Biomedical subjects

M D Boyle

Publications and source records attributed to M D Boyle.

At least 37 records · Page 2Linked to original sources

PubMed1998

Role of emm and mrp genes in the virulence of group A streptococcal isolate 64/14 in a mouse model of skin infection.

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

Animals↗

PubMed1998

Clot formation by group A streptococci.

Group A streptococci of several different M serotypes can cause human plasma to clot in nutrient-poor media. Addition of glucose to the medium prevents clot formation. Once formed, clots are stable for several days and can be lysed on addition of exogenous streptokinase or urokinase. Clot lysis can also be achieved by addition of glucose to a clot containing wild-type group A streptococci but not clots containing an isogenic mutant in which the ska gene was inactivated.

Blood Coagulation↗

PubMed1997

Inactivation of single genes within the virulence regulon of an M2 group A streptococcal isolate result in differences in virulence for chicken embryos and for mice.

An M2 streptococcal isolate and isogenic mutants in which either the emm or mrp gene was insertionally inactivated were tested for virulence using either a mouse model or a chicken embryo model. The results of the studies using the mouse model demonstrated that neither the emm nor mrp gene products had a significant effect on virulence when mice were challenged via the i.p. route. However, when the bacteria were injected into the skin the emm gene product was identified as a virulence factor. In parallel studies in the chicken embryo model the mrp gene product was found to be a major virulence factor, while a minor contribution to virulence could also be attributed to the emm gene product. The importance of these gene products to virulence was noted when the chicken embryo were injected either i.v or when the bacteria were placed on top of the chorioallantoic membrane. The direct comparison of a single wild type group A organism and its paired isogenic mutants in two animal models suggests that different combinations of bacterial factors are required to overcome host defense strategies associated with different animal species.

Animals↗

PubMed1997

Identification of key gene products required for acquisition of plasmin-like enzymatic activity by group A streptococci.

Group A streptococci incubated in human plasma can acquire a plasmin-like enzymatic activity. This process involves at least two bacterial proteins and two human protein cofactors. In this study, the key bacterial proteins were identified by using a series of isogenic mutants of group A isolate, CS101. These studies confirm a key role for the secreted plasminogen activator, streptokinase, and identify the major surface fibrinogen-binding protein as the product of the mrp gene. The requirement for human fibrinogen and plasminogen as key cofactors was also confirmed.

Bacterial Proteins↗

PubMed1997

Detection of streptococcal class I M protein in psoriasis by confocal immunofluorescent microscopy.

Epidemiological evidence implicates Streptococcus pyogenes (group A) infection as a common triggering stimulus for psoriasis. Unequivocal demonstration of streptococcal antigens in psoriatic skin has been difficult due to cross-reactive antigens in both normal human tissue and group A streptococci, which complicate immunohistological analysis. In this study cryostat sections of involved psoriatic skin were stained with monoclonal antibody 111-15504 to group A streptococci. The epitope recognized by this antibody was found to be specific for group A streptococci and is associated with class I M protein. Streptococcal antigens were found in the dermal papillae and epidermis of psoriatic skin lesions of 20 out 38 patients. These findings indicate that specific S. pyogenes antigen, associated with class I M protein, is often present in psoriatic lesions. Such an antigen, originating from focal infection elsewhere could be responsible for T-lymphocyte inflammatory responses triggering the development of psoriatic lesions.

Antibodies, Monoclonal↗

PubMed1997

Plasminogen activation by invasive human pathogens.

In this review the interaction between invasive human pathogens expressing plasmin(ogen) receptors and/or producing plasminogen activators with the human plasmin(ogen) system is described. Evidence is presented for multiple mechanisms by which human pathogens can acquire a surface bound form of plasmin that cannot be regulated by host serpins. The potential importance of these pathways in providing the organisms with the ability to cross tissue barriers is discussed.

Bacterial Infections↗

PubMed1996

Identification of an amino acid signature sequence predictive of protein G-inhibitable IgG3-binding activity in group-A streptococcal IgG-binding proteins.

Sequence comparison of six known group-A streptococcal IgG-binding proteins, sharing the common property of protein G-inhibitable IgG3-binding-activity, identified a highly conserved 35-amino-acid (aa) sequence (74-100% similarity) within an EQ-rich central conserved core region of each protein. A search of aa sequence databases identified four additional proteins with > 50% similarity to this consensus sequence. All of these proteins demonstrated protein G-inhibitable IgG3-binding activity. Taken together, these results identify a signature sequence that predicts the presence of a protein G-inhibitable IgG3-binding domain(s) in group-A streptococcal IgG-binding proteins.

Amino Acid Sequence↗

PubMed1996

Different alleles of the fcrA/mrp gene of Streptococcus pyogenes encode M-related proteins exhibiting an identical immunoglobulin-binding pattern.

The majority of group A streptococci (GAS, Streptococcus pyogenes) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong either to the emm or the emm-related (fcrA/mrp and enn) gene family and are located in close proximity on the GAS genome, where they form part of the vir regulon. In the present study analysis of sequence data of the 5' terminal portions of the fcrA/mrp genes from GAS isolates representing 37 different M serotypes led to a classification of six different types. Thus, although fcrA/mrp genes exhibit an allelic polymorphism, they do not display the high degree of N-terminal sequence diversity found among emm genes. The nucleotide sequences of the fcrA/mrp genes from 3 GAS isolates, belonging to serotypes M8, M9, and M13 and representing newly characterized fcrA/mrp gene types, are reported. Analysis of the Ig-binding properties of recombinant FcrA/Mrp8, 9, and 13 proteins, demonstrated a similar Ig-binding profile being reactive with human IgG subclasses 1, 2, and 4. This pattern is identical to that previously described for other recombinant fcrA/mrp4, 49, 64/14 and 76 gene products, indicating that this property is not affected by the N-terminal variability. Evidence for recombination between an fcrA/mrp and an mga gene was observed in an M-type 33 strain isolate providing further support for the concept of gene rearrangement contributing to the diversity of vir regulon gene products.

Amino Acid Sequence↗

PubMed1996

M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes.

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsonophagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains. Subsequent analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive controls attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co-incubation. Kinetic data suggested that attachment to granulocytes proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.

Antigens, Bacterial↗

PubMed1996

Role of staphylokinase in the acquisition of plasmin(ogen)-dependent enzymatic activity by staphylococci.

In this study, the role of the staphylococcal plasminogen activator, staphylokinase (SAK), was analyzed for its ability to mediate acquisition of cell-associated plasmin-like activity by staphylococci in the presence of a source of human plasminogen. A panel of staphylococcal strains isolated from humans was tested for the presence of the SAK gene, secretion of the plasminogen activator, and the ability to acquire enzymatic activity when incubated with purified human plasminogen or serum. When SAK was compared with the eukaryotic plasminogen activators, urokinase and tissue plasminogen activator, only SAK could mediate acquisition of cell-associated enzymatic activity by staphylococci without first generating significant fluid-phase plasmin. These studies provide evidence for a novel mechanism by which SAK-producing S. aureus can acquire an unregulatable host plasmin-like activity that might contribute to their invasive potential.

Base Sequence↗

PubMed1996

Properties of IgG-binding proteins expressed by Streptococcus pyogenes isolates are predictive of invasive potential.

Recent clinical Streptococcus pyogenes isolates of the M1 serotype can be grouped according to the IgG-binding properties of their M proteins. One group expressed an IgG-binding M1 protein reactive with human IgG1, IgG2, IgG3, and IgG4 (type IIo); the other expressed a protein with predominant reactivity with human IgG3 alone (type IIb). Both IgG-binding protein phenotypes were equally resistant to phagocytosis in human blood; however, when they were injected into a skin air sac on outbred CD1 mice, all mice injected with M1 isolates of the type IIo phenotype were dead within 70 h, while only 40% of those injected with M1 isolates of the type IIb phenotype died within the same period. Bacteria recovered from the spleens of animals that died after injection with type IIb phenotype isolates demonstrated a change in their IgG-binding profile and were indistinguishable, in vitro or in vivo, from isolates displaying the type IIo phenotype.

Animals↗

PubMed1996

Evidence for independent binding domains within a group A streptococcal type IIo IgG-binding protein.

The gene for a type IIo IgG-binding protein has previously been cloned and sequenced. The approximately 60,000 M(r) recombinant gene product binds all four human IgG subclasses and fibrinogen. Treatment of this recombinant protein with CNBr results in generation of a series of fragments. One fragment, an approximately 32,000 M(r) polypeptide, binds IgG1, IgG2, and IgG4 but neither IgG3 nor fibrinogen. N-terminal amino sequencing of this fragment indicated that this was an internal fragment of the protein starting at amino acid 186 of the mature protein. These findings provide evidence for two distinct domains for binding IgG1, IgG2, and IgG4 and binding IgG3 within a single bacterial IgG-binding protein.

Amino Acid Sequence↗

PubMed1995

Variation of multifunctional surface binding proteins--a virulence strategy for group A streptococci?

Variation in surface antigens has been well recognized as a mechanism by which pathogenic organisms can avoid elimination and remain as potential pathogens in immunocompetent individuals. A variety of viral and parasitic organisms elude the immune system by varying their surface antigenic structures. Other persistent human pathogens, for example group A streptococci, are associated with cyclic variation in the severity of infections without any major change in their surface antigenic structures. Recent analysis of group A streptococcal proteins, in particular surface M and M-like proteins, has documented the existence of an array of multifunctional surface proteins which have the ability to bind to a variety of normal human plasma proteins, extracellular matrix components and human cells. The ability to change the functional activities of these surface molecules by genetic recombination among members of a closely related M protein supergene family has now been reported. In this paper, the potential importance of generating functional heterogeneity in surface binding proteins of group A streptococcus is discussed. The role of these proteins in enabling an organism to sense its environment and express the appropriate virulence factors is proposed as an explanation for the periodic changes in the frequency and severity of invasive group A streptococcal infections that can occur in the absence of a toxic-shock-like syndrome.

Antigens, Bacterial↗

PubMed1995

Characterization of a gene coding for a type IIo bacterial IgG-binding protein.

Two antigenic classes of non-immune IgG-binding proteins can be expressed by group A streptococci. One antigenic group of proteins is recognized by an antibody prepared against the product of a cloned fcrA gene (anti-FcRA). In this study, the immunogen used to prepare the antibody that defines the second antigenic class was shown to be the product of the emm-like (emmL) gene of M serotype 55 group A isolate, A928. The emmL55 gene expressed in E. coli produced an M(r) approximately 58,000 molecule which bound human IgG1, IgG2, IgG3 and IgG4, as well as horse, rabbit and pig IgG in a non-immune fashion. These properties are characteristic of the previously described type IIo IgG-binding protein isolated from this strain. In addition, the recombinant protein was reactive with human serum albumin and fibrinogen. The emmL 55 gene sequence was analysed and found to have the organization and sequence characteristics of a typical class I emm-like gene.

Amino Acid Sequence↗

PubMed1995

Characterization of a type II'o group A streptococcal immunoglobulin-binding protein.

The opacity factor positive M type 2 group A streptococcal isolate, A207, expresses a unique functional type II'o IgG-binding protein which reacts with all four human IgG subclasses and rabbit IgG. In order to determine the gene product or products responsible for this activity, three genes of the vir regulon from this isolate were cloned, expressed and analysed. The fcr A2 gene coded for a protein binding hyman IgG1, IgG2 and IgG4 but not IgG3. The enn2 gene coded for a protein reacting exclusively with human IgA, while the emmL2 gene product bound IgG1, IgG2, IgG3 and IgG4 as well as rabbit but not horse or pig IgG. The IgG3-binding activity of the EmmL2 protein was functionally indistinguishable from the Form 1 IgG3-binding activity present in heat extracts of group A isolate A207.

Animals↗

PubMed1995

Analysis of the interaction of group A streptococci with fibrinogen, streptokinase and plasminogen.

Group A streptococci demonstrate a number of distinct ways to interact with the human fibrinolytic system to acquire unregulatable cell-surface enzymatic activity. Interactions between bacteria, fibrinogen, streptokinase and plasminogen resulted in acquisition of cell-associated enzymatic activity that can lyse fibrin clots despite the presence of the major physiological plasmin inhibitor, alpha 2-antiplasmin. Western blot analysis of extracted streptococcal surface proteins suggested that binding of fibrinogen to M or M-related proteins mediated the capture of streptokinase-plasminogen complexes to the bacteria. The enzymatic complex formed by reaction of bacteria with fibrinogen, streptokinase and plasminogen was found to be more stable in human plasma than pre-formed plasmin bound directly to the same bacteria strain.

Aminocaproic Acid↗

PubMed1995

A role for fibrinogen in the streptokinase-dependent acquisition of plasmin(ogen) by group A streptococci.

Acquisition of plasmin(ogen) by group A streptococci occurs by two distinct pathways. In addition to the well-characterized direct interaction of plasmin with cell-surface receptors on group A streptococci, a second pathway dependent on streptokinase and a nonplasminogen factor(s) present in human plasma was identified. The role of streptokinase in the second pathway was not merely as a plasminogen activator, since substitution of the plasminogen activator urokinase did not result in the capture of plasmin(ogen) by bacteria in the presence of plasminogen-depleted plasma. However, if streptokinase was added to plasmin that had been generated by treatment of plasminogen with urokinase, the ability of the bacteria to capture plasmin in the presence of plasminogen-depleted plasma was restored. Fibrinogen present in human plasma was identified as the major factor required for streptokinase-dependent uptake of plasmin(ogen) by group A streptococci.

Fibrinogen↗

PubMed1995

Protection of mice from group A streptococcal skin infection by interleukin-12.

It has been shown that interleukin (IL)-12 induces cell-mediated immunity and provides significant protection against intracellular organisms. The ability of this cytokine to enhance immunity in a mouse model of group A streptococcal skin infection was studied. Outbred CD1 mice were injected for 3 consecutive days with 0.1 microgram of recombinant murine IL-12 before or after challenge with strain 64/14 group A streptococci. In both cases, in vivo IL-12 treatment significantly decreased the rate of death after infection and increased survival over the period of experimental observation. Thus, IL-12 may be useful for treatment of gram-positive bacterial infections. The time course of the experiments suggests that IL-12 is acting in this model system to enhance natural, rather than acquired, immunity.

Animals↗