Characterization of a factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans.

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of 125I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16,000-21,000 daltons, bound strongly to CM-Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125I-labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present in partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for beta-core protein, a cleavage product of the beta subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified beta-core protein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to beta-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species.

Binding sites for LH in Candida albicans: comparison with the mammalian corpus luteum LH receptor.

We have described recently the presence of binding sites for human LH (hLH) and human chorionic gonadotrophin (hCG) in microsomal and cytosol fractions prepared from the dimorphic, pathogenic fungus, Candida albicans. We have now compared the properties of Candida LH/hCG-binding sites with those of the ovine luteal LH receptor. Sheep luteal LH-binding sites were associated with luteal membranes, and little or no binding activity was present in cytosol fractions. In contrast, significant LH/hCG-binding activity was present in Candida cytosol. Moreover, there were marked differences in sensitivity to inhibition by metal ions, association and dissociation rates, and affinity constants between sheep and Candida LH-binding sites. Scatchard plots of 125I-labelled hLH binding to sheep luteal receptors demonstrated a single high-affinity component (association constant (Ka) 0.3 litres/pmol) which was displaceable by hCG. In contrast, Scatchard plots of binding to Candida microsomes and cytosol fractions demonstrated two components, one with high affinity (Ka 0.18 litres/pmol) and low capacity and a second site with lower affinity (Ka 4 litres/nmol) and high capacity, both of which were displaceable by unlabelled hCG. Gel permeation chromatography of cytosol demonstrated two distinct peaks of LH-binding activity with approximate molecular weights of greater than 1,000,000 and 30,000-50,000. Scatchard plots of 125I-labelled hLH binding to the higher molecular weight peak demonstrated a single, high-affinity LH-binding site (Ka 0.18 litres/pmol), whereas the lower molecular weight fraction contained both high- (Ka 0.17 litres/pmol) and low-affinity (Ka 4 litres/nmol) LH-binding sites. Both partially purified and highly purified hCG and hLH preparations displaced binding of 125I-labelled hLH and hCG to sheep luteal LH receptors at similar concentrations. 125I-labelled hLH/hCG binding to Candida membranes was also displaceable by low levels (ng) of partially purified hCG preparations, but much higher levels (micrograms) of highly purified hLH and hCG were required. This paradoxical observation suggested the presence of radiolabelled contaminants, or damaged forms induced during radioiodination of hormone tracers, which can bind more strongly to Candida membranes than unlabelled hCG and hLH but which do not bind to sheep LH receptors. However, no evidence for hLH tracer contaminants with differential binding to Candida and sheep luteal receptors was obtained following gel exclusion chromatography or fractionation on Concanavalin A-Sepharose. (Although three distinct 125I-labelled hLH fractions were resolved on Concanavalin A-Sepharose, presumably reflecting differences in their carbohydrate compositions, all three tracer peaks bound equivalently to both Candida membranes and ovine luteal LH receptors).(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental staphylococcal infections in newborn mice: inhibition of weight gain as an index of virulence.

We attempted to evaluate the neonatal mouse model as an indicator of the virulence of staphylococcal strains freshly isolated from human patients in hospital. In preliminary studies with two previously characterised clinical isolates of Staphylococcus aureus and one of S. epidermidis, three indices of infection were studied. These were: mortality rate, multiplication of organisms in the skin, and the effect on weight gain. Of these inhibition of normal weight grain by mice given subcutaneous injections when 3 days old was the most convenient and easily applied test. At a challenge dose of 10(6) c.f.u., the multiplication of organisms in the skin was correlated with the inhibition of normal weight gain. Weight gain was used to compare the virulence of a small series of clinical isolates from different types of staphylococcal infection. Strains isolated from severe infections caused a greater inhibition of weight gain than did strains from milder infections or environmental sources.

Association between histocompatability antigens (HLA) and nasal carriage of Staphylococcus aureus.

We investigated the association between phenotypes of histocompatability antigen (HLA) and nasal carriage of Staphylococcus aureus in two populations--healthy laboratory workers and patients attending an outpatients' clinic. When data from the two sources were pooled, it was evident that the presence of HLA-DR3 was associated with carriage, and the presence of HLA-DR2, HLA-DR1 and HLA-Bw35 with lack of carriage. However, since each person may have two antigenic specificities encoded at the HLA-A, the HLA-B, and the HLA-DR loci, the carriage of the organism was analysed for paired combinations of the more frequent phenotypes. For example, the lack of carriage evident with HLA-DR1 was more marked with the DR1-A11 and DR1-B7 combinations while the predisposition towards carriage shown with HLA-DR3 was more marked with the DR3-DR5 combination. The importance of the analysis of antigen combinations is discussed in relation to association of single antigens with carriage of S. aureus.

Novel inhibitors of fungal protein synthesis produced by a strain of Graphium putredinis. Isolation, characterisation and biological properties.

The isolation and structure determination of 6 analogues of the fungal protein synthesis inhibitor GR135402, from Graphium putredinis, is described. The relative potencies of the compounds as protein synthesis inhibitors and as in vitro antifungal agents provide interesting insights into the structure-activity relationships in this series.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Candida albicans gastrointestinal colonization and invasion in the mouse: effect of antibacterial dosing, antifungal therapy and immunosuppression.

Infant mice infected with Candida albicans by the oral-intragastric route became colonized in the gut and were persistently colonized into adulthood. Faecal levels of Candida were correlated with total gastrointestinal Candida and provided a useful means of detecting yeast overgrowth or elimination. Antibacterial agents promoting Candida overgrowth when given by the oral or parenteral route included ceftriaxone, augmentin and cefoperazone. Ceftizoxime had less effect. Ceftazidime and latamoxef produced raised levels only by the oral route. Gentamicin, vancomycin and metronidazole did not affect the Candida levels. Dosing with some antibacterials promoted an increase in gastrointestinal Candida and invasion to a greater extent than immunosuppression. Antifungal therapy to reduce gastrointestinal colonization was investigated using amphotericin B, nystatin, ketoconazole, intraconazole and fluconazole. Fluconazole was most effective at reducing faecal Candida.

Hormonal factors in vaginal candidiasis in rats.

The hormonal status of rats affected vaginal infection with Candida albicans. Four hours after infection viable counts were higher and germ tubes were longer in those animals in estrous than in other animals. However, the infection was not maintained with the change in epithelial cell type which occurred as part of the estrous cycle. Estrogen dosing following ovariectomy predisposed toward infection, while progesterone dosing did not. In rats injected with progesterone, germ tube clumping was seen, leukocytes were present, and elimination occurred before hyphal growth was evident. In rats injected with estrogen, however, infection was maintained, with hyphal growth extending throughout the cornified epithelial layer. Vaginal washings from rats dosed with estrogen promoted elongation of germ tubes in vitro to a greater extent than washings from other rats. Preincubation of blastospores in progesterone and subsequent infection of rats in pseudoestrous promoted clumping of germ tubes in the vagina. Strains of C. albicans varied in their virulence, which correlated with their ability to produce germ tubes in vitro. Loss of virulence occurred on subculture of a clinical isolate.

Antifungal properties in a novel series of triazino[5,6-b]indoles.

GR99060 and GR99062 are representatives of a series of 1,2,4-triazino[5,6-b]indole compounds. This series possessed broad-spectrum antifungal activity in vitro. The MIC ranges of the two compounds were as follows: 0.25 to 4 micrograms/ml for Candida albicans, 0.25 to 16 micrograms/ml for Candida sp., 1 to 8 micrograms/ml for Aspergillus spp., and 0.25 to 16 micrograms/ml for Cryptococcus neoformans. GR99062 was metabolized in vitro by a mouse liver microsomal preparation, while GR99060 was stable. GR99060 was efficacious in a murine model of systemic candidiasis by oral or parenteral administration, although no clear dose-response was achieved, suggesting that other factors adversely affected the compound's in vivo activity.

Inflammatory reactions to staphylococcal protein A in mice.

To throw more light on the role of Protein A (a cell-wall component of most strains of S. aureus), in infection and inflammation, due to this organism the immediate inflammatory reaction has been studied in hairless and hairless/obese mice after s.c. injection of the protein into the footpad following various forms of immunization or pretreatment (described). Non-immunized mice showed an inflammatory reaction to Protein A, as judged by swelling, reaching a peak 2 h after injection. This might have been due to a nonspecific interaction between certain mouse Igs and Protein A. When specific antibody levels were raised by prior immunization or infection, the swelling was greatly increased. No delayed reaction was seen at 24 or 48 h, nor was a positive patch test obtained. The difference in results seen in mice and other animals may be due partly to the fact that intradermal injections are not possible in the mouse, or because in the mouse, unlike other subjects which have been used, histamine does not play a part. Mice do not show anaphylactic shock and this may be a function of the class of murine Igs interacting with Protein A. Further studies on these factors are required.

Some effects of plasmids coding for antibiotic resistance on the virulence of Staphylococcus aureus.

The relative virulence of pairs of staphylococci differing in resistance plasmid content has been studied using the neonatal mouse weight gain test. Both clinical and laboratory strains were used which had undergone genetic manipulation, either curing for loss of plasmids or transduction for gain of plasmids. A difference in virulence was detected between two variants of S. aureus NCTC 8325 possessing different plasmids coding for penicillinase. However in most cases any form of genetic manipulation seemed to reduce the virulence of the staphylococcus. In the case of NCTC 9789 (PS 80) which was originally an epidemic strain, curing of a plasmid coding for cadmium resistance resulted in reduced virulence but original virulence could not be restored by transduction of the plasmid into the cured derivative.

Ketoconazole in experimental vaginal candidosis in rats.

Rats in pseudoestrous were treated subcutaneously with ketoconazole at 25 mg/kg twice a day. In both uninfected and infected rats ketoconazole inhibited the cornification of the vaginal epithelium. Thus, ketoconazole, in addition to having an antifungal effect, may aid in the removal of candida by inhibiting the epithelial conditions suitable for hyphal invasion.

6,7-disubstituted 2,4-diaminopteridines: novel inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Four novel, disubstituted diaminopteridines have been identified which antagonize the uptake of a folate precursor (para-aminobenzoic acid) by rat-derived Pneumocystis carinii maintained in short-term axenic culture at concentrations ranging from 4.5 to 26 microM. The compounds were at least 10 to 100 times more active than trimethoprim in this assay. None of these entities exhibited toxicity to mammalian cell lines at < 100 microM. The same structures also caused significant inhibition of Toxoplasma gondii tachyzoite replication within Madin-Darby bovine kidney cells at concentrations ranging from 0.1 to 10 microM. Three of the structures (GR92754, AH10639, and AH2504) were at least an order of magnitude more potent than the standard anti-T. gondii agent, pyrimethamine. All three entities were also significantly more potent and selective than pyrimethamine as inhibitors of T. gondii dihydrofolate reductase (DHFR), with 50% inhibitory concentrations within the range of 0.018 to 0.033 microM. One of these compounds, 6,7-dibutyl-2,4-diaminopteridine (GR92754), was also a potent and selective inhibitor of P. carinii DHFR (50% inhibitory concentration, 0.082 microM). GR92754 is the first DHFR inhibitor described that exhibits greater potency, selectivity, and intracellular activity against both organisms than any of the DHFR agents used clinically, namely, trimethoprim, pyrimethamine, and trimetrexate. This information could provide the starting point for examination of the pharmacokinetic and therapeutic potential of GR92754 and related chemical entities with animal models.

Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species.

The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.

Receptor-mediated elevation of adenylate cyclase by luteinizing hormone in Candida albicans.

Human luteinizing hormone (hLH) and the GTP analogue guanosine 5'-O-(3-thio)triphosphate stimulated morphogenesis in the dimorphic fungal pathogen Candida albicans. hLH bound specifically to subcellular fractions from C. albicans and stimulated adenylate cyclase activity in C. albicans microsomes. We provide the first demonstration of guanine-nucleotide-binding proteins (G-proteins) in C. albicans, and propose that the stimulation of C. albicans morphogenesis by hLH is mediated by a receptor-coupled adenylate cyclase system involving G-proteins.

A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the "ura-blaster" protocol described for Saccharomyces [Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.

Sordarins: A new class of antifungals with selective inhibition of the protein synthesis elongation cycle in yeasts.

GR135402, a sordarin derivative, was isolated in an antifungal screening program. GR135402, sordarin, and derivatives of both compounds were evaluated for their ability to inhibit cell-free translational systems from five different pathogenic fungi (Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Cryptococcus neoformans). The activity profile of GR135402 is extended to other chemical compounds derived from sordarin. Experimental results indicate that sordarin analogs exert their antifungal effects by specifically inhibiting the protein synthesis elongation cycle in yeasts but do not affect protein synthesis machinery in mammalian systems. Intrinsically resistant strains owe their resistance to differences in the molecular target of sordarins in these strains. Preliminary studies performed to elucidate the mode of action of this new class of antifungal agents have shown that the putative target of sordarins is one of the protein synthesis elongation factors.