Molecular markers for discriminating Streptococcus pyogenes and S. dysgalactiae subspecies equisimilis.

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus, a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Characterisation of the urease gene cluster in Bordetella bronchiseptica.

Bordetella bronchiseptica is a common ureolytic mammalian respiratory pathogen. The urease operon of this organism is encoded within an 8.9 kb DNA fragment which contains the structural genes (ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to other urease genes. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes, nickel donation to the urease active site, and prevention of nickel incorporation until correct formation of the active site, respectively. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may potentially be involved in nickel transport. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (BbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. LysR proteins have been shown to regulate urease in Klebsiella aerogenes (NAC), and catalase in Escherichia coli (OxyR), which offers the intracellular bacterium protection from phagolysosome damage. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was found 27 bp upstream of the urease promoter in B. bronchiseptica. We hypothesise that BbuR controls urease expression which is involved in protection of intracellular B. bronchiseptica from phagolysosomal damage. Comparison of the urease promoter regions of B. bronchiseptica, Bordetella parapertussis ATCC15311 and the urease-negative strain B. pertussis Tohama I revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the putative bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself.

Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract.

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.

Prospecting for new group A streptococcal vaccine candidates.

BACKGROUND & OBJECTIVES: Most group A streptococcal (GAS) vaccine strategies focused on the surface M protein of the GAS. However, vaccine based on M protein have some drawbacks. In the present study, we used two approaches to identify new proteins and peptides that may have utility as vaccine candidates. METHODS: A whole gel elution procedure was used to separate GAS surface antigens into 9 size fractionated pools. Mice were vaccinated with each pool and antibody titre, opsonic ability and protective capacity measured. In an alternative approach BioInformatics was used to identify putative GAS surface proteins. Peptides from within these proteins were then selected on the basis of predicted antigenicity or location. These peptides were conjugated to keyhole lymphocyanin (KLH) and immunogenicity measured in a mouse model. RESULTS: One pool of GAS surface proteins (approximately 29kDa) induced antibodies that were both opsonic and potentially protective. Immunoflourescent microscopy demonstrated that these antibodies bound to the surface of M1 GAS. Amino acid sequencing subsequently identified superoxide dismutase as the major antigen in this pool. A BioInformatic search of the M1 GAS genome and subsequent analysis identified several peptides that fulfilled criteria as potential vaccine candidates. Each peptide when conjugated to KLH was able to induce a strong antibody response. INTERPRETATION & CONCLUSION: Several new antigens were identified that may have potential as vaccine targets. A future GAS vaccine may have multiple peptide epitopes, providing protection against multiple GAS strains.

Updated model of group A Streptococcus M proteins based on a comprehensive worldwide study.

Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.

Characterization of the proteinase specified by varicella-zoster virus gene 33.

Varicella-zoster virus (VZV) genes 33 and 33.5 are predicted to encode the VZV proteinase and its substrate (the assembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33.5 was co-expressed with the VZV proteinase, processing of the VZV33.5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33.5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.

Estimating medical industry impacts on a regional economy.

The authors demonstrate a large-scale, computerized simulation model to estimate the economic impacts of the regional health services industry on a seven-county region in Northeast Minnesota, known as the Arrowhead Region. The model, known as SIMLAB, was developed at the University of Minnesota. SIMLAB is designed to simulate the economic performance of a subnational region. Three scenarios were developed: one to illustrate a regional economy functioning normally, one to show how the area's economy would operate in the absence of its entire health services industry, and one to show the regional economic impacts of the closing of a single major hospital within the region. The first two scenarios were compared, with the differences between them representing the impact estimates for the region. The findings give a graphic demonstration of how vitally important health services are to the region. Without the health sector, the region's total gross output for all industries is estimated to fall by more than $1 billion by 1990. A comparison of the first and third scenarios demonstrates the sensitivity of SIMLAB to smaller but still significant economic dislocations, such as the closing of a single major health care facility.

Molecular analysis of the bvg-repressed urease of Bordetella bronchiseptica.

Bordetella bronchiseptica is a ureolytic mammalian respiratory pathogen. We have investigated the regulation of urease in B. bronchiseptica and the potential role of this enzyme in eukaryotic invasion and intracellular survival. Our results indicate urease is a bordetella virulence repressed gene. Urease activity in virulent B. bronchiseptica BB7865 is up-regulated from basal levels by 5 gl1 magnesium sulphate at 37 degrees C. At 30 degrees C, urease activity remained at basal levels, even in the presence on magnesium sulphate, suggesting a second temperature dependent mechanism of urease regulation was also operating. Urease was not inducible by 10 mM urea nor up-regulated in nitrogen limiting conditions. To evaluate the role of urease in intracellular invasion and survival urease-negative mutants of B. bronchiseptica BB7865 and B. bronchiseptica BB7866 were created by transposon mutagenesis, and compared to the urease-positive parental strains in a HeLa cell invasion assay. We demonstrate that increasing the concentration of urea in the assay increased survival of the urease-positive but not urease-negative strains after 24 h, suggesting that urease does have a role in intracellular survival. Partial DNA sequence analysis of an 11.0 kb EcoRI DNA fragment encoding urease activity revealed an open reading frame containing 50%, 45%, 45%, and 41% homology to the UreA urease subunit protein of Klebsiella aerogenes, Proteus vulgaris, Helicobacter pylori and Proteus mirabilis respectively. We also show Bordetella pertussis to contain sequences homologous with a DNA probe containing the gene encoding UreA of B. bronchiseptica indicating the possible presence of cryptic urease genes in this species.

Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins.

The ability of two novel synthetic compounds to inhibit the HIV protease-mediated processing of HIV-1 precursor polyproteins was investigated in an in vitro gag-protease mixed lysate assay system and in an assay using recombinant baculoviruses engineered to express the HIV-1 gag and pol genes in cultured insect cells. With the in vitro mixed lysate assay we have shown that both compounds at 1 microM can completely inhibit the HIV-1 and HIV-2 protease-mediated release of p24 from the HIV-1 gag precursor at pH 5.5 and pH 7.0. In the intracellular baculovirus system these compounds were shown to inhibit the protease-mediated maturation of gag and also the excision of the protease moiety from its precursor.

In vitro activity of hepatitis B virus polymerase: requirement for distinct metal ions and the viral epsilon stem-loop.

Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.

Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations.

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.