PubMed Health⌕ Search

Biomedical subjects

John L Telford

Publications and source records attributed to John L Telford.

At least 19 recordsLinked to original sources
PubMed2007

BibA: a novel immunogenic bacterial adhesin contributing to group B Streptococcus survival in human blood.

By the analysis of the recently sequenced genomes of Group B Streptococcus (GBS) we have identified a novel immunogenic adhesin with anti-phagocytic activity, named BibA. The bibA gene is present in 100% of the 24 GBS strains analysed. BibA-specific IgG were found in human sera from normal healthy donors. The putative protein product is a polypeptide of 630 amino acids containing a helix-rich N-terminal domain, a proline-rich region and a canonical LPXTG cell wall-anchoring domain. BibA is expressed on the surface of several GBS strains, but is also recovered in GBS culture supernatants. BibA specifically binds to human C4-binding protein, a regulator of the classic complement pathway. Deletion of the bibA gene severely reduced the capacity of GBS to survive in human blood and to resist opsonophagocytic killing by human neutrophils. In addition, BibA expression increased the virulence of GBS in a mouse infection model. The role of BibA in GBS adhesion was demonstrated by the impaired ability of a bibA knockout mutant strain to adhere to both human cervical and lung epithelial cells. Furthermore, we calculated that recombinant BibA bound to human epithelial cells of distinct origin with an affinity constant of approximately 10(-8) M for cervical epithelial cells. Hence BibA is a novel multifunctional protein involved in both resistance to phagocytic killing and adhesion to host cells. The identification of this potential new virulence factor represents an important step in the development of strategies to combat GBS-associated infections.

Adhesins, Bacterial↗
PubMed2007

Transcriptional and proteomic profiles of group B Streptococcus type V reveal potential adherence proteins associated with high-level invasion.

Group B Streptococcus (GBS) is an opportunistic organism that can harmlessly colonize the human gut, vagina, and rectum but can also cause pneumonia, sepsis, and meningitis in neonates born to colonized mothers. We have shown previously that growth rate and oxygen level regulate the ability of GBS to invade eukaryotic cells in vitro. Herein we extend and expand on these observations to show that GBS type V, an emergent serotype, grown in a chemostat at a cell mass-doubling time (t(d)) of 1.8 h with oxygen invaded human ME-180 cervical epithelial cells in large numbers compared with those grown at the same t(d) without oxygen or at a slower t(d) of 11.0 h. The fact that several GBS type V cell wall-associated and membrane proteins were expressed exclusively under the invasive growth condition prompted an investigation, using genomics and proteomics, of all upregulated genes and proteins. Several proteins with potential roles in adherence were identified, including an undefined surface antigen (SAG1350), a lipoprotein (SAG0971), penicillin-binding protein 2b (SAG0765), glyceraldehyde-3-phosphate dehydrogenase (SAG0823), and an iron-binding protein (SAG1007). Mouse antisera to these five proteins inhibited binding of GBS type V to ME-180 cells by > or =85%. Recombinant undefined surface antigen (SAG1350), lipoprotein (SAG0971), and penicillin-binding protein 2b (SAG0765) each bound to ME-180 cells in a dose-dependent fashion, confirming their ability to act as ligands. Collectively, these data increase the number of potential GBS adherence factors and also suggest a role for these surface-associated proteins in initial pathogenic events.

Bacterial Adhesion↗
PubMed2006

Use of Lactococcus lactis expressing pili from group B Streptococcus as a broad-coverage vaccine against streptococcal disease.

Recent data indicate that the human pathogen group B Streptococcus (GBS) produces pilus-like structures encoded in genomic islands with similar organization to pathogenicity islands. On the basis of the amino acid sequence of their protein components, 3 different types of pili have been identified in GBS, at least 1 of which is present in all isolates. We recently demonstrated that recombinant pilus proteins protect mice from lethal challenge with GBS and are thus potential vaccine candidates. Here, we show that GBS pilin island 1, transferred into the nonpathogenic microorganism Lactococcus lactis, leads to pilus assembly. We also show that systemically or mucosally delivered Lactococcus expressing pilin island 1 protects mice from challenge with GBS isolates carrying pilus 1. Furthermore, lactococci engineered to express hybrid pili containing GBS pilus 1 and pilus 2 components confer protection against strains expressing either of the 2 pilus types. These data pave the way to the design of pilus-based, multivalent live vaccines against streptococcal pathogens.

Animals↗
PubMed2006

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

Amino Acid Sequence↗
PubMed2006

Pili in gram-positive pathogens.

Most bacterial pathogens have long filamentous structures known as pili or fimbriae extending from their surface. These structures are often involved in the initial adhesion of the bacteria to host tissues during colonization. In gram-negative bacteria, pili are typically formed by non-covalent interactions between pilin subunits. By contrast, the recently discovered pili in gram-positive pathogens are formed by covalent polymerization of adhesive pilin subunits. Evidence from studies of pili in the three principal streptococcal pathogens of humans indicates that the genes that encode the pilin subunits and the enzymes that are required for the assembly of these subunits into pili have been acquired en bloc by the horizontal transfer of a pathogenicity island.

Bacterial Adhesion↗
PubMed2006

Identification of novel genomic islands coding for antigenic pilus-like structures in Streptococcus agalactiae.

We have recently reported the presence of covalently linked pilus-like structures in the human pathogen, Group B Streptococcus (GBS). The pilus operon codes for three proteins which contain the conserved amino acid motif, LPXTG, associated with cell wall-anchored proteins together with two genes coding for sortase enzymes. Analysis of the eight sequenced genomes of GBS has led to the identification of a second, related genomic island of which there are two variants, each containing genes coding for proteins with LPXTG motifs and sortases. Here we show that both variant islands also code for pilus-like structures. Furthermore, we provide a thorough description and characterization of the genomic organization of the islands and the role of each protein in the assembly of the pili. For each pilus, polymerization of one of the three component proteins is essential for incorporation of the other two proteins into the pilus structure. In addition, two sortases are required for complete pilus assembly, each with specificity for one of the pilus components. A component protein of one of the newly identified pili is also a previously identified protective antigen and a second component of this pilus is shown to confer protection against GBS challenge. We propose that pilus-like structures are important virulence factors and potential vaccine candidates.

Adhesins, Bacterial↗
PubMed2006

The cell-specific phenotype of the polymorphic vacA midregion is independent of the appearance of the cell surface receptor protein tyrosine phosphatase beta.

There are two alleles, m1 and m2, of the midregion of the vacuolating cytotoxin gene (vacA) of Helicobacter pylori which code for toxins with different cell specificities. Here we describe the construction of five chimeric strains in which regions of vacA were exchanged between the two genotypes. By analyzing the toxicity of these strains for HeLa and RK13 cells we have confirmed that a 148-amino-acid region determines the phenotypic differences between the two forms of the protein and that this entire region is important for cytotoxicity. Furthermore, we have used our chimeric strains to investigate whether variations in the midregion of VacA have an effect on phorbol 12-myristate 13-acetate (PMA)-induced VacA sensitivity in HL-60 cells. The PMA-induced VacA sensitivity of HL-60 cells has been previously associated with the appearance of the cell surface receptor protein tyrosine phosphatase beta (RPTPbeta). Our data indicate that both the m1 and m2 forms of VacA are able to utilize RPTPbeta, and the cell-specific phenotype of the midregion is independent of the presence of RPTPbeta. It appears that another as-yet-unidentified receptor exists in HL-60 cells that accounts for the m2 phenotype in this cell line. Also, by studying the effect of PMA on levels of RPTPbeta in other cell lines and toxicity of VacA in these cell lines we have shown that RPTPbeta does not play a major role in the vacuolation of HeLa cells.

Amino Acid Sequence↗
PubMed2006

A Helicobacter pylori vacuolating toxin mutant that fails to oligomerize has a dominant negative phenotype.

Most Helicobacter pylori strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Delta49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Delta49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Delta49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.

Bacterial Proteins↗
PubMed2005

Group B Streptococcus crosses human epithelial cells by a paracellular route.

Colonization of the colon and vagina is thought to be important in the pathogenesis of group B Streptococcus (GBS) infection. However, little is known about the strategies used by GBS to translocate through the epithelial barrier during the onset of disease. We used differentiated epithelial cells grown on transwell inserts as a model of the epithelial barrier. Bacterial translocation occurred without a detectable decrease in transepithelial resistance. Whereas acapsular GBS was better able to adhere to and invade epithelial cells, the percentage of bacteria translocating across the epithelial monolayer was independent of the presence of the capsule. Transmission electron microscopy showed the intimate association of GBS with intercellular junctions and the capacity to cross the monolayer by a paracellular mechanism. This process consisted of an active and transient opening of cell junctions. Indeed, GBS was preferentially found along the cell perimeter, where it colocalized with junctional protein complexes.

Bacterial Capsules↗
PubMed2005

Group A Streptococcus produce pilus-like structures containing protective antigens and Lancefield T antigens.

Although pili have long been recognized in Gram-negative pathogens as important virulence factors involved in adhesion and invasion, very little is known about extended surface organelles in Gram-positive pathogens. Here we report that Group A Streptococcus (GAS), a Gram-positive human-specific pathogen that causes pharyngitis, impetigo, invasive disease, necrotizing fasciitis, and autoimmune sequelae has long, surface-exposed, pilus-like structures composed of members of a family of extracellular matrix-binding proteins. We describe four variant pili and show that each is recognized by a specific serum of the Lancefield T-typing system, which has been used for over five decades to characterize GAS isolates. Furthermore, we show that immunization of mice with a combination of recombinant pilus proteins confers protection against mucosal challenge with virulent GAS bacteria. The data indicate that induction of a protective immune response against these structures may be a useful strategy for development of a vaccine against disease caused by GAS infection.

Animals↗
PubMed2005

Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial "pan-genome".

The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae, the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for approximately 80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Amino Acid Sequence↗
PubMed2005

Identification of a universal Group B streptococcus vaccine by multiple genome screen.

Group B Streptococcus (GBS) is a multiserotype bacterial pathogen representing a major cause of life-threatening infections in newborns. To develop a broadly protective vaccine, we analyzed the genome sequences of eight GBS isolates and cloned and tested 312 surface proteins as vaccines. Four proteins elicited protection in mice, and their combination proved highly protective against a large panel of strains, including all circulating serotypes. Protection also correlated with antigen accessibility on the bacterial surface and with the induction of opsonophagocytic antibodies. Multigenome analysis and screening described here represent a powerful strategy for identifying potential vaccine candidates against highly variable pathogens.

Animals↗
PubMed2005

Genome analysis reveals pili in Group B Streptococcus.

Pili are essential virulence factors in many Gram-negative bacteria; however, they have not been described in most important Gram-positive pathogens. While screening the sequence of multiple genomes of Group B Streptococcus, we identified protective antigens that formed high molecular weight polymers. Immunogold electron microscopy revealed that the structures have a pilus-like form. These large structures have gone unrecognized in decades of studies of Group B Streptococcus.

Animals↗
PubMed2005

Defective Vav expression and impaired F-actin reorganization in a subset of patients with common variable immunodeficiency characterized by T-cell defects.

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by impaired antibody production, which is in many instances secondary to defective T-cell function (T-CVID). We have previously identified a subset of patients with T-CVID characterized by defective T-cell receptor (TCR)-dependent protein tyrosine phosphorylation. In these patients, ZAP-70 fails to be recruited to the TCR as the result of impaired CD3zeta phosphorylation, which is, however, not dependent on defective Lck expression or activity. Here we show that neither Fyn nor CD45 is affected in these patients. On the other hand, T-CVID T cells show dramatic defects in the Vav/Rac pathway controlling F-actin dynamics. A significant deficiency in Vav protein was indeed observed; in 3 of 4 patients with T-CVID, it was associated with reduced VAV1 mRNA levels. The impairment in Vav expression correlated with defective F-actin reorganization in response to TCR/CD28 co-engagement. Furthermore, TCR/CD28-dependent up-regulation of lipid rafts at the cell surface, which requires F-actin dynamics, was impaired in these patients. The actin cytoskeleton defect could be reversed by reconstitution of Vav1 expression in the patients' T cells. Results demonstrate an essential role of Vav in human T cells and strongly suggest Vav insufficiency in T-CVID.

Actins↗
PubMed2005

Immune subversion by Helicobacter pylori.

To maintain prolonged colonization of the human gastric mucosa, Helicobacter pylori must avoid both innate and adaptive immune responses. During its long coexistence with humans, it has evolved complex strategies to maintain a mild inflammation of the gastric epithelium while limiting the extent of immune effector activity. Severe disease, associated with bacterial colonization, might reflect loss of this control. Several mechanisms and the bacterial factors involved in immune subversion have, in recent years, been elucidated, thus opening the possibility of a better understanding of the pathogenicity of this microorganism.

Antigen Presentation↗
PubMed2004

Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism.

Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.

Bacterial Adhesion↗
PubMed2003

The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms.

Helicobacter pylori toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.

Active Transport, Cell Nucleus↗