Protein-Protein Interactions within Type III Secretion System-Dependent Pili of Rhizobium sp . Strain NGR 234

Many gram-negative bacteria employ type III secretion systems (T3SS) to deliver virulence “effector” proteins directly into eukaryotic cells, which then modulate cellular functions in the host. A common feature of these secretion systems is the T3SS-dependent extracellular appendages that link bacteria to their hosts, where they probably act as conduits for the effector proteins. The appendages of animal pathogens, also called needle complexes, are mostly composed of many copies of a single, small ( 10-kDa) protein. When the needle comes in contact with a host cell, synthesis of a translocation pore composed of different bacterial proteins (termed translocators) occurs in the plasma membrane of the target cell. Formation of the pore is essential for delivery of the effector proteins. Extracellular appendages of the animal pathogen Yersinia sp. have been well characterized (7); the needle is composed of YscF, and three translocators have been identified (LcrV, YopB, and YopD). YopB and YopD contain hydrophobic domains and can insert into erythrocyte membranes; LcrV does not possess such domains, but it is absolutely required for the assembly of functional pores (15). LcrV forms a distinct structure at the tip of the YscF needle, upon which the YopBD translocation pore is assembled (16). YscF and LcrV can interact. LcrV is probably distal to YscF since YscF needles still form if there is a mutation in lcrV, although the translocation of effectors is blocked. Phytopathogens produce much longer pilus-like structures, called Hrp pili, and their assembly is dependent upon the hypersensitive response and pathogenicity (Hrp) gene cluster. Hrp pili are also composed of multiple copies of a single, small protein (9, 17). T3SS of phytopathogens are also thought to form translocation pores in the plant plasma membrane. The translocating protein HrpF of Xanthomonas campestris pv. vesicatoria has hydrophobic domains and has been shown to insert into lipid bilayers (6). Furthermore, mutation of hrpF yields a phenotype characteristic of translocators; protein secretion can occur in vitro from cell cultures but not in vivo into host cells (18). Purification of Hrp pili from Ralstonia solanacearum yielded a second protein (PopF1) that copurified with the major subunit, HrpY. PopF1 is homologous to HrpF and functions as a translocator in R. solanacearum. Although this situation seems to be analogous to the situation in animal pathogens, it should be noted that HrpY and PopF1 did not interact in a yeast two-hybrid binding assay (14). Nitrogen-fixing symbiotic bacteria, collectively called rhizobia, utilize a variety of signal molecules that govern the range of legumes with which they interact (3). Certain rhizobia also possess functional T3SS, and proteins secreted by these systems (nodulation outer proteins [Nops]) are another determinant of host range (12). For example, in Rhizobium species strain NGR234 at least six T3SS-secreted Nops have been identified: NopA, NopB, NopC, NopL, NopP, and NopX (1, 8, 13, 19–21). Depending upon the legume host, abolition of Nop secretion by NGR234 can improve or block symbiotic interactions. Electron microscopic methods showed that T3SS-dependent pili of NGR234 are mostly composed of NopA but also contain NopB and NopX (8, 19). T3SS-dependent pili have also been observed in Rhizobium fredii USDA257, where they are also composed of several proteins. Characterization of pili using antibodies against Nops identified two of these proteins as NopB and NopX (10, 11). Based upon its relative abundance and because it has secondary structural features similar to those of other pilus subunits, NopA is the major component of NGR234 T3SS pili (none of the major pilus subunits from different bacterial species share any amino acid similarity). As NopX has significant homology to HrpF and PopF, it could also be a translocating protein, and its copurification with NGR234 T3SS pili mirrors that of PopF1 in R. solanacearum. The presence of NopB in pilus extracts is intriguing, however. Although BLAST searches with NopB revealed significant homology only to other rhizobial (NopB-like) proteins, the carboxy terminus of NopB has some homology to FlgK, a linker of two distinct flagellum proteins, the hook and filament (which are synthesized by related T3SS). Perhaps the role of NopB might be as a coupling protein within the NGR234 pili. We propose three simple models (Fig. 1) to explain the positions of NopA, NopB, and NopX within the pili. NopX, as a potential * Corresponding author. Mailing address: LBMPS, Université de Genève, Sciences III, 30 quai Ernest-Ansermet, 1211 Genève 4, Switzerland. Phone: 41 22 379 3108. Fax: 41 22 379 3009. E-mail: william . broughton@bioveg.unige. ch. † Present address: LIPM, UMR CNRS-INRA 2594/441, BP 52627, 31326 Castanet Tolosan Cedex, France. ‡ Present address: School of Life Sciences, SunYat-Sen (Zhongshan) University, 135 Xingangxi Road, Guangzhou, 510275, China. Published ahead of print on 2 November 2007.