INTERACTIONS OF THE COMPONENTS OF THE GENERAL SECRETION PATHWAY - ROLE OF PSEUDOMONAS-AERUGINOSA TYPE-IV PILIN SUBUNITS IN COMPLEX-FORMATION AND EXTRACELLULAR PROTEIN SECRETION

The general secretion pathway (GSP), found in a wide range of bacteria , is responsible for extracellular targeting of a subset of proteins f rom the periplasm, In Pseudomonas aeruginosa, the GSP requires the par ticipation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homolog ues of PilA, the major subunit of type IV pili, The interaction betwee n the pilin-like Xcp proteins was investigated using bifunctional cros slinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT -containing complex, This complex was shown to consist of XcpT/PilA he terodimers, The role of PilA in the GSP was examined, using P. aerugin osa mutants in the pilA gene, or in rpoN, a gene regulating pilA expre ssion, Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells, The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other comp onents of the secretion machinery, nor did it require the pilus biogen esis factors PilB and PilC. The dimeric XcpT/PilA complex was also for med in a pilD mutant, which lacks the leader peptidase enzyme, demonst rating that the leader peptide at the N-terminus or PilA or XcpT did n ot have to be removed for the dimerization to occur, XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When express ion of XcpT is increased, its homodimers, as well as XcpT/XcpW heterod imers, can be detected, Finally, an oligohistidine-tagged XcpT was sho wn to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. T he results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the s ecretion apparatus. Alternatively, these may represent dynamic interme diates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretio n argues for a critical role played by PilA in two related processes d uring P. aeruginosa infections: formation of an adhesive pilus organel le and secretion of exoenzymes.