Incorporation of metabolic enzymes to improve predictivity of reporter gene assay results for estrogenic and anti-androgenic activity.

Identification and monitoring of so-called endocrine-disrupting compounds has received ample attention; both the OECD and the United States Environmental Protection Agency (US EPA) have designed tiered testing approaches, involving in vitro bioassays to prioritize and partly replace traditional animal experiments. Since the estrogen (ER) and androgen (AR) receptor are frequent targets of endocrine disrupting chemicals, bioassays detecting interaction with these receptors have a high potential to be of use in risk assessment of endocrine active compounds. However, in many bioassays in vivo hepatic metabolism is not accounted for, which hampers extrapolation to the in vivo situation. In the present study, we have developed a metabolic module using rat liver S9 as an add-on to human cell-based reporter gene assays. The method was applied to reporter gene assays for detection of (anti-) estrogens and (anti-) androgens, but can be extended to cell-based reporter gene assays covering a variety of endpoints related to endocrine disruption.

Incorporation of a metabolizing system in biodetection assays for endocrine active substances.

The use of in vitro assays is important for the biodetection of endocrine active substances (EAS), reducing and replacing the in vivo studies required for regulatory assessment. However, this approach often fails to take into account the role of biotransformation on the activity of the test substances. A method incorporating an S9 metabolic system into the CALUX-reporter gene assays for estrogen receptor α- and anti-androgen receptor -mediated activities has been developed. Methoxychlor, which is known to exhibit increased estrogenic and anti-androgenic activities after biotransformation, was used to set up the method in ERa and anti-AR CALUX. For the anti-androgenic assay, stanozolol was used as a competing agonist not metabolized by S9. The method was first applied in both agonist and antagonist modes to methoxychlor and bisphenol A, as positive and negative controls, respectively. Then, benzo(a)pyrene and flutamide were also tested for their potential of bioactivation. Co-treatment with S9 successfully increased the ERα agonist and AR antagonist potency of methoxychlor; no change was observed for bisphenol A. Incubation with S9 also enhanced the anti-androgenic activity of flutamide. Interestingly, the metabolism of benzo(a)pyrene by the S9 resulted in an increased estrogen receptor-mediated transcriptional activation; any increase in the potency was only minor. It is likely that both enzyme kinetics and metabolite stability have influenced these effects, which would affect the composition of the final metabolite mixture. Together these results demonstrate the relevance of including biotransformation in in vitro bioassays for the detection of EAS.

Household transmission of leptospira infection in urban slum communities.

BACKGROUND: Leptospirosis, a spirochaetal zoonotic disease, is the cause of epidemics associated with high mortality in urban slum communities. Infection with pathogenic Leptospira occurs during environmental exposures and is traditionally associated with occupational risk activities. However, slum inhabitants reside in close proximity to environmental sources of contamination, suggesting that transmission during urban epidemics occurs in the household environment. METHODS AND FINDINGS: A survey was performed to determine whether Leptospira infection clustered within households located in slum communities in the city of Salvador, Brazil. Hospital-based surveillance identified 89 confirmed cases of leptospirosis during an outbreak. Serum samples were obtained from members of 22 households with index cases of leptospirosis and 52 control households located in the same slum communities. The presence of anti-Leptospira agglutinating antibodies was used as a marker for previous infection. In households with index cases, 22 (30%) of 74 members had anti-Leptospira antibodies, whereas 16 (8%) of 195 members from control households had anti-Leptospira antibodies. Highest titres were directed against L. interrogans serovars of the Icterohaemorrhagiae serogroup in 95% and 100% of the subjects with agglutinating antibodies from case and control households, respectively. Residence in a household with an index case of leptospirosis was associated with increased risk (OR 5.29, 95% CI 2.13-13.12) of having had a Leptospira infection. Increased infection risk was found for all age groups who resided in a household with an index case, including children <15 years of age (P = 0.008). CONCLUSIONS: This study identified significant household clustering of Leptospira infection in slum communities where recurrent epidemics of leptospirosis occur. The findings support the hypothesis that the household environment is an important transmission determinant in the urban slum setting. Prevention therefore needs to target sources of contamination and risk activities which occur in the places where slum inhabitants reside.

Evaluation of the EIE-IgM-Leptospirose assay for the serodiagnosis of leptospirosis.

Access to low-cost, effective diagnosis for leptospirosis is urgently needed in developing countries. The EIE-IgM-Leptospirose, a kit produced for public health laboratories in Brazil, was shown to have a sensitivity of 76% (77 of 102 patients) and 100% (102 of 102 patients) during acute and convalescent-phase leptospirosis, respectively, and a specificity of 93-100% (total healthy and patient control subjects evaluated, 486). These findings indicate that the assay will be useful for diagnosis of this emerging infectious disease in Brazil and other developing countries.

Association of a novel high molecular weight, serine-rich protein (SrpA) with fibril-mediated adhesion of the oral biofilm bacterium Streptococcus cristatus.

The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 +/- 19 nm long with longer, less abundant fibrils 403 +/- 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 +/- 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 +/- 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.

Dental plaque formation.

Dental plaque is a complex biofilm that accumulates on the hard tissues (teeth) in the oral cavity. Although over 500 bacterial species comprise plaque, colonization follows a regimented pattern with adhesion of initial colonizers to the enamel salivary pellicle followed by secondary colonization through interbacterial adhesion. A variety of adhesins and molecular interactions underlie these adhesive interactions and contribute to plaque development and ultimately to diseases such as caries and periodontal disease.

Natural transformation of Streptococcus crista.

Over the years Streptococcus gordonii (sanguis) Challis has become the workhorse of genetic manipulations for the sanguis group of oral streptococci. This is because strain Challis was shown in early studies to be highly naturally competent for transformation. However, Challis is not usually the most appropriate strain to use in studies which focus on oral microbial adherence. We report that other members of the newly reorganized sanguis group, particularly within the species S. crista, display reasonable transformation frequencies, with both plasmid and chromosomal DNA, if transformed at the appropriate time during the growth curve. The ability to transform S. crista may be especially important for genetic studies of biological properties that appear to be limited to these specific streptococcal strains.

scbA from Streptococcus crista CC5A: an atypical member of the lraI gene family.

A new member of the lraI family of putative adhesin genes was cloned, from Streptococcus crista CC5A, and sequenced. The gene, scbA appears to be part of an ABC transport operon and encodes a putative peptide of 34.7 kDa. The protein contains a signal sequence with residues 17 to 21 (L-A-A-C-S) matching the consensus sequence for the prolipoprotein cleavage site of signal peptidase II. ScbA is 57 to 93% identical, at the amino acid level, with the five previous sequenced members of the LraI family. Surprisingly, ScbA does not exhibit adhesion properties characteristic of the other LraI proteins. Strain CC5A bound poorly to saliva-coated hydroxyapatite and did not coaggregate with Actinomyces naeslundii PK606. An scbA insertion-duplication mutation that abolished expression (of ScbA was created. There was no difference in fibrin binding between this mutant and wild-type CC5A. Since it is possible that ScbA could play a role in corncob formation between S. crista and Fusobacterium nucleatum, this property was examined. The mutant strain retained the ability to form corncobs. On the basis of the lack of adhesin properties it appears that ScbA is an atypical member of the LraI family.

Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916.

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of "corncobs", a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.

Molecules of Streptococcus gordonii that bind to Porphyromonas gingivalis.

Interbacterial binding is considered an important colonization mechanism for many of the organisms that inhabit dental plaque. Porphyromonas gingivalis, a periodontal pathogen, can adhere to species that comprise early plaque, such as Streptococcus gordonii. In this study, the molecules of S. gordonii G9B that mediate binding to P. gingivalis were investigated. Biotinylated surface molecules of S. gordonii were extracted and mixed with P. gingivalis cells. Interactive streptococcal components were identified by SDS-PAGE of the P. gingivalis cells followed by electroblotting, and visualization of the adsorbed streptococcal molecules with streptavidin-alkaline phosphatase. S. gordonii molecules of 45 kDa and a doublet of 62/60 kDa were observed to bind to P. gingivalis. Polyclonal antibodies raised to the 62/60 kDa proteins inhibited the binding interaction. These antibodies demonstrated an antigenic relationship between the 62/60 kDa molecules and the 45 kDa protein. Both molecules were also antigenically related to, and may be breakdown products of, a larger molecule of 170 kDa which is antigenically related to the P1 antigen of S. mutans. Cloning and expression in Enterococcus faecalis of the gene for the P1-like molecule from S. gordonii M5 resulted in a phenotype that expressed the 62/60 kDa and 45 kDa antigens and was capable of binding to P. gingivalis. These results suggest that a P1-like molecule in S. gordonii is involved in adherence to P. gingivalis. Processing of the P1-like molecule into smaller fragments of 62/60 kDa and 45 kDa may be required for binding activity.

Involvement of Porphyromonas gingivalis fimbriae in adherence to Streptococcus gordonii.

Adherence of Porphyromonas gingivalis to early plaque bacteria, such as Streptococcus gordonii, is considered an important colonization mechanism. The molecules that mediate this interspecies binding have not been determined. Fimbriae were prepared from P. gingivalis 33277 by mild agitation, ammonium sulfate precipitation and DEAE-Sepharose chromatography. In a nitrocellulose blot adherence assay, purified fimbriae inhibited S. gordonii G9B-P. gingivalis 33277 binding by up to 54%. In addition, fimbriae bound to S. gordonii cells in a dot-blot assay. Incubation of fimbriae with S. gordonii cells followed by washing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotting and probing with P. gingivalis antibodies also revealed that the fimbriae bind to S. gordonii. In contrast, S. gordonii did not interact with fimbriae that were first subjected to SDS-PAGE and electroblotting or deposited on a nitrocellulose membrane, suggesting that conformational determinants of the fimbriae may be important in binding. The results indicate that binding between P. gingivalis and S. gordonii is mediated, at least in part, by the porphyromonads' fimbriae.

Characterization of the adherence of Porphyromonas gingivalis to oral streptococci.

Adherence of Porphyromonas gingivalis to strains of Streptococcus sanguis and Streptococcus mitis deposited on nitrocellulose paper was investigated. A variety of laboratory strains and clinical isolates of P. gingivalis bound to both S. sanguis and S. mitis. Binding of P. gingivalis to all but one of the streptococci was not inhibited by salivary molecules. Pretreatment of P. gingivalis with periodate and pretreatment of S. sanguis and S. mitis with pronase decreased binding, suggesting that adherence may be mediated by a protein on the streptococci interacting with a carbohydrate on P. gingivalis. Binding was not inhibited by a selection of simple sugars. The ability to adhere to early plaque bacteria such as S. sanguis and S. mitis may be important in the colonization of the mouth by P. gingivalis.

Salivary-agglutinin-mediated adherence of Streptococcus mutans to early plaque bacteria.

Interspecies binding is important in the colonization of the oral cavity by bacteria. Streptococcus mutans can adhere to other plaque bacteria, such as Streptococcus sanguis and Actinomyces viscosus, and this adherence is enhanced by saliva. The salivary and bacterial molecules that mediate this interaction were investigated. Salivary agglutinin, a mucinlike glycoprotein known to mediate the aggregation of many oral streptococci in vitro, was found to mediate the adherence of S. mutans to S. sanguis or A. viscosus. Adherence of S. mutans to saliva- or agglutinin-coated S. sanguis and A. viscosus was inhibited by antibodies to the bacterial agglutinin receptor. Expression of the S. sanguis receptor (SSP-5) gene in Enterococcus faecalis increased adhesion of this organism to saliva- or agglutinin-coated S. sanguis and A. viscosus. This interaction could be inhibited by antibodies to the agglutinin receptor. The results suggest that salivary agglutinin can promote adherence of S. mutans to S. sanguis and A. viscosus through interactions with the agglutinin receptor on S. mutans.

Adherence of mutans streptococci to other oral bacteria.

Adherence of mutans streptococci to strains of Actinomyces viscosus, Streptococcus sanguis, and Streptococcus mitis immobilized on a nitrocellulose membrane was measured. Strains of Streptococcus mutans, S. sobrinus, and S. rattus bound in a lactose-independent manner to a variety of the actinomyces and streptococci. Most of these reactions could proceed in the presence of whole saliva although adherence of S. rattus BHT to the streptococci was inhibited by salivary molecules. In contrast, adherence of S. mutans 10449 and KPSK2 to A. viscosus, S. sanguis, and S. mitis was enhanced by salivary molecules. S. mutans KPSK2, S. sobrinus OMZ 176, and S. rattus FA-1 binding to A. viscosus NC3 and S. sanguis G9B exhibited saturation kinetics. Adherence to A. viscosus NC3 was of a higher avidity than adherence to S. sanguis G9B. Attachment of S. mutans KPSK2 to S. sanguis G9B and of S. mutans OMZ 176 to A. viscosus NC3 and S. sanguis G9B was inhibited by heat treatment of the mutans streptococci. Attachment of S. mutans KPSK2 to A. viscosus NC3 and of S. rattus FA-1 to A. viscosus NC3 and S. sanguis G9B was unaffected by heat. These observations suggest that the mutans streptococci can adhere to a variety of early plaque bacteria by several distinct mechanisms. Such interactions may be important in the colonization of tooth surfaces by the mutans streptococci.

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin.

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.

Cloning and expression of an adhesin antigen of Streptococcus sanguis G9B in Escherichia coli.

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.

Characterization of an adhesion antigen of Streptococcus sanguis G9B.

An antigen possessing the attributes of an adhesion has been identified in Streptococcus sanguis G9B. Cell surface components were extracted from G9B and a spontaneously occurring nonadherent mutant of G9B, strain Adh-, with a 2 mM barbital buffer, pH 8.6. The extract of G9B but not of Adh- absorbed more than 80% of the adhesion-inhibitory activity of anti-G9B immunoglobulin G (IgG). Immunoblots revealed 80- and 52-kilodalton (kDa) antigens present in the G9B extract but not in the Adh- extract. Absorption of anti-G9B IgG with Adh- and G9B barbital extracts showed a correlation between the loss of the 80- and 52-kDa antibodies and the loss of adhesion-inhibitory activity. An antibody prepared against the 80-kDa antigen excised from sodium dodecyl sulfate-polyacrylamide gels recognized the 80- and 52-kDa antigens and another antigen of 62 kDa but did not inhibit adhesion. However, an antibody from an electroblot containing the native protein from which the 80-kDa and related antigens were derived (the 80-kDa antigen complex) inhibited adhesion to the same extent as anti-G9B IgG. Periodate oxidation of the G9B barbital extract modified the 80-kDa antigen complex and resulted in the loss of 40% of its absorbing activity. The barbital extract also contained an endogenous enzyme responsible for producing the 62- and 52-kDa antigens from the 80-kDa protein and which, under optimal conditions, degraded the antigen completely, resulting in the loss of antibody-absorbing activity. The 80-kDa antigen complex has a molecular mass of more than 200 kDa in native polyacrylamide gels and a pI of 4.1 to 4.8. These observations suggest that the adhesion antigen in S. sanguis G9B is a large glycoprotein from which an 80-kDa antigen complex is derived.