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.