Portal de Periódicos da CAPES

Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins

2000; Springer Nature; Volume: 19; Issue: 22 Linguagem: Inglês

10.1093/emboj/19.22.5989

1460-2075

Egbert Hoiczyk, Andreas Roggenkamp, Marisa Reichenbecher, Andrei N. Lupas, Jürgen Heesemann,

Streptococcal Infections and Treatments

Article15 November 2000free access Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins Egbert Hoiczyk Egbert Hoiczyk Present address: Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA Search for more papers by this author Andreas Roggenkamp Andreas Roggenkamp Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Marisa Reichenbecher Marisa Reichenbecher Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Andrei Lupas Andrei Lupas SmithKline Beecham Pharmaceuticals UP1345, 1250 South Collegeville Road, Collegeville, PA, 19426-0989 USA Search for more papers by this author Jürgen Heesemann Corresponding Author Jürgen Heesemann Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Egbert Hoiczyk Egbert Hoiczyk Present address: Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA Search for more papers by this author Andreas Roggenkamp Andreas Roggenkamp Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Marisa Reichenbecher Marisa Reichenbecher Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Andrei Lupas Andrei Lupas SmithKline Beecham Pharmaceuticals UP1345, 1250 South Collegeville Road, Collegeville, PA, 19426-0989 USA Search for more papers by this author Jürgen Heesemann Corresponding Author Jürgen Heesemann Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany Search for more papers by this author Author Information Egbert Hoiczyk2, Andreas Roggenkamp1, Marisa Reichenbecher1, Andrei Lupas3 and Jürgen Heesemann 1 1Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, Ludwig Maximilians University Munich, Pettenkoferstraße 9a, D-80336 München, Germany 2Present address: Laboratory of Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA 3SmithKline Beecham Pharmaceuticals UP1345, 1250 South Collegeville Road, Collegeville, PA, 19426-0989 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5989-5999https://doi.org/10.1093/emboj/19.22.5989 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The non-fimbrial adhesins, YadA of enteropathogenic Yersinia species, and UspA1 and UspA2 of Moraxella catarrhalis, are established pathogenicity factors. In electron micrographs, both surface proteins appear as distinct 'lollipop'-shaped structures forming a novel type of surface projection on the outer membranes. These structures, amino acid sequence analysis of these molecules and yadA gene manipulation suggest a tripartite organization: an N-terminal oval head domain is followed by a putative coiled-coil rod and terminated by a C-terminal membrane anchor domain. In YadA, the head domain is involved in autoagglutination and binding to host cells and collagen. Analysis of the coiled-coil segment of YadA revealed unusual pentadecad repeats with a periodicity of 3.75, which differs significantly from the 3.5 periodicity found in the Moraxella UspAs and other canonical coiled coils. These findings predict that the surface projections are formed by oligomers containing right- (Yersinia) or left-handed (Moraxella) coiled coils. Strikingly, sequence comparison revealed that related proteins are found in many proteobacteria, both human pathogenic and environmental species, suggesting a common role in adaptation to specific ecological niches. Introduction Many pathogenic bacteria assemble multifunctional proteinaceous structures on their surface that serve as adhesins and/or defense shields against the bactericidal attack of the host. Bacterial adhesins can be divided into three groups: (i) pili, forming hair-like structures, 2–10 nm in diameter; (ii) thin fibers called aggregative pili or curli; and (iii) non-pilus-associated adhesins, which are monomeric or oligomeric proteins anchored to the outer membrane (Hultgren et al., 1993). Typical members of the latter group of non-pilus-associated adhesins are the invasin (Inv) and the virulence plasmid (pYV)-encoded Yersinia adhesin (YadA) of the enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis, and the ubiquitous surface proteins UspA1 and UspA2 of Moraxella catarrhalis. In Y.enterocolitica, adhesion is crucial for pathogenicity (reviewed in Cornelis et al., 1998; Aepfelbacher et al., 1999). For the initial step of invasion of the intestinal mucosa, Inv is required for interaction with β1-integrins of M cells (Isberg and Tran von Nhieu, 1995). After invasion, YadA predominates as adhesin in infected tissue. YadA mediates adherence to epithelial cells (Heesemann and Grüter, 1987), to professional phagocytes (Roggenkamp et al., 1996) and extracellular matrix (ECM) proteins (Emödy et al., 1989; Schulze-Koops et al., 1992, 1993; Flügel et al., 1994). YadA also protects the bacterium against complement and defensin lysis (Balligand et al., 1985; Pilz et al., 1992; Visser et al., 1996). Moreover, YadA is involved in autoagglutination, a phenomenon occurring after growth in tissue culture medium at 37°C (Skurnik et al., 1984). The biological function of the UspAs in Moraxella is not characterized as well. Nevertheless, the UspA proteins seem to share certain functional aspects with YadA: (i) UspA1 is essential for cell attachment; and (ii) UspA2 mediates serum resistance (Aebi et al., 1998; Lafontaine et al., 2000). Sequencing of yadA genes of diverse Yersinia predicted polypeptides of 41–44 kDa, depending on species and serotype (Skurnik and Wolf-Watz, 1989), whereas uspA1 and uspA2 sequences of M.catarrhalis O35E predicted 83 and 60 kDa proteins, respectively (Aebi et al., 1997). In SDS–PAGE, all these proteins form heat-stable aggregates with an apparent mol. wt of 160–250 kDa, suggesting oligomerization of 3–4 monomers in YadA (Tamm et al., 1993; Mack et al., 1994; Gripenberg-Lerche et al., 1995) or 2–3 monomers in the UspAs (Cope et al., 1999). Variously mutated forms of YadA have been constructed to correlate functional features of the adhesin with different regions within the sequence of the YadA poly peptide. Truncation of the C-terminus abrogated functional expression of YadA and the formation of oligomeric forms (Tamm et al., 1993), whereas the deletion of amino acid residues 29–81 (YadAΔ29–81) led to the loss of only one function, i.e. adherence to neutrophils (Roggenkamp et al., 1996). The abrogation of binding to ECM and HeLa cells could be obtained without impairing the other functions of YadA by the replacement of histidine residues His156 and 159 with tyrosine residues (YadA-2; Roggenkamp et al., 1995). Even more severe distortions (loss of collagen binding and autoagglutination) of the YadA functions resulted from the deletion of the hydrophobic stretch of residues 83–104 (Tamm et al., 1993). Recently, it was also shown that NSVAIG-S motifs in the N-terminal half of the YadA molecule are involved in collagen binding (Tahir et al., 2000). Analysis of the UspA1 and UspA2 amino acid sequence indicated that the two proteins are only 43% identical. Both UspAs contain an internal segment of 140 amino acid length with 93% identity, which is present in all disease-associated Moraxella strains tested so far (Aebi et al., 1997). Furthermore, both proteins are predicted to possess internal regions with a high coiled coil probability (Cope et al., 1999). However, detailed functional mapping studies, as for YadA, are still missing for UspAs. Despite some effort, the structural organization of this class of adhesin remains poorly characterized. Previous electron microscope studies of Y.enterocolitica cells reported very inconsistent results of a tack-like or 50–70 nm long fibrillar structure of YadA (Lachica et al., 1984; Kapperud et al., 1985, 1987; Zaleska et al., 1985). Therefore, we used electron microscopy to analyze the cell envelopes of Y.enterocolitica serotype 08 and 03 (producing 41 and 44 kDa YadA, respectively) and M.catarrhalis O35E, as well as isogenic mutants with abrogated YadA or UspA1/UspA2 production. Moreover, we purified YadA and genetically modified YadA oligomers in order to establish a structure–function relationship for these surface molecules. Our refined investigation of the cell envelopes demonstrated that YadA and the UspAs form 'lollipop'-shaped surface projections that have not been described before. Comparisons with sequence data from ongoing genome projects show that proteins related to YadA and UspA1/UspA2 are found in many proteobacteria, both free-living and pathogenic, thus indicating the existence of a novel class of adhesins in proteobacteria. Results Structure of the cell envelopes of Y.enterocolitica Yersinia enterocolitica serotype O8, strain WA-314 and its plasmid-less derivative WA-C were grown in tissue culture medium at 37°C to stationary phase (optimal condition for yadA expression), harvested and then cryosubstituted. Cross-sections of the envelope profiles of the isogenic cells appeared quite different (Figure 1). Proceeding from inside, the typical Gram-negative cell wall was covered with an additional layer on the top of the outer membrane, resembling a canopy of 23 nm width (Figure 1A). This extracellular layer was anchored to the outer membrane by pillar-like stalks, thereby forming a 'quasi-periplasmic space' between the heads of the projections and the outer membrane, so far only described for archaebacteria (Peters et al., 1995). In conventionally prepared cells, this structure was either completely lost or collapsed upon preparation, resulting in a 'fuzzy' surface coat (results not shown). The canopy-like surface layer was not seen with strain WA-C (Figure 1B), suggesting that the projections of WA-314 are composed of YadA molecules. This could be confirmed by immunoelectron microscopy, by labeling of thin sections of Y.enterocolitica WA-314 cells with a purified monoclonal antibody against YadA (Figure 2). Figure 1.Electron micrographs showing cross-sections of the isogenic pair of Y.enterocolitica (serotype O8) grown in RPMI medium at 37°C. (A) The cell envelope of the virulence plasmid-harboring strain WA-314 is covered with a canopy-like layer formed by YadA. (B) Note the absence of this additional surface structure on the cells of the plasmid-cured strain WA-C. Bar, 100 nm. Download figure Download PowerPoint Figure 2.Electron micrograph of aldehyde-fixed and immunogold-labeled Y.enterocolitica strain WA-314. The cells were embedded in Epon and thin sectioned. Labeling was carried out first with anti-YadA IgG and secondly with gold-labeled (10 nm) goat anti-mouse IgG. The labeling is almost exclusively associated with the YadA surface coat of the cells. Bar, 100 nm. Download figure Download PowerPoint Ultrastructure of isolated native or mutated YadA oligomers In order to define better the molecular architecture of the filigree components of the canopy-like layer, whole cells or isolated cell envelopes were negatively stained. As can be seen in Figure 3B and D, the cell surface of WA-314 was densely covered with 'lollipop'-shaped surface projections that are absent on the surface of WA-C (Figure 3A and C). The individual YadA 'lollipops' had an overall length of ∼23 nm, consisting of a stem-like segment of ∼18 nm (stalk domain) and a bulky head domain of ∼5 nm. The size and shape of these projections not only matches the dimension of the YadA layer in thin sections, but also easily explains the canopy-like appearance of this layer in the sections seen in Figure 1A. Figure 3.Ultrastructural analysis of negatively stained whole cells (A and B) and isolated cell envelopes (C and D) of the isogenic pair of Y.enterocolitica serotype O8. (A) Smooth surface of the plasmid-cured derivative WA-C. (B) Appearance of the cell surface of the plasmid-harboring strain WA-314, which is densely covered with 'lollipop'-shaped YadA oligomers. (C) Cell envelopes of WA-C. (D) Cell envelopes of WA-314. The 'lollipop' structure of single YadA oligomers can easily be seen with a length of ∼23 nm. Bars, 50 nm. Download figure Download PowerPoint To further substantiate these results, we investigated four genetically modified YadA mutants of the serotype O8 strain WA-314, and the YadA of the serotype O3 strain Y-108 (Figure 4; Table I). The previously constructed N-terminal-truncated mutant YadAΔ29–81 showed a 50% decrease in the size of the head domain in comparison with the wild-type YadA, whereas the mutant YadA-2 (H156→Y/H159→Y substitutions result in the loss of binding to HeLa cells and ECM) forms 'lollipop' structures indistinguishable from wild-type YadA (Figure 4A, B and D). In order to obtain more information about the stalk domain of the molecule, internally truncated mutants of YadA strain WA-314 were studied. Predictions of the coiled-coil structure derived from the amino acid sequence of the YadA of Y.enterocolitica serotype O8 suggested that seven pentadecad repeats probably constitute the stalk domain (see below on the amino acid sequence analysis of YadA and related proteins, and Figure 5). YadAΔ184–266 and YadAΔ184–318 lack 40 amino acids of the putative head domain plus three (YadAΔ184–266) or six (YadAΔ184–318) of the seven pentadecad repeats. As can be seen in Figure 4F, the YadAΔ184–266 mutant still formed 'lollipop'-shaped projections, although with a stalk nearly half the length of the wild-type stalk. Compared with this, the further truncated YadAΔ184–318 did not show electron microscopically detectable surface projections, but autoagglutinated and reacted with YadA-specific antibodies, indicating surface exposure (result not shown). The hypothesis that the pentadecad repeats indeed constitute the stalk of the surface projection could be further substantiated by studying the YadA of Y.enterocolitica serotype O3 strain Y-108. This YadA sequence carries nine instead of the seven 15-residue repeats found in YadA of the serotype O8 strain. In agreement with this prediction, the YadA stalk of serotype O3 is found to be about a quarter longer than the stalk of the O8 strain (Figure 4E). Finally, the yadA null mutant strain WA(pYV08-A-0) expressing the plasmid-encoded type III protein secretion system was used as a negative control, and the Escherichia coli DH5α (pUC-A-1, pB8-5) carrying the cloned yadA gene and its transcriptional activator gene virF as a positive control. The yadA null mutant showed a smooth surface lacking 'lollipop'-like projections, similar to strain WA-C in Figure 3A and C, whereas the E.coli transformant showed patches of densely packed 'lollipops' on the surface identical to those structures seen with Y.enterocolitica strain WA-314 (data not shown). Additionally, we studied Y.enterocolitica serotype O8 strain 8081, which is distinct in genotype and lipopolysaccharide (LPS) composition (smooth form) from that of strain WA-314 (semi-rough form) (Gaede and Heesemann, 1995). In the electron microscope, both O8 serotypes formed surface projections indistinguishable from one another (data not shown). In conclusion, the structural predictions from the yadA sequences could be confirmed by electron microscopy. Figure 4.Comparison of the ultrastructure of YadA oligomers obtained from cell envelopes of various strains (except C). (A) Wild-type YadA of strain WA-314. (B) N-terminal-truncated YadAΔ29–81. Note the pronounced decrease in the size of the head domain of the oligomers. (C) The extraction of YadA with low pH glycine buffer does not change the ultrastructure of the 'lollipop'-shaped oligomers formed after dialysis. (D) YadA-2 obtained from strain WA(pYV08-A-2), which no longer binds collagen, but has the same appearance as the wild type and still shows the zipper-like interaction important for the autoagglutination of the cells. (E) YadA of Y.enterocolitica serotype O3 strain Y-108. The YadA of this serotype contains nine instead of seven pentadecad repeats in its sequence and therefore has a somewhat longer stalk. (F) This internally deleted mutant YadAΔ184–266 has lost three of the seven pentadecad repeats. Note the decrease in the length of its stalk. Bars, 50 nm. Download figure Download PowerPoint Figure 5.Simple illustration of the way YadA molecules may be arranged in a 'lollipop'-shaped structure (based on data from electron microscopy and amino acid sequence analysis). Left: schematic diagram of the YadA molecule with the N- and C-termini. Amino acid 26 indicates the cleavage site of the signal peptide. The predicted domain structures forming the head, the stalk and the membrane anchor, respectively, are indicated (see also Figures 8 and 9). Right: for simplicity, folding of only two YadA molecules into a 'lollipop'-shaped structure is shown, forming one-half (tetramer) or two-thirds (trimer) of the adhesin. The length of the stalk (right-handed coiled coil) and the diameter of the head are indicated in nanometers. The β-strands of the C-terminal region may form a β-barrrel within the outer membrane. Download figure Download PowerPoint Table 1. Bacterial strains used in this study Strain Description Reference Y.enterocolitica WA-314 clinical isolate, serotype O8, carrying the virulence plasmid pYVO8, YadA+ of O8 Heesemann et al. (1983) WA-C plasmid-cured derivative of WA-314 Heesemann et al. (1983) WA(pYVO8-A-0) WA-C, harboring pYVO8 with a Km-GenBlock insertion in yadA, YadA− Roggenkamp et al. (1995) WA(pYVO8-A-2) WA-C, harboring pYVO8 with integrated yadA-2 mutant gene, YadA-2+(H156Y, H159Y) Roggenkamp et al. (1995) WA(pYVO8-AΔ29–81) WA-C, harboring pYVO8 with integrated yadAΔ29–81, YadAΔ29–81+ Roggenkamp et al. (1996) WA(pYVO8-AΔ184–266) WA-C, harboring pYVO8 with integrated yadAΔ184–266, YadAΔ184–266+ this study WA(pYVO8-AΔ184–318) WA-C, harboring pYVO8 with integrated yadAΔ184–318, YadAΔ184–318+ this study Y-108 clinical isolate, serotype O3, carrying the pYVO3, YadA+ of O3 Heesemann et al. (1983) 8081 clinical isolate, serotype O8, carrying pYVO8, YadA+ Skurnik and Wolf-Watz (1989) E.coli DH5α(pUC-A-1, pB8-5) DH5α harboring the yadA gene of pYVO8 in pUC13 vector and the transcriptional activator gene virF for yadA expression in pRK290 vector, respectively, YadA+ Roggenkamp et al. (1996) M.catarrhalis 035E clinical isolate, UspA1+, UspA2+ Aebi et al. (1997) 035E12 derivative of 035E, UspA1−, UspA2− Aebi et al. (1998) Characterization of purified YadA of strain WA-314 YadA was extracted from whole cells or cell membranes by acid glycine buffer (pH 2.5). After dialysis of this extract, 'lollipop'-shaped vesicles could be visualized (Figure 4C). When 'lollipop'-covered small vesicles (see Materials and methods were incubated at room temperature with 1% Octyl-PoE, the 'lollipops' started to assemble into larger multilayered aggregates. In these multilayered aggregates, the YadA oligomers interact with each other via their hydrophobic membrane anchor domain, forming sheets. Once such sheets are formed, they attach to each other via their hydrophobic membrane domain side and in a zipper-like fashion via the tips of the 'lollipops', respectively (Figure 6A and B). The zipper-like interaction indicated that a more proximal part of the oval head domain mediates autoagglutination, as can be shown with whole cells (Figure 6C). Figure 6.Visualization of different characteristic features of YadA of strain WA-314. (A) After incubation of small YadA-covered vesicles with Octyl-POE, the oligomers start to aggregate with each other. The head domains interact in a zipper-like fashion, whereas the anchor domain forms membrane-like structures such as vesicles or sheets. (B) Prolonged incubation results in the formation of large, even macroscopically visible aggregates. (C) Electron micrograph of a cross-section of autoagglutinated Yersinia cells. The interaction of the cells is mediated by an interaction of the YadA layers on the cell surfaces. (D) SDS–PAGE (lane 1) and immunoblotting of purified YadA aggregates of WA-314. Lane 1, oligomeric YadA, Coomassie Blue stain; lane 2, molecular weight markers (kilodaltons); lanes 3 and 4, immunoblot showing oligomeric YadA and partially disintegrated YadA, respectively (monomer 41 kDa). Download figure Download PowerPoint In these experiments, successful purification of YadA could be achieved by simply picking up large aggregates with tweezers. SDS–PAGE and immunoblotting using YadA-specific monoclonal antibody confirmed that the aggregates were composed of oligomeric YadA (Figure 6D). The monomeric YadA band appeared after partial disintegration of the oligomeric form by short boiling in sample buffer. These results demonstrate that purified and detergent-solubilized YadA forms multilayered membranes containing well-ordered arrays of closely packed YadA protein, resembling that of the bacterial outer membrane. Solubilization experiments followed by electron microscope evaluation revealed that non- or zwitterionic detergents (e.g. 2% Triton X-100, 2% Genapol X-80, 2% Sulfobetaine, 2% sodium deoxycholate) or low concentrations of chaotropes (e.g. 4 M urea) did not affect the integrity of the YadA oligomers at all. However, anionic detergents (e.g. 2% SDS), high concentrations of chaotropes (e.g. 8 M urea) or low pH (<3) led to disintegration of YadA vesicles and 'lollipop'-shaped structure (results not shown). Ultrastructure of non-pilus-associated surface proteins in other proteobacteria: M.catarrhalis UspAs Results of sequence comparison revealed that 'lollipop'-shaped surface projections should also be present in members of proteobacteria other than Yersinia. As further examples, whole cells of M.catarrhalis O35E were negatively stained. After growth of M.catarrhalis O35E on blood agar at 37°C, the surface of these microorganisms was found to be densely covered with 'lollipop'-shaped surface projections that could not be seen in a uspA1/uspA2 double mutant (Figure 7). While the shape of the projections matched the architecture of the YadA projections, a major difference could be detected in the stalk of the 'lollipops'. The length of the rod-like segments of the UspA1/UspA2 projections was more than three times longer than the stalk of YadA, measuring up to ∼60 nm, whereas the globular head domains of YadA and UspA1/UspA2 were about the same size. The examination of UspA1 and UspA2 single knockout mutants showed that both proteins formed 'lollipop'-shaped surface projections, although their total number per cell was lower and UspA2 had somewhat shorter stalks than UspA1 (data not shown). Figure 7.Ultrastructure of surface projections in Moraxella. (A) Surface of M.catarrhalis strain O35E expressing its ubiquitous surface proteins UspA1 and UspA2. The surface of the cells is covered with 'lollipop'-shaped projections formed by the two related proteins. (B) Surface of the Moraxella O35E uspA1/uspA2 double mutant. (C) Isolated cell envelope of M.catarrhalis O35E showing the long rod-like stalk and the head domain of UspA1 and UspA2. Bars, 50 nm. Download figure Download PowerPoint Amino acid sequence analysis of YadA and related proteins Sequence analysis of YadA, UspA1 and UspA2 showed that their stalks are most likely formed by extended coiled-coil domains (Figure 8A). However, the coiled-coil-forming probabilities for YadA were surprisingly low, prompting us to search for unusual features in this sequence. A search for repetitive sequence patterns in the putative coiled-coil segment using Fast Fourier analysis (McLachlan and Stewart, 1976) revealed a strong 15-residue periodiocity with the highest harmonic peak at 3.75 (15/4) (Figure 8B) resulting from a set of degenerate 15-residue repeats recognizable in the sequence (Figure 9B). Secondary structure prediction and hydrophobic moment analysis suggested that the entire repeat region forms a strongly amphipathic α-helix, in agreement with the coiled-coil analysis. The observed periodicity of 3.75 residues per turn is significantly larger than the 3.5–3.6 typically observed in left-handed coiled coils (Seo and Cohen, 1993) or the 3.67 postulated for right-handed coiled coils (Peters et al., 1996). Structurally, it is most compatible with a tightly supercoiled right-handed coiled coil having a pitch of 11.5 nm, a pitch angle of ∼20° and a length of ∼17.5 nm, as compared with 18 nm measured in electron micrographs (see Figure 5). In contrast, the main periodicity of the UspA1 and UspA2 stalk sequences is 3.52, suggesting a canonical left-handed coiled coil. Figure 8.(A) Coiled-coil-forming probabilities for YadA of Y.enterocolitica serotype O8 strain 8081, and for UspA1 and UspA2 of M.catarrhalis. Probabilities were calculated in Coils 2.2 using the MTK matrix and a 28 residue window. The C-terminal peak in all three graphs corresponds to the coiled-coil segment shown in Figure 9C, which is conserved in all proteins with a YadA-like membrane anchor. (B) Fast Fourier analysis of hydrophobic residues in the putative head and stalk sequences. The head sequence shows peaks near 14 and 14/6 residues, and the stalk sequence shows peaks at all the main harmonics of 15. Download figure Download PowerPoint Figure 9.Multiple sequence alignment of YadA and related proteins. Organisms are arranged in the order shown in Table II. Sequences from complete genomes are labeled by their gene denotations and the start and end residues are numbered, except where the gene is frameshifted. Sequences from unfinished genomes are named, where necessary, by their contig number. The DDBJ/EMBL/GenBank accession numbers are shown at the end of (C). (A) Degenerate repeats of the putative YadA head sequence and their representation in related proteins. The consensus residues of the approximate 14-residue repeat are highlighted in red. Sequences showing this repeat pattern invariably end in a conserved neck sequence. (B) Pentadecad repeats of the putative YadA stalk sequence and their representation in related proteins. Consensus residues are highlighted in blue. (C) Conserved sequence elements of the YadA family of proteins, consisting of the neck sequence, a left-handed coiled-coil segment and a membrane anchor formed by four transmembrane β-strands. In proteins with a more extended stalk, this is inserted after the neck sequence in a continuation of the coiled coil. Conserved residues of the neck region are highlighted in blue, hydrophobic core residues of the coiled-coil segment in green and membrane-oriented residues of the transmembrane β-strands in red. A nearly invariant glycine residue that is conserved in an equivalent position in eight-stranded porins and autotransporters is shown in blue. The C-terminal β-strands of eight-stranded porins are represented by OmpA of E.coli (Eco OmpA), Ail of Y.enterocolitica (Yen Ail) and OpaA of Neisseria gonorrhoeae (Ngo OpaA); and of autotransporters by AidA of E.coli (Eco AidA) and the Bordetella pertussis pertactin P.68 (Bpe P.68). The secondary structure for the YadA family is the consensus prediction obtained from the Jpred server (h = α-helix; s = β-strand), and for OmpA it is the membrane-embedded part of the β-strands observed in the crystal structure (Pautsch and Schulz, 1998). Download figure Download PowerPoint Fast Fourier analysis of the putative YadA head sequence also revealed a periodic structure, with a repeating pattern of ∼14 residues (Figure 8B). In the sequence, this was recognizable as a succession of degenerate repeats containing an alternating pattern of branched chain aliphatic and small residues, followed by a position consisting mainly of Ala, Gly, Ser or Thr (Figure 9A). The same periodicity and repeat pattern were found in the head sequence of UspA1, but not of UspA2. Secondary structure prediction suggested that this repeat region consists primarily of β-strands. Sequence comparisons between YadA, UspA1 and UspA2 showed that all three sequences have a similar C-terminal domain (Figure 9C). In addition, YadA and UspA1 have similar head sequences, and UspA1 and UspA2 have similar stalk sequences, giving UspA1 the appearance of a mosaic protein. Searches in DDBJ/EMBL/GenBank and in the unfinished genomes database at NCBI yielded a surprising number of sequences that were clearly related to YadA and UspAs, from a phylogenetically diverse set of free-living and pathogenic proteobacteria (Table II). Several of these sequences appear to be frameshifted (including the yadA of Yersinia pestis), suggesting that they represent pseudogenes. The similarity was most pronounced in a short sequence element that is found in YadA between the head and stalk repeats, and that we therefore named the 'neck' (Figure 9A). Abo