A novel gingipain regulatory gene in Porphyromonas gingivalis mediates host cell detachment and inhibition of wound closure.

The black pigmentation-related genes in Porphyromonas gingivalis are primarily involved in regulating gingipain functions. In this study, we identified a pigmentation-related gene, designated as pgn_0361. To characterize the role of pgn_0361 in regulating P. gingivalis-mediated epithelial cell detachment and inhibition of wound closure, PgΔ0361, an isogenic pgn_0361-defective mutant strain, and PgΔ0361C, a complementation strain, were constructed using P. gingivalis ATCC 33277. The gingipain and hemagglutination activities, as well as biofilm formation, were examined in all three strains. The effect of P. gingivalis strains on epithelial cell detachment was investigated using the HO-1-N-1 and Ca9-22 epithelial cell lines. The inhibition of wound closure by heat-killed P. gingivalis cells and culture supernatant was analyzed using an in vitro wound closure assay. Compared to the wild-type strain, the PgΔ0361 strain did not exhibit gingipain or hemagglutination activity but exhibited enhanced biofilm formation. Additionally, the PgΔ0361 strain exhibited attenuated ability to detach the epithelial cells and to inhibit wound closure in vitro. Contrastingly, the culture supernatant of PgΔ0361 exhibited high gingipain activity and strong inhibition of wound closure. The characteristics of PgΔ0361C and wild-type strains were comparable. In conclusion, the pgn_0361 gene is involved in regulating gingipains. The PGN_0361-defective strain exhibited reduced virulence in terms of epithelial cell detachment and inhibition of wound closure. The culture supernatant of the mutant strain highly inhibited wound closure, which may be due to high gingipain activity.

Comparative analysis of virulence factors & biotypes of Gardnerella vaginalis isolated from the genital tract of women with & without bacterial vaginosis.

BACKGROUND & OBJECTIVES: : Bacterial vaginosis (BV) involves the presence of a thick vaginal multispecies biofilm, where Gardnerella vaginalis is the predominant species. The reason for an increase in the number of G. vaginalis which are usually present as normal flora of the female genital tract in cases of BV, is not known. Hence, the objective of the present study was to compare the biotypes and virulence factors of G. vaginalis isolated from the genital tract of women with and without BV. METHODS: : High vaginal swabs collected from 811 women of reproductive age were cultured. G. vaginalis isolates were biotyped and tested for adherence to vaginal epithelial cells, biofilm formation, agglutination of human red blood cells (RBCs), protease production, phospholipase production and surface hydrophobicity. RESULTS: : Of the isolates from women with BV, 83.3 per cent (60/72) showed good adherence, 78.4 per cent (58/74) produced biofilm, 82.9 per cent (63/76) produced phospholipase, 67.1 per cent (51/76) produced protease, 77.3 per cent (58/75) were positive for surface hydrophobicity and 61.6 per cent (45/73) were positive for haemagglutination of human RBC. In case of G. vaginalis from non-BV women, 25 per cent (15/60) isolates showed good adherence, 18.4 per cent (9/49) biofilm production, 35 per cent (21/60) phospholipase, 36.6 per cent (22/60) protease, 41.7 per cent (25/60) surface hydrophobicity and 10.1 per cent (6/59) agglutination of human RBCs. Maximum number of isolates belonged to biotypes 6, 2 and 3. Biotype 3 was more associated with non-BV rather than BV; biotype 6, 2 and 1 were more associated with cases of BV. Maximum virulence factors were expressed by biotypes 6, 2 and 1. INTERPRETATION & CONCLUSIONS: : Virulence factors were more expressed by G. vaginalis isolates obtained from women with BV rather than from non-BV. Biotypes 6, 2 and 1 were more associated with cases of BV and expressed maximum virulence factors.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes.

Sequence analysis of the hypervariable region in hmtp210 of Avibacterium paragallinarum.

The hmtp210 gene of Avibacterium paragallinarum, the causative agent of infectious coryza, encodes an outer-membrane hemagglutinin (HA) that plays an essential role in pathogenicity. A hypervariable region within this HA, which is highly antigenic, is proposed as a candidate for recombinant vaccine production. Nonetheless, little is known about its genetic variability. We performed sequencing analysis of the hmtp210 hypervariable region in 16 clinical isolates from Costa Rica and compared them with 4 vaccine strains and the hmtp210 sequences available in public databases. Except for isolate ApCR12, all isolates showed high identity with reference vaccine strains 0083 and H18. Better genetic characterization of the hypervariable region of hmtp210 is necessary to develop better immunogenic strategies and improved molecular typing methods.

MN typing discrepancies based on GYPA-B-A hybrid.

BACKGROUND AND OBJECTIVES: Gene conversion events between GYPA and GYPB or GYPA and GYPE are facilitated by the close chromosomal proximity and high degree of sequence homology and can lead to the formation of GP hybrid genes. Discrepant results between blood group genotyping and haemagglutination in 22 random blood donors induced molecular characterization. MATERIALS AND METHODS: Sequence analysis of GYPA exons 1-7 and GYPB exons 1-5 was performed for gDNA and cDNA. The linkage of the nucleotide alterations was defined by haplotype separation. RESULTS: DNA analysis demonstrated a normal GYPA haplotype (GYPA*N n = 20, GYPA*M n = 2) with an altered GP hybrid nucleotide sequence in trans. A GYPB homologue sequence of minimal 10-bp encompassing intron 1 and exon 2 was translated into GYPA, accounting for an amino acid substitution from arginine to glutamic acid at position 13 (38 C>A). Genomic DNA analysis demonstrated the cis-linkage of the hybrid nucleotide sequence with each GYPA(Ser20, Gly24) (n = 20) associated with the expression of M and GYPA(Leu20, Glu24) (n = 2) encoding the N phenotype. The serologic data indicate that the changes do not affect the expression of a normal M and N antigen. cDNA sequences confirmed the gDNA results and furthermore identified a heterozygous deletion of GYPB exon 2 in all probands. CONCLUSION: The results document a GYPA-B-A hybrid gene, probably produced via a single unequal homologous recombination event. A segmental transfer of GYPB seems most likely accounting for the allelic dropout.

Quantitative assessment of Mycoplasma hemadsorption activity by flow cytometry.

A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

Apoptosis of granulocytes and lymphocytes in peripheral blood in rabbits infected with haemagglutinating and non-haemagglutinating antigenic variants of the RHD (rabbit haemorrhagic disease) virus.

This paper attempts to study the dynamics of apoptosis of granulocytes and lymphocytes in peripheral blood in rabbits infected with haemagglutinating (Vt97, Triptis, Hartmannsdorf) and non-haemagglutinating (Pv97, 9905 RHDVa) antigenic variants of the RHD virus. The pathogenicity of those antigenic variants was also assessed by recording the mortality of the infected animals. The animals were infected with antigenic variants and blood was sampled at hour 0, 4, 8, 12, 24, 36 p.i. and the percentage of apoptotic granulocytes and lymphocytes was measured with the use of flow cytometry. The results of the study showed that apoptosis is included during RHDV infection, as the number of apoptotic granulocytes and lymphocytes increases throughout the experiment; depending on the antigenic variant, apoptosis joins in at 4-8-12 h p.i. and lasts until 24-36 h p.i. Furthermore, the mortality of rabbits infected with the examined strains of RHD virus varied from 30% to 100%. This study performed for the first time in this manner, indicates the importance of apoptosis during infection with the RHD virus.

Novel RHD alleles with weak hemagglutination and genetic Exon 9 diversity: weak D Types 45.1, 75, and 76.

BACKGROUND: Molecular variant RHD allele analysis is best complemented by detailed characterization of the associated D phenotype. STUDY DESIGN AND METHODS: Variant D types were characterized using molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen quantification. RESULTS: We discovered three novel weak D types termed weak D Types 45.1, 75, and 76 with RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches; antigen densities of approximately 1.990, 900, and 240 D sites per red blood cell were found, respectively. Adsorption-elution technique-supported D epitope mapping of these three weak D types demonstrated the expression of all tested D epitopes. Initial molecular typing of the three investigated samples by RHD gene exon scanning polymerase chain reaction using sequence-specific priming yielded a negative reaction for A1193 located in RHD Exon 9 and could be explained by specific mutations for weak D Types 45.1 (C818T, G1195A), 75 (G1194C), and 76 (A1215C). CONCLUSION: All novel weak D types expressed all tested D epitopes. It is of interest that for weak D Types 45.1, 75, and 76, similar alleles with a maximal divergence of one amino acid only, that is, weak D Types 45, 41, and 68, respectively, have been reported so far.

Proteomic analysis of a twin-arginine translocation-deficient mutant unravel its functions involved in stress adaptation and virulence in fish pathogen Edwardsiella tarda.

Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E. tarda was identified previously. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in the Tat system did not affect the morphology and biofilm formation in E. tarda, but did affect motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g. macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E. tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n = 61), shedding light on the Tat system associated with virulence and stress-associated processes in E. tarda.

A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(ß1-3)Gal(α1-4), and Gal(α1-4)GalNAc(ß1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

A BCAM0223 mutant of Burkholderia cenocepacia is deficient in hemagglutination, serum resistance, adhesion to epithelial cells and virulence.

Burkholderia cepacia complex (Bcc) bacteria are a problematic group of microorganisms causing severe infections in patients with Cystic Fibrosis. In early stages of infection, Bcc bacteria must be able to adhere to and colonize the respiratory epithelium. Although this is not fully understood, this primary stage of infection is believed to be in part mediated by a specific type of adhesins, named trimeric autotransporter adhesins (TAAs). These homotrimeric proteins exist on the surface of many gram negative pathogens and often mediate a number of critical functions, including biofilm formation, serum resistance and adherence to an invasion of host cells. We have previously identified in the genome of the epidemic clinical isolate B. cenocepacia J2315, a novel cluster of genes putatively encoding three TAAs (BCAM0219, BCAM0223 and BCAM0224). In this study, the genomic organization of the TAA cluster has been determined. To further address the direct role of the putative TAA BCAM0223 in B. cenocepacia pathogenicity, an isogenic mutant was constructed via insertional inactivation. The BCAM0223::Tp mutant is deficient in hemagglutination, affected in adherence to vitronectin and in biofilm formation and showed attenuated virulence in the Galleria mellonella model of infection. Moreover, the BCAM0223::Tp mutant also showed a significant reduction in its resistance to human serum as well as in adherence, but not in invasion of, cultured human bronchial epithelial cells. Altogether these results demonstrate that the BCAM0223 protein is a multifunctional virulence factor that may contribute to the pathogenicity of B. cenocepacia.

Structural analysis of effector functions related motifs, complement activation and hemagglutinating activities in Lama glama heavy chain antibodies.

Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains.

Red cells from the original JAL+ proband are also DAK+ and STEM+.

BACKGROUND: The low-prevalence Rh antigen, JAL, was named after the index case, Mr. J. Allen. Based on reactivity of seven multi-specific sera with his RBCs, it was apparent that they express at least one additional low-prevalence antigen. The purpose of this study was to investigate the other low-prevalence antigen(s) on J. Allen's RBCs. METHODS: Blood samples and reagents were from our collections. Hemagglutination and DNA analyses were performed by standard methods. RESULTS: Our DNA analyses confirmed the presence of RHCE*ceS(340T) in J. Allen and revealed the presence of RHCE*ceBI (ce 48C, 712G, 818T, 1132G) and RHD*DOL (509T, 667T). RBCs from J. Allen were agglutinated by anti-JAL, anti-STEM, and anti-DAK. Two of the reactive multi-specific sera reported in the original paper reacted with RBCs from J. Allen, and with RBCs from four other people with RHCE*ceBI, including the original STEM+ index case (P. Stemper) but not with RBCs with the DIIIa, DAK+ phenotype. We conclude that they contain anti-STEM. CONCLUSION: J.Allen's RBCs express the low-prevalence Rh antigens, JAL, V/VS (extremely weakly), STEM, and DAK. The presence of JAL on the variant Rhce, RhceJAL (16Cys, 114Trp, 245Val), STEM on the variant Rhce, RhceBI (16Cys, 238Val, 273Val, 378Val), and DAK on the variant RhD (170Thr, 223Val), encoded by RHD*DOL in trans to RHCE*ceBI is consistent with expression of these antigens. When J. Allen RBCs are used to detect and identify an anti-JAL, it is important to remember that they also express STEM and DAK.

QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda.

The inter-kingdom communication with the mammalian hosts mediated by autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE), and transduced by two-component systems QseBC has recently been described. As a fish pathogen and opportunistic pathogen for human beings, Edwardsiella tarda develops surface structures such as flagellar and fimbriae to cause different hemagglutination phenotypes and serotypes and initiate pathogen-host recognition and invasion process. E. tarda survives within macrophages in fish using type III secretion system (TTSS). Here, the genes of E. tarda two-component system, qseB and qseC, were found to be co-transcribed. Phylogenetic analysis indicated that evolution of QseC strongly correlated to different host niches. Compared with the wild type and their complemented strains, ΔqseB and ΔqseC mutants exhibited significant impaired flagellar motilities. Mammalian Epi was able to stimuli the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in ΔqseB but repressed in ΔqseC. Disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements EseB and EsaC, and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence-related surface structures (flagellum, fimbriae and secretion system) and that hormone might stimulate the virulence of the pathogen in fish.

New mutations in C1GALT1C1 in individuals with Tn positive phenotype.

Tn polyagglutination results from inactivating mutations in C1GALT1C1, an X-borne gene encoding a core 1 beta3-galactosyltransferase-specific molecular chaperone (cosmc) required for the functioning of T-synthase (beta 1,3-galactosyltransferase), a glycosyltransferase essential for the correct biosynthesis of O-glycans. This study found novel inactivating mutations (Glu152Lys, Ser193Pro and Met1Ile) in the coding sequence of C1GALT1C1 in three Tn positive individuals and a complete lack of C1GALT1C1 cDNA expression was observed in an additional Tn positive individual. In addition, expression of ST6GALNAC1, which encodes (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 and gives rise to sialyl-Tn antigen, was present at comparable levels in normal and Tn-positive human erythroblasts. Expression studies of wild-type and Tn positive C1GALT1C1 cDNA in the Jurkat cell line confirmed that the amino acid substitutions observed in Tn are inactivating. Analysis of the transcriptome of cultured normal and Tn positive erythroblasts revealed numerous differences in gene expression. Reduced transcript levels for fatty acid binding protein 5 (FABP5) and plexin D1 (PLXND1), and increased levels for aquaporin 3 (AQP3) were confirmed by quantitative real-time polymerase chain reaction. These data show that alteration of O-glycan structures resulting from T-synthase deficiency is accompanied by altered expression of a wide variety of genes in erythroid cells.

Biological and immunological relations among human parvovirus B19 genotypes 1 to 3.

The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by approximately 10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.

Subcloning of the 200-kDa Porphyromonas gingivalis antigen gene and inhibition of hemagglutination by an antibody against the recombinant protein.

Porphyromonas gingivalis is a major etiologic agent of periodontitis and exhibits hemagglutinating and adherence activities. We previously succeeded in molecular cloning the 200-kDa cell-surface antigenic protein (200-k AP), designated pMD101, that is recognized in sera from periodontitis patients, and identified the 200-k AP as a hemagglutinin A (HagA) derivative. HagA is one of the hemagglutinins known to be a useful vaccine against periodontitis. HagA has four large, contiguous, direct repeats and the repeat unit is believed to contain the hemagglutinin domain. Because production of 200-k AP was low in the Escherichia coli host, it was difficult to obtain large amounts of recombinant protein. In this study, we attempt to subclone the gene encoding the useful antigen from pMD101 in an effort to obtain large quantities. A subclone, designated pMD160, encoding a fusion protein of 80-kDa HagA and maltose-binding protein was successfully constructed, and the novel clone produced relatively large amounts of recombinant protein. DNA nucleotide sequences of the pMD160 insert demonstrated that the 80-kDa protein contained a short hemagglutinin motif and a direct repeat unit region. The recombinant protein was purified to homogeneity and rabbit antiserum was raised. The antibody was capable of inhibiting the hemagglutinating activity of P. gingivalis. These findings suggest that novel 80-kDa HagA derivative proteins can be produced efficiently from E. coli hosts and these may be useful in developing immunotherapy against periodontitis infected by P. gingivalis.

Altered glycosylation leads to Tr polyagglutination.

BACKGROUND: Polyagglutination refers to red blood cells (RBCs) that are agglutinated by a high proportion of ABO-matched adult sera but not by cord sera. Polyagglutinable RBCs have been associated with microbial infection, myeloproliferative disorders, and myelodysplasia. Lectins aid in the identification of polyagglutination. CASE STUDY: A Hispanic male infant with mild hemolytic anemia, a "Bernard-Soulier-like" syndrome, intermittent neutropenia, mitral valve regurgitation, ligament hyperlaxity, and mild mental retardation was studied. The patient's Group O RBCs were polyagglutinable; they were agglutinated by normal human sera, several lectins [including Arachis hypogea, Salvia sclarea, Salvia horminum, Glycine max, Ulex europaeus, Griffonia simplicifolia I, and Gr. simplicifolia II], and some monoclonal antibodies. His RBCs were not agglutinated by cord sera, Dolichos biflorus, or Phaseolus lunatus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the RBC membranes followed by staining with periodic acid-Schiff stain showed markedly reduced staining of glycophorins A and B. Staining with Coomassie brilliant blue revealed that Band 3 has a faster mobility than normal. CONCLUSIONS: Collectively, the results suggest that the patient's RBCs have a reduction in N-acetylneuraminic acid on both N- and O-glycans, exposing, respectively, beta1,4-galactosidase and beta1,3-galactosidase. The patient likely has an altered glycosyltransferase that results in defective glycosylation in RBCs and other cell lineages. This type of polyagglutination was named Tr.

Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites.

Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.