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Structural Diversity in the Type IV Pili of Multidrug-resistant Acinetobacter*

Open AccessPublished:September 15, 2016DOI:https://doi.org/10.1074/jbc.M116.751099
      Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter. Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.

      Introduction

      Type IV pili are extracellular adhesive appendages primarily comprising a single protein subunit, called the major pilin, which is assembled into a narrow (∼6–9-nm) helical fiber of variable length (up to 2.5 μm) (
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      ). One or more other proteins, called minor pilins, are also incorporated into the fiber at low levels. All pilins contain an N-terminal signal sequence followed by an ∼30-amino acid hydrophobic α-helix resembling a transmembrane domain (the α1-N domain). This is, in turn, followed by a soluble ∼15-kDa globular domain referred to as the pilin headgroup; the hydrophobic helical regions are buried together in the center of the fiber, whereas portions of the C-terminal headgroup are exposed (
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      Type IV pilin proteins: versatile molecular modules.
      ,
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      Type IV pilus structure and bacterial pathogenicity.
      ).
      Type IV pili are found in both Gram-negative (
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      ,
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      Sequencing of the gene encoding the major pilin of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli and evidence that CFA/III is related to type IV pili.
      ) and Gram-positive (
      • Piepenbrink K.H.
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      Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile.
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      ) bacteria as well as Archea (
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      Diversity, assembly and regulation of archaeal type IV pili-like and non-type-IV pili-like surface structures.
      ). They are involved in a wide range of processes, including twitching motility (
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      ), horizontal gene transfer (
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      DNA transformation leads to pilin antigenic variation in Neisseria gonorrhoeae.
      ), host-cell adhesion (
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      • Yanagisawa T.
      • Kim K.S.
      • Yokoyama S.
      • Ohnishi M.
      Meningococcal PilV potentiates Neisseria meningitidis type IV pilus-mediated internalization into human endothelial and epithelial cells.
      ), and microcolony/biofilm formation (
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      ). This functional diversity is reflected in the sequence of the pilin proteins that typically have little or no sequence identity beyond the hydrophobic portion of the N-terminal α-helix. This lack of sequence identity is apparent even in cases where there is high structural similarity.
      In contrast, within a given species, the minor pilins are typically well conserved. Only the major pilin is highly variable (
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      ,
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      The frequency and rate of pilin antigenic variation in Neisseria gonorrhoeae.
      • Toma C.
      • Kuroki H.
      • Nakasone N.
      • Ehara M.
      • Iwanaga M.
      Minor pilin subunits are conserved in Vibrio cholerae type IV pili.
      ) and then only in those regions left exposed in the assembled pilus (
      • Blank T.E.
      • Zhong H.
      • Bell A.L.
      • Whittam T.S.
      • Donnenberg M.S.
      Molecular variation among type IV pilin (bfpA) genes from diverse enteropathogenic Escherichia coli strains.
      ). This sequence diversity may result from diversifying selection as a mechanism by which to avoid detection by the host immune system (
      • Maldarelli G.A.
      • De Masi L.
      • von Rosenvinge E.C.
      • Carter M.
      • Donnenberg M.S.
      Identification, immunogenicity, and cross-reactivity of type IV pilin and pilin-like proteins from Clostridium difficile.
      ). However, such diversity can also be found in species whose life cycle is primarily environmental (
      • Aagesen A.M.
      • Häse C.C.
      Sequence analyses of type IV pili from Vibrio choleraeVibrio parahaemolyticusVibrio vulnificus.
      ). Glycosylation is an additional source of variability in some type IV pilins; O-linked glycosylation has been observed in multiple strains of both Pseudomonas aeruginosa (
      • Allison T.M.
      • Conrad S.
      • Castric P.
      The group I pilin glycan affects type IVa pilus hydrophobicity and twitching motility in Pseudomonas aeruginosa 1244.
      ,
      • Smedley 3rd, J.G.
      • Jewell E.
      • Roguskie J.
      • Horzempa J.
      • Syboldt A.
      • Stolz D.B.
      • Castric P.
      Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function.
      • Voisin S.
      • Kus J.V.
      • Houliston S.
      • St-Michael F.
      • Watson D.
      • Cvitkovitch D.G.
      • Kelly J.
      • Brisson J.R.
      • Burrows L.L.
      Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with Mycobacterium-like α-1,5-linked d-Araf oligosaccharides.
      ) and Neisseria (
      • Aas F.E.
      • Vik A.
      • Vedde J.
      • Koomey M.
      • Egge-Jacobsen W.
      Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure.
      ,
      • Power P.M.
      • Seib K.L.
      • Jennings M.P.
      Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli.
      ). Additional glycosylation sites have been found in class II strains of Neisseria meningitidis where they have been hypothesized to play a role in immune evasion (
      • Gault J.
      • Ferber M.
      • Machata S.
      • Imhaus A.F.
      • Malosse C.
      • Charles-Orszag A.
      • Millien C.
      • Bouvier G.
      • Bardiaux B.
      • Péhau-Arnaudet G.
      • Klinge K.
      • Podglajen I.
      • Ploy M.C.
      • Seifert H.S.
      • Nilges M.
      • et al.
      Neisseria meningitidis type IV pili composed of sequence invariable pilins are masked by multisite glycosylation.
      ).
      Among the many genera of Gram-negative bacteria that express type IV pili is Acinetobacter, a coccobacillus that is widely distributed in nature and can be isolated from the environment in the soil and in water as well as from a variety of mammalian hosts (
      • Falagas M.E.
      • Kopterides P.
      Risk factors for the isolation of multi-drug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: a systematic review of the literature.
      ,
      • Peleg A.Y.
      • Seifert H.
      • Paterson D.L.
      Acinetobacter baumannii: emergence of a successful pathogen.
      ). Several species of Acinetobacter, chiefly Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, and Acinetobacter pittii, are collectively referred to as the A. calcoaceticus-baumannii (Acb)
      The abbreviations used are: Acb, A. calcoaceticus-baumannii; MBP, maltose-binding protein; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MPD, 2-methyl-2,4-pentanediol; PAK, P. aeruginosa strain K; OTase, oligosaccharyltransferase; r.m.s.d., root mean square deviation.
      complex and constitute an increasingly common source of nosocomial infections (
      • Dijkshoorn L.
      • Nemec A.
      • Seifert H.
      An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii.
      ,
      • Jones A.
      • Morgan D.
      • Walsh A.
      • Turton J.
      • Livermore D.
      • Pitt T.
      • Green A.
      • Gill M.
      • Mortiboy D.
      Importation of multidrug-resistant Acinetobacter spp infections with casualties from Iraq.
      • Harding C.M.
      • Kinsella R.L.
      • Palmer L.D.
      • Skaar E.P.
      • Feldman M.F.
      Medically relevant Acinetobacter species require a type II secretion system and specific membrane-associated chaperones for the export of multiple substrates and full virulence.
      ). Although reports of infections by A. baumannii predominate in the literature, phenotypic similarity makes it difficult to differentiate between related Acinetobacter species (
      • Chang H.C.
      • Wei Y.F.
      • Dijkshoorn L.
      • Vaneechoutte M.
      • Tang C.T.
      • Chang T.C.
      Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region.
      ), and in model organisms, all four species have been found to be infectious (
      • Antunes L.C.
      • Visca P.
      • Towner K.J.
      Acinetobacter baumannii: evolution of a global pathogen.
      ).
      Like other Acinetobacter species, A. baumannii lacks flagella but exhibits twitching motility, which is dependent on type IV pili (
      • Harding C.M.
      • Tracy E.N.
      • Carruthers M.D.
      • Rather P.N.
      • Actis L.A.
      • Munson Jr., R.S.
      Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility.
      ). Type IV pili are also required for its natural transformation (
      • Harding C.M.
      • Tracy E.N.
      • Carruthers M.D.
      • Rather P.N.
      • Actis L.A.
      • Munson Jr., R.S.
      Acinetobacter baumannii strain M2 produces type IV pili which play a role in natural transformation and twitching motility but not surface-associated motility.
      ,
      • Wilharm G.
      • Piesker J.
      • Laue M.
      • Skiebe E.
      DNA uptake by the nosocomial pathogen Acinetobacter baumannii occurs during movement along wet surfaces.
      ), but their role in other biological processes is unclear. Virstatin, a known inhibitor of type IV pilus formation in Vibrio cholerae, was shown to both reduce type IV pilus expression and inhibit biofilm formation in A. baumannii (
      • Nait Chabane Y.
      • Mlouka M.B.
      • Alexandre S.
      • Nicol M.
      • Marti S.
      • Pestel-Caron M.
      • Vila J.
      • Jouenne T.
      • Dé E.
      Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii.
      ). In another study, no correlation could be demonstrated between antigenic variation in the A. baumannii major pilin, pilA, and biofilm formation in vitro (
      • Eijkelkamp B.A.
      • Stroeher U.H.
      • Hassan K.A.
      • Papadimitrious M.S.
      • Paulsen I.T.
      • Brown M.H.
      Adherence and motility characteristics of clinical Acinetobacter baumannii isolates.
      ). More recently, Oh and Choi (
      • Oh M.H.
      • Choi C.H.
      Role of LuxIR homologue AnoIR in Acinetobacter nosocomialis and the effect of virstatin on the expression of anoR gene.
      ) reported that deletion of a LuxR-type regulator, AnoR, reduces both biofilm formation and surface motility in A. nosocomialis ATCC 17903. In addition to variation in the sequence of pilA, some Acinetobacter strains utilize an O-oligosaccharyltransferase to specifically glycosylate the major pilin at a C-terminal serine with the major capsule polysaccharide repeat unit (
      • Harding C.M.
      • Nasr M.A.
      • Kinsella R.L.
      • Scott N.E.
      • Foster L.J.
      • Weber B.S.
      • Fiester S.E.
      • Actis L.A.
      • Tracy E.N.
      • Munson Jr, R.S.
      • Feldman M.F.
      Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins.
      ,
      • Hu D.
      • Liu B.
      • Dijkshoorn L.
      • Wang L.
      • Reeves P.R.
      Diversity in the major polysaccharide antigen of Acinetobacter baumannii assessed by DNA sequencing, and development of a molecular serotyping scheme.
      ). These post-translational modifications are independent of the more general protein O-glycosylation system common to many gammaproteobacteria (including P. aeruginosa and Dichelobacter nodosus) (
      • Iwashkiw J.A.
      • Seper A.
      • Weber B.S.
      • Scott N.E.
      • Vinogradov E.
      • Stratilo C.
      • Reiz B.
      • Cordwell S.J.
      • Whittal R.
      • Schild S.
      • Feldman M.F.
      Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.
      • Lees-Miller R.G.
      • Iwashkiw J.A.
      • Scott N.E.
      • Seper A.
      • Vinogradov E.
      • Schild S.
      • Feldman M.F.
      A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii.
      ,
      • Cagatay T.I.
      • Hickford J.G.
      Glycosylation of type-IV fimbriae of Dichelobacter nodosus.
      • DiGiandomenico A.
      • Matewish M.J.
      • Bisaillon A.
      • Stehle J.R.
      • Lam J.S.
      • Castric P.
      Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity.
      ). However, Harding et al. (
      • Harding C.M.
      • Nasr M.A.
      • Kinsella R.L.
      • Scott N.E.
      • Foster L.J.
      • Weber B.S.
      • Fiester S.E.
      • Actis L.A.
      • Tracy E.N.
      • Munson Jr, R.S.
      • Feldman M.F.
      Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins.
      ) reported that pilin C-terminal glycosylation is not required for either competence or twitching motility.
      To understand the basis for the variability in sequence and glycosylation of Acinetobacter PilA, we have resolved the x-ray crystal structures of the major type IV pilin from three members of the Acb complex, strains ACICU and BIDMC 57 of A. baumannii and strain M2 of A. nosocomialis. In these three structures, we observe structural divergence independent of species within Acinetobacter. We demonstrate that Acinetobacter type IV pili promote host-cell adhesion in a manner independent of C-terminal glycosylation. We also provide evidence that the structural variation of Acinetobacter pilins is underpinned by functional differentiation.

      Discussion

      From an evolutionary standpoint, the x-ray crystal structures reported here pose three questions for us. Why have the major pilins of A. baumannii diverged? Why are some, but not all, PilA proteins C-terminally glycosylated? And why do the major pilins from A. baumannii ACICU and BIDMC 57 resemble their counterparts from other bacterial species (P. aeruginosa and D. nodosus, respectively) more closely than they do each other?
      The presence of close homologs to both PilAACICU and PilABIDMC57 in all four species that make up the Acb complex strongly implies that the divergence in pilA predates the divergence of A. baumannii and A. nosocomialis. This, combined with the similarities between PilAACICU and PilAPAK and between PilABIDMC57 and FimA (serotype A), suggest that the divergence in Acinetobacter pilA is not due to functionally neutral diversifying selection, as is thought to be the case in Neisseria pilE, but instead due to functionally divergent evolution.
      Determining which selective pressures favor a PilAACICU/PilAPAK-like structure over that of PilABIDMC57 and FimA (or vice versa) is more difficult, but possibilities include altered binding specificity and stability under different environmental conditions. We note that both Acinetobacter and Pseudomonas inhabit a wide range of environments and that both genera as well as Dichelobacter can be isolated from soil. Differing types of soil or solid surfaces may favor one structure over another.
      Another possibility is that some Acinetobacter type IV pilus systems are optimized for one function (horizontal gene transfer, twitching motility, or adherence) over another. Direct comparisons of twitching motility between A. nosocomialis M2 (cluster I) and A. baumannii ATCC 17978 (cluster III) do show somewhat greater motility for M2 (supplemental Fig. 4), but this complex process is impacted by many factors in addition to the sequence, structure, and function of PilA.
      The related question of why Acinetobacter pilA genes have diverged into glycosylated and non-glycosylated forms is complicated by the fact that no functional gain or defect has been attributed to the C-terminal glycan in Acinetobacter, and both tfpO− and tfpO+ strains have been shown to be infectious (
      • Jacobs A.C.
      • Thompson M.G.
      • Black C.C.
      • Kessler J.L.
      • Clark L.P.
      • McQueary C.N.
      • Gancz H.Y.
      • Corey B.W.
      • Moon J.K.
      • Si Y.
      • Owen M.T.
      • Hallock J.D.
      • Kwak Y.I.
      • Summers A.
      • Li C.Z.
      • et al.
      AB5075, a highly virulent isolate of Acinetobacter baumannii, as a model strain for the evaluation of pathogenesis and antimicrobial treatments.
      ,
      • Jones C.L.
      • Clancy M.
      • Honnold C.
      • Singh S.
      • Snesrud E.
      • Onmus-Leone F.
      • McGann P.
      • Ong A.C.
      • Kwak Y.
      • Waterman P.
      • Zurawski D.V.
      • Clifford R.J.
      • Lesho E.
      Fatal outbreak of an emerging clone of extensively drug-resistant Acinetobacter baumannii with enhanced virulence.
      ). Similar results were obtained for the ΔtfpO mutant of P. aeruginosa 1244, which was also found to be equally susceptible to phage attachment (
      • Smedley 3rd, J.G.
      • Jewell E.
      • Roguskie J.
      • Horzempa J.
      • Syboldt A.
      • Stolz D.B.
      • Castric P.
      Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function.
      ). Also arguing against a functional role for C-terminal glycans is the lack of correlation between polysaccharide and polypeptide composition; for example, PilA proteins from A. baumannii ATCC 19606 and A. nosocomialis M2 are 93% identical, but the major polysaccharide glycans from these strains are completely unrelated (supplemental Fig. 5) (
      • Harding C.M.
      • Nasr M.A.
      • Kinsella R.L.
      • Scott N.E.
      • Foster L.J.
      • Weber B.S.
      • Fiester S.E.
      • Actis L.A.
      • Tracy E.N.
      • Munson Jr, R.S.
      • Feldman M.F.
      Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins.
      ,
      • Scott N.E.
      • Kinsella R.L.
      • Edwards A.V.
      • Larsen M.R.
      • Dutta S.
      • Saba J.
      • Foster L.J.
      • Feldman M.F.
      Diversity within the O-linked protein glycosylation systems of Acinetobacter species.
      ). Taken together, these findings imply that even gross alterations to the exposed surface of the major pilin have little functional impact and suggest that some or all of these binding events may occur not through the major pilin but rather through the minor pilins.
      It was this lack of observable phenotype that led us to search for alternative explanations for the prevalence of tfpO-mediated glycosylation in Acinetobacter. Previous work in P. aeruginosa 1244, demonstrating that a ΔtfpO mutant was more vulnerable to phagocytosis mediated by opsonization (
      • Tan R.M.
      • Kuang Z.
      • Hao Y.
      • Lau G.W.
      Type IV pilus of Pseudomonas aeruginosa confers resistance to antimicrobial activities of the pulmonary surfactant protein-A.
      ,
      • Tan R.M.
      • Kuang Z.
      • Hao Y.
      • Lee F.
      • Lee T.
      • Lee R.J.
      • Lau G.W.
      Type IV pilus glycosylation mediates resistance of Pseudomonas aeruginosa to opsonic activities of the pulmonary surfactant protein A.
      ), implied that C-terminal glycosylation formed an obstacle to binding by host immune proteins. Our quantification of the ability of Acinetobacter C-terminal glycans to mask their conjugate polypeptides shows that over 25% of the PilA surface area available for binding is occluded. C-terminal glycosylation should, therefore, offer an advantage provided that the glycan surface is less vulnerable to binding by antibodies or other opsonins.
      The evolutionary distance between A. baumannii BIDMC 57 (and A. nosocomialis M2) and D. nodosus suggests that the close resemblance between their respective pilin proteins is the result of convergent evolution. Although the functional benefit of this fold to the soil gammaproteobacteria found in class I of the alignment in Fig. 6 remains to be determined, it seems unlikely that the absence of a C-terminal disulfide bond in 12 of the 14 cluster I sequences is due to chance. We speculate that the cluster I fold may be advantageous in an anaerobic environment.
      A further implication of the diversity in Acinetobacter type IV pilins is the challenge it poses for vaccine development. Because of their abundance in the extracellular space, type IV pili are obvious candidates for subunit vaccines and have been successfully used as such for other bacteria, including D. nodosus (
      • Bhardwaj V.
      • Dhungyel O.
      • de Silva K.
      • Whittington R.J.
      Investigation of immunity in sheep following footrot infection and vaccination.
      ,
      • Korpi F.
      • Irajian G.
      • Mahadavi M.
      • Motamedifar M.
      • Mousavi M.
      • Laghaei P.
      • Raei N.
      • Behrouz B.
      Active immunization with recombinant PilA protein protects against Pseudomonas aeruginosa infection in a mouse burn wound model.
      ). However, in Acinetobacter, the combination of variability in the PilA polypeptide with variation in polysaccharide structure in many strains may present a significant barrier to inducing a robust and durable immune response.
      In conclusion, the results presented here reveal that type IV pili in Acinetobacter have diverged in a manner unrelated to the genetic divergence of species within the Acb complex and that similarities in type IV pili cross species, genus, and family lines. These data reinforce the principle that functional requirements determine protein structure while allowing considerable variation in sequence. These data also imply that three distinct functional classes of type IV pili exist in Acinetobacter and other soil gammaproteobacteria.

      Author Contributions

      K. H. P. conducted most of the experiments, analyzed the results, and wrote the paper. E. L. conducted the cell binding measurements. C. M. H. created the mutant Acinetobacter strains used in this study (with the aid of R. S. M.) and provided valuable experimental input and commentary during writing. J. W. L. and X. Z. performed the glycan conformational simulations. C. A. R. conducted the in vitro biofilm formation experiments. S. E. G., M. F. F., J. J. G., and E. J. S. helped to coordinate the study and write the paper.

      Acknowledgments

      We thank the staff at Argonne National Laboratory Advanced Photon Source, General Medical Sciences and Cancer Institutes Structural Biology Facility, beam lines 23ID-D and 23ID-B, and the staff at Stanford Synchrotron Radiation Lightsource, beam line 12-2, for technical assistance with x-ray data collection. We also thank Dr. Angela Wilks for the use of the circular dichroism spectrophotometer.

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