The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates N-linked glycosylation of the Haemophilus influenzae HMW1 adhesin

The Haemophilus influenzae HMW1 adhesin is an important virulence exoprotein that is secreted via the two-partner secretion pathway and is glycosylated at multiple asparagine residues in consensus N-linked sequons. Unlike the heavily branched glycans found in eukaryotic N-linked glycoproteins, the modifying glycan structures in HMW1 are mono-hexoses or di-hexoses. Recent work demonstrated that the H. influenzae HMW1C protein is the glycosyltransferase responsible for transferring glucose and galactose to the acceptor sites of HMW1. An Actinobacillus pleuropneumoniae protein designated ApHMW1C shares high-level homology with HMW1C and has been assigned to the GT41 family, which otherwise contains only O-glycosyltransferases. In this study, we demonstrated that ApHMW1C has N-glycosyltransferase activity and is able to transfer glucose and galactose to known asparagine sites in HMW1. In addition, we found that ApHMW1C is able to complement a deficiency of HMW1C and mediate HMW1 glycosylation and adhesive activity in whole bacteria. Initial structure-function studies suggested that ApHMW1C consists of two domains, including a 15-kDa N-terminal domain and a 55-kDa C-terminal domain harboring glycosyltransferase activity. These findings suggest a new subfamily of HMW1C-like glycosyltransferases distinct from other GT41 family O-glycosyltransferases.     

Translocator proteins in the two-partner secretion family have multiple domains

The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, we characterized the functional domains of the Haemophilus influenzae HMW1B protein, a TpsB protein that interacts with the H. influenzae HMW1 adhesin. Using c-Myc epitope tag insertions and cysteine substitution mutagenesis, we discovered that HMW1B contains an N-terminal surface-localized domain, an internal periplasmic domain, and a C-terminal membrane anchor. Functional and biochemical analysis of the c-Myc epitope tag insertions and a series of HMW1B deletion constructs demonstrated that the periplasmic domain is required for secretion of HMW1 and that the C-terminal membrane anchor (HMW1B-(234-545)) is capable of oligomerization and pore formation. Similar to our observations with HMW1B, examination of a Bordetella pertussis TpsB protein called FhaC revealed that the C terminus of FhaC (FhaC-(232-585)) is capable of pore formation. We speculate that all TpsB proteins have a modular structure, with a periplasmic domain that interacts with the cognate TpsA protein and with pore forming activity contained within the C terminus.     

The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase,an enzyme involved in lipooligosaccharide biosynthesis

Non-typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non-typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co-immunoprecipitation and yeast two-hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1-mediated adherence, thus revealing a novel mechanism by which glycosylation influences cell-cell interactions.     

Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a periplasmic intermediate and requires the HMWB and HMWC proteins

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H . influenzae HMW1 and HMW2 non-pilus adhesins mediate attachment to human epithelial cells, an essential step during colonization. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involves cleavage of a 441-amino-acid N-terminal fragment. In the present study, we investigated the pathway for the secretion of HMW1 and HMW2. Cell fractionation experiments and cryoimmunoelectron microscopy demonstrated that a periplasmic intermediate occurs, suggesting involvement of the Sec machinery. Additional analysis revealed that, ultimately, the proteins are partially released from the surface of the organism. Studies with Escherichia coli harbouring plasmid subclones extended earlier findings and suggested that the secretion of HMW1 requires accessory proteins designated HMW1B and HMW1C, while the secretion of HMW2 requires proteins called HMW2B and HMW2C. Further analysis established that HMW1B/HMW1C and HMW2B/HMW2C are interchangeable, an observation consistent with the high degree of homology between HMW1B and HMW2B and between HMW1C and HMW2C. Additional studies of the hmw1 locus indicated that HMW1B is located in the outer membrane and serves to translocate HMW1 across the outer membrane. In the absence of HMW1B, HMW1 remains unprocessed and is degraded in the periplasmic space, at least in part by the DegP protease. Mutagenesis of an HMW1 N-terminal motif shared with other secreted proteins resulted in diminished processing and extracellular release, suggesting interaction of this motif with the HMW1B protein. Continued investigation of the HMW1 and HMW2 adhesins may provide general insights into protein secretion and bacterial pathogenesis.     

常见致病菌糖基转移酶的结构与功能

糖基转移酶(glycosyltransferases,GTs)将糖基从活化的供体转移到糖、脂、蛋白质和核酸等受体,其参与的蛋白质糖基化是最重要的翻译后修饰(post-translational modifications,PTMs)之一。近年来越来越多的研究证明,糖基转移酶与致病菌毒力密切相关,在致病菌的黏附、免疫逃逸和定殖等生物学过程中发挥关键作用。目前,已鉴定的糖基转移酶根据其蛋白质三维结构特征分为3种类型GT-A、GT-B和GT-C,其中常见的是GT-A和GT-B型。在致病菌中发挥黏附功能的糖基转移酶,在结构上属于GT-B或GT-C型,对致病菌表面蛋白质(黏附蛋白、自转运蛋白等)进行糖基化修饰,在致病菌黏附、生物被膜的形成和毒力机制发挥具有重要作用。糖基转移酶不仅参与致病菌黏附这一感染初始过程,其中属于GT-A型的一类致病菌糖基转移酶会进入宿主细胞,通过糖基化宿主蛋白质影响宿主信号传导、蛋白翻译和免疫应答等生物学功能。本文就常见致病菌糖基转移酶的结构及其糖基化在致病机制中的作用进行综述,着重介绍了特异性糖基化高分子量(high-molecular-weight,HMW)黏附蛋白的糖基转移酶、针对富丝氨酸重复蛋白(serine-rich repeat proteins,SRRP)糖基化修饰的糖基转移酶、细菌自转运蛋白庚糖基转移酶(bacterial autotransporter heptosyltransferase,BAHT)家族、N-糖基化蛋白质系统和进入宿主细胞发挥毒力作用的大型梭菌细胞毒素、军团菌(Legionella)葡萄糖基转移酶以及肠杆菌科的效应子NleB。为揭示致病菌中糖基转移酶致病机制的系统性研究提供参考,为未来致病菌的诊断、药物设计研发以及疫苗开发等提供科学依据和思路。     

Solution structure of Alg13: the sugar donor subunit of a yeast N-acetylglucosamine transferase

The solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae, is presented. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel beta sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.     

The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial two-partner secretion

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92 A of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed beta-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.     

Fold recognition analysis of glycosyltransferase families: further members of structural superfamilies

Glycosyltransferases (GTs) are diverse enzymes organized into 65 families. X-ray crystallography and in silico studies have shown many of these to belong to two structural superfamilies: GT-A and GT-B. Through application of fold recognition and iterated sequence searches, we demonstrate that families 60, 62, and 64 may also be grouped into the GT-A fold superfamily. Analysis of conserved acidic residues suggests that catalytic sites are better conserved in superfamily GT-B than in GT-A. Although 26% and 29% of GT families may now be confidently placed in superfamilies GT-A and GT-B, respectively, the remaining 45% of families bear no discernible resemblance to either superfamily, which, given the sensitivity of modern fold recognition methods, suggests the existence of novel structural scaffolds associated with GT activity. Furthermore, bioinformatics studies indicate the apparent ease with which mechanism-inverting or retaining-may change during evolution.     

The crystal structure of DR2241 from Deinococcus radiodurans at 1.9 A resolution reveals a multi-domain protein with structural similarity to chelatases but also with two additional novel domains

A unique family of proteins have been identified in the Deinococcus genus with an N-terminal cobalamin (vitamin B(12)) chelatase domain denoted CbiX and an additional unique C-terminal domain with unknown function. Here we report the first crystal structure from this new family of proteins with the structure of Deinococcus radiodurans protein DR2241. The structure reveals a multi-domain protein where domains A (residues 1-132) has the same fold as the small CbiX (CbiX(S)), domains A and B (residues 1-272) follow the chelatase super-family fold and the two additional unique domains C and D have no structural homologues. Domain D harbours the sequence motifs CxxC and CxxxC, in which DR2241 gives the first evidence that these motifs bind a [4Fe-4S] iron-sulphur cluster. In solution there are indications of multimeric forms, and in the crystallographic asymmetric unit a tetramer is found where domains C and D are involved in stabilising the tetrameric assembly.     

Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.     

Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1-48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-mediated adherence, arguing that the C-terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram-negative bacterial exoproteins.     

Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism

T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.     

X-ray crystal structure of leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a convergence of metal ion-independent glycosyltransferase mechanism

Leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Galbeta1-3[GlcNAcbeta1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Galbeta1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Galbeta1-3GalNAc at 2.0 and 2.7A resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Galbeta1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside diphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.     

Backbone assignments for the SPOUT methyltransferase MTT<Subscript><Emphasis Type="Italic">Tm</Emphasis></Subscript>, a knotted protein from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The SPOUT family of methyltransferase proteins is noted for containing a deep trefoil knot in their defining backbone fold. This unique fold is of high interest for furthering the understanding of knots in proteins. Here, we report the ¹H, ¹³C, ¹⁵N assignments for MTT _Tm , a canonical member of the SPOUT family. This protein is unique, as it is one of the smallest members of the family, making it an ideal system for probing the unique properties of the knot. Our present work represents the foundation for further studies into the topology of MTT _Tm , and understanding how its structure affects both its folding and function.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

Crystal structure of banana lectin reveals a novel second sugar binding site

Banana lectin (Banlec) is a dimeric plant lectin from the jacalin-related lectin family. Banlec belongs to a subgroup of this family that binds to glucose/mannose, but is unique in recognizing internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. Here we present the crystal structures of Banlec alone and with laminaribiose (LAM) (Glcbeta1, 3Glc) and Xyl-beta1,3-Man-alpha-O-Methyl. The structure of Banlec has a beta-prism-I fold, similar to other family members, but differs from them in its mode of sugar binding. The reducing unit of the sugar is inserted into the binding site causing the second saccharide unit to be placed in the opposite orientation compared with the other ligand-bound structures of family members. More importantly, our structures reveal the presence of a second sugar binding site that has not been previously reported in the literature. The residues involved in the second site are common to other lectins in this family, potentially signaling a new group of mannose-specific jacalin-related lectins (mJRL) with two sugar binding sites.     

Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria meningitidis W and Y Defines a New Sialyltransferase Family

Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)_n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)_n in NmY. The capsule polymerases, SiaD_W and SiaD_Y, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399–762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1–398) and C-terminal (aa 763–1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database).     

Structure of the Escherichia coli heptosyltransferase WaaC: binary complexes with ADP and ADP-2-deoxy-2-fluoro heptose

Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.     

Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non-typable Haemophilus influenzae (NTHI), mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to 70–75% of NTHI strains. NTHI strains that lack HMW1/ HMW2-like proteins remain capable of efficient attachment to cultured human epithelial cells, suggesting the existence of additional adhesion molecules. We reasoned that characterization of high-molecular-weight immunogenic proteins from an HMW1/HMW2-deficient strain might identify additional adhesion proteins. A genomic library was prepared in λEMBL3 with chromosomal DNA from non-typable Haemophilus strain 11, a strain that lacks HMW1/HMW2-like proteins. The library was screened immunologically with convalescent serum from a child naturally infected with strain 11, and phage clones expressing high-molecular-weight recombinant proteins were identified by Western blot analysis. One clone was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non-adherent Escherichia coli strain DH5α with plasmids containing the genetic locus encoding this protein gave rise to E. colitransformants that adhered avidly to Chang conjunctival cells. Subcloning and mutagenesis studies localized the DNA conferring the adherence phenotype to a 4.8 kbp fragment, and nucleotide sequence analysis further localized the gene encoding the adhesion protein to a 3.3 kbp open reading frame predicted to encode a protein of 114kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2-deficient non-typable H. influenzae strains. In contrast, the hia gene was not present in any of 23 non-typable H. influenzae strains which expressed HMW1/HMW2-like proteins. Identification of this second family of high-molecular-weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HMW1/HMW2-like proteins and Hia-like proteins which would be protective against disease caused by most or all non-typable H. influenzae     

Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis

Glycosyltransferases (GTs) are among the largest groups of enzymes found and are usually classified on the basis of sequence comparisons into many families of varying similarity (CAZy systematics). Only two different Rossman-like folds have been detected (GT-A and GT-B) within the small number of established crystal structures. A third uncharacterized fold has been indicated with transmembrane organization (GT-C). We here use a method based on multivariate data analyses (MVDAs) of property patterns in amino acid sequences and can with high accuracy recognize the correct fold in a large data set of GTs. Likewise, a retaining or inverting enzymatic mechanism for attachment of the donor sugar could be properly revealed in the GT-A and GT-B fold group sequences by such analyses. Sequence alignments could be correlated to important variables in MVDA, and the separating amino acid positions could be mapped over the active sites. These seem to be localized to similar positions in space for the alpha/beta/alpha binding motifs in the GT-B fold group structures. Analogous, active-site sequence positions were found for the GT-A fold group. Multivariate property patterns could also easily group most GTs annotated in the genomes of Escherichia coli and Synechocystis to proper fold or organization group, according to benchmarking comparisons at the MetaServer. We conclude that the sequence property patterns revealed by the multivariate analyses seem more conserved than amino acid types for these GT groups, and these patterns are also conserved in the structures. Such patterns may also potentially define substrate preferences.