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Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

2016; Elsevier BV; Volume: 291; Issue: 38 Linguagem: Inglês

10.1074/jbc.m116.738377

1083-351X

Tiffany L. Leighton, Daniel H. Yong, P. Lynne Howell, Lori L. Burrows,

Bacterial Genetics and Biotechnology

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo. Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces. Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo. Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces. Many bacteria, including Pseudomonas aeruginosa, use type IV pili (T4P) 3The abbreviations used are: T4P, type IV pili; OM, outer membrane; IM, inner membrane; β-ME, β-mercaptoethanol; T2S, type II secretion; TMS, transmembrane segment; FRT, FLP recombination target; Cb, carbenicillin; Gm, gentamicin. 3The abbreviations used are: T4P, type IV pili; OM, outer membrane; IM, inner membrane; β-ME, β-mercaptoethanol; T2S, type II secretion; TMS, transmembrane segment; FRT, FLP recombination target; Cb, carbenicillin; Gm, gentamicin. for surface attachment/adhesion, biofilm formation, and twitching motility (1Bradley D.E. A function of Pseudomonas aeruginosa PAO polar pili: twitching motility.Can. J. Microbiol. 1980; 26: 146-154Crossref PubMed Scopus (242) Google Scholar2Burrows L.L. Weapons of mass retraction.Mol. Microbiol. 2005; 57: 878-888Crossref PubMed Scopus (170) Google Scholar, 3Burrows L.L. Pseudomonas aeruginosa twitching motility: type IV pili in action.Annu. Rev. 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Orthologous components of the alignment subcomplexes in the two systems (PilMN/GspL, PilO/GspM, and PilP/GspC) have limited sequence identity, but atomic resolution structures revealed a high degree of structural similarity, and all three components form equimolar complexes with a ratio of 1:1:1 (18Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (61) Google Scholar, 20Tammam S. Sampaleanu L.M. Koo J. Sundaram P. Ayers M. Chong P.A. Forman-Kay J.D. Burrows L.L. Howell P.L. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.Mol. Microbiol. 2011; 82: 1496-1514Crossref PubMed Scopus (54) Google Scholar, 26Abendroth J. Bagdasarian M. Sandkvist M. Hol W.G. 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Previous work in P. aeruginosa showed that PilP fails to interact with a PilO homodimer and requires both PilN and PilO for stability (17Ayers M. Sampaleanu L.M. Tammam S. Koo J. Harvey H. Howell P.L. Burrows L.L. PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin.J. Mol. Biol. 2009; 394: 128-142Crossref PubMed Scopus (97) Google Scholar, 20Tammam S. Sampaleanu L.M. Koo J. Sundaram P. Ayers M. Chong P.A. Forman-Kay J.D. Burrows L.L. Howell P.L. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.Mol. Microbiol. 2011; 82: 1496-1514Crossref PubMed Scopus (54) Google Scholar). Although PilO crystallized as a homodimer (18Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (61) Google Scholar), complementation of a pilO mutant with a plasmid-encoded copy of the gene led to formation of predominantly PilO homodimers at the cost of PilN stability, and thus T4P function (17Ayers M. Sampaleanu L.M. Tammam S. Koo J. Harvey H. Howell P.L. Burrows L.L. PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin.J. Mol. Biol. 2009; 394: 128-142Crossref PubMed Scopus (97) Google Scholar). In Thermus thermophilus, purified T4P alignment subcomplex components were estimated by negative stain cryoelectron microscopy to be in a ratio of 2:2:2 for PilM/PilN/PilO, where initial PilN homodimers in complex with PilM were disrupted by the addition of PilO, ultimately forming PilNO heterodimers (29Karuppiah V. Collins R.F. Thistlethwaite A. Gao Y. Derrick J.P. 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Pineau C. Effantin G. Robert X. Shevchik V.E. Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.PLoS One. 2013; 8: e79562Crossref PubMed Scopus (24) Google Scholar). This mechanism was proposed to propagate movement from the cytoplasmic to periplasmic regions of the T2S system machinery, when the ATPase GspE, equivalent to T4P extension ATPase PilB, interacted with the cytoplasmic domain of GspL, equivalent to PilM (34Lallemand M. Login F.H. Guschinskaya N. Pineau C. Effantin G. Robert X. Shevchik V.E. Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.PLoS One. 2013; 8: e79562Crossref PubMed Scopus (24) Google Scholar). The interaction itself, or the hydrolysis of ATP and resulting conformational changes in GspE, could induce a corresponding change in GspL, transduced to GspM through their interaction. Previous work in the T2S system and, more recently, the T4P system provided evidence for interactions between the equivalents of PilM and the cytoplasmic ATPase, PilB, either directly or in conjunction with the platform protein PilC (38Arts J. de Groot A. Ball G. Durand E. El Khattabi M. Filloux A. Tommassen J. Koster M. Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF).Microbiology. 2007; 153: 1582-1592Crossref PubMed Scopus (32) Google Scholar39Robert V. Filloux A. Michel G.P. Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa.FEMS Microbiol. Lett. 2005; 252: 43-50Crossref PubMed Scopus (30) Google Scholar, 40Bischof L.F. Friedrich C. Harms A. Søgaard-Andersen L. van der Does C. The type IV pilus assembly ATPase PilB of Myxococcus xanthus interacts with the inner membrane platform protein PilC and the nucleotide binding protein PilM.J. Biol. 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To test this idea, we introduced Cys substitutions at predicted interfaces between PilNO and PilOO, and we used non-reducing SDS-PAGE to observe dimer formation. Covalent PilN and PilO homodimers, as well as PilNO heterodimers, were captured in vivo under physiological conditions. Depending on the location, disulfide bonding disrupted the normal function of the T4P system, inhibiting motility. Deletion of the motor ATPases had no effect on the dimerization state of PilN and PilO, arguing against a link between motor function and conformational switching, but loss of either PilM or PilP specifically blocked PilNO heterodimer formation. These data provide new insights into the architecture and dynamics of the T4P system. Our PilO homodimer structure (Protein Data Bank code 2RJZ) (18Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (61) Google Scholar) was used to identify candidate residues predicted to be within appropriate distances for disulfide bond formation (Fig. 1A, top). In the PilO model, two relevant interfaces were identified as follows: the core, comprising mainly contacts between the α4-helix and β4-strand; and the coiled coils, between the α1- and α2-helices (18Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (61) Google Scholar). In the PilOΔ68 model, the coiled-coils are truncated and folded back, because they are not anchored in the IM. Because of the potentially artificial nature of that particular interaction interface, we focused on residues in the core region previously shown to be important for function (42Leighton T.L. Dayalani N. Sampaleanu L.M. Howell P.L. Burrows L.L. A novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations.J. Bacteriol. 2015; 197: 2229-2238Crossref PubMed Scopus (25) Google Scholar). Residues in the α4-helix and β4-strand of the PilO homodimer model with an estimated distance between β-carbons of less than 8 Å were selected for mutagenesis (Fig. 1A, bottom) (37Bass R.B. Butler S.L. Chervitz S.A. Gloor S.L. Falke J.J. Use of site-directed cysteine and disulfide chemistry to probe protein structure and dynamics: applications to soluble and transmembrane receptors of bacterial chemotaxis.Methods Enzymol. 2007; 423: 25-51Crossref PubMed Scopus (76) Google Scholar, 43Careaga C.L. Falke J.J. Thermal motions of surface α-helices in the d-galactose chemosensory receptor. Detection by disulfide trapping.J. Mol. Biol. 1992; 226: 1219-1235Crossref PubMed Scopus (218) Google Scholar, 44Careaga C.L. Falke J.J. Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies.Biophys. J. 1992; 62: 209-216Abstract Full Text PDF PubMed Scopus (76) Google Scholar). All attempts to purify soluble P. aeruginosa PilN for structural studies, or to solve an x-ray crystal structure of the PilNO heterodimer, have so far been unsuccessful. The structure of a periplasmic portion of PilN from T. thermophilus is available (29Karuppiah V. Collins R.F. Thistlethwaite A. Gao Y. Derrick J.P. Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: E4638-E4647Crossref PubMed Scopus (50) Google Scholar); however, due to the low primary sequence identity with PilN from P. aeruginosa, as well as a different arrangement of secondary structure elements (Thermus PilN has an extra α-helix inserted in the first αββ motif), we were unable to generate a high confidence model using that structural template. P. aeruginosa PilN is predicted to have a structural organization similar to PilO, and thus we used the truncated P. aeruginosa PilO homodimer structure (18Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (61) Google Scholar) to generate a high confidence Phyre2 (45Kelley L.A. Sternberg M.J. Protein structure prediction on the Web: a case study using the Phyre server.Nat. Protoc. 2009; 4: 363-371Crossref PubMed Scopus (3555) Google Scholar) model of a PilNΔ57 monomer, and we used the PilO homodimer interface to model a PilNO heterodimer using the same core-core interface (Fig. 1B, top) (42Leighton T.L. Dayalani N. Sampaleanu L.M. Howell P.L. Burrows L.L. A novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations.J. Bacteriol. 2015; 197: 2229-2238Crossref PubMed Scopus (25) Google Scholar). This model was used to design a series of single Cys substitutions in the α4-helix and β4-strand of either PilN or PilO (Fig. 1B, bottom). The PilNO model has been partially validated in previous work (42Leighton T.L. Dayalani N. Sampaleanu L.M. Howell P.L. Burrows L.L. A novel role for PilNO in type IV pilus retraction revealed by alignment subcomplex mutations.J. Bacteriol. 2015; 197: 2229-2238Crossref PubMed Scopus (25) Google Scholar), but for this study, we included a negative control Cys pair (PilNR142C/PilOA158C) at a Cβ distance of over 12 Å, considered too far for disulfide bond formation (37Bass R.B. Butler S.L. Chervitz S.A. Gloor S.L. Falke J.J. Use of site-directed cysteine and disulfide chemistry to probe protein structure and dynamics: applications to soluble and transmembrane receptors of bacterial chemotaxis.Methods Enzymol. 2007; 423: 25-51Crossref PubMed Scopus (76) Google Scholar, 44Careaga C.L. Falke J.J. Structure and dynamics of Escherichia coli chemosensory receptors.