ-driven type-III protein export as suggested by Minamino et al.[21]( see also Fig. S7). FliH binds to both the ATPase FliI and the cytoplasmic C-ring, thereby localizing secretion substrates to the export gate for efficient unfolding by FliI prior to a Δψ or ΔpH-driven translocation process. In the absence of FliJ, the inner membrane export apparatus can still function as a less-efficient ΔpH-driven protein-proton antiporter and the secretion process is facilitated by substrate unfolding via the FliHI complex. The non-essential substrate unfolding is not occurring in a fliHI mutant strain, however the export gate operates in the highly-efficient Δψ export mode, which might be able to energize substrate unfolding as well. The switch from early to late( flagellin-like) substrate secretion is a bottleneck in the process of flagellar bioassembly. The importance of this step in flagellar bioassembly was evidenced by the fact that we observed assembly of single flagellar filaments of wildtype lengths and longer in our fliHIJ deletion mutants( Fig. 6). It has been demonstrated before that the inner membrane ring complex, the C-ring and integral membrane components of the fT3SS assemble normally in the absence of the fliHIJ complex[38],[39]. Thus, the fliHIJ mutants analyzed here presumably assemble a physiological number of secretion system complexes. Only upon completion of the hook-basal-body structure, the secretion apparatus undergoes its secretion substrate specificity switch. In the absence of the FliHIJ complex, flagellar protein export is strongly impaired and thus HBB completion and the switch to filament-type secretion is a rare event. However, there appears to exist a mechanism for localization of the available late flagellar substrates to the single hook-basal-body that is in a late-export-competent state since we observed filament lengths that were as long or longer than the wildtype in our fliHIJ mutants( Fig. 6). An increase in PMF then likely assists in efficient export of late secretion substrates and thereby overcomes the requirement of the FliHIJ ATPase complex for late substrate secretion – similar to the related scenario where excess substrates bypass the requirement of the FliHIJ ATPase complex. Our findings also have important implications for the evolution of the bacterial flagellum and type-III secretion systems. As mentioned above, the cytoplasmic components of the type-III secretion system share strong homology to components of the FOF1 ATP synthase and it has been proposed that the flagellum was derived from a proto FOF1 ATP synthase where ATP hydrolysis might have energized the export process. The present results would indicate that a proto F1-ATPase was added to a primordial type-III export system( which might have been proton-powered) with the evolutionary benefit of facilitating the export process. This made the bacterial type-III secretion apparatus to the highly efficient protein-export system of the contemporary flagellum and injectisome. In summary, type-III protein export is a process of great complexity and involves a substantial number of checkpoints to ensure the correct order of export. A reasonable, general proposal for the export mechanism of bacterial type-III secretion systems is that the respective ATPase complexes bind to substrate proteins, shuttle the substrates to the base of the growing structure, energize chaperone release and substrate unfolding in an ATP-dependent manner and present the substrate to the membrane-embedded export apparatus components for efficient PMF-dependent secretion.
The type-III protein secretion apparatus is a complex nanomachine responsible for secretion of building blocks and substrate proteins of the flagellum and the virulence-associated injectisome needle complex of many gram-negative pathogens . Type-III secretion systems utilize the energy of the proton motive force and ATP hydrolysis of a cytoplasmic ATPase to drive substrate export . The cytoplasmic components of the secretion system share strong homology to the FoF1 ATP synthase and it is thought that the flagellum was derived from a proto FoF1-ATP synthase where ATP hydrolysis energized the export process . Here , we report the dispensability of ATPase activity for the type-III protein export process in Salmonella . This finding has important implications for the evolution of the bacterial flagellum and type-III secretion systems , suggesting that a proto ATPase was added to a primordial proton-powered type-III export system with the evolutionary benefit of facilitating the export process .