In the dimorphic fungus Cad Chitin is a fibrous polymer off-1,4-N-acetylglucosamine that constitutes a major structural component of the cell wall of many species of fungi, including those species that are pathogenic in humans. Because chitin is not found in mammals and its biosynthesis is normally essential for the shape and viability of the fungal cell (1, 2), chitin synthesis is an attractive target for the design of antifungal drugs. The most common medical mycosis is caused by Candida albicans, which is a diploid organism with no sexual cycle. This fungus is capable ofdimorphic growth where growth can occur by unicellular budding or filamentous hyphal extension and branch formation (3). The hyphal cell wall has three to five times the chitin content of the yeast cell wall (4, 5) and the cells have up to 10 times the in vivo chitin synthase activity (6). Three genes encoding chitin synthases have been cloned and sequenced in C. albicans (7,8,37). Two encode chitin synthase zymogens (CHSI and CHS2), homologous to the CHS genes of Saccharomyces cerevisiae. The third chitin synthase gene, CHS3, is homologous to the S. cerevisiae CSD2 (9) gene (also called CALI) and is apparently the structural gene for an enzyme, which in S. cerevisiae, does not require proteolysis for its activity. Northern blot analysis of the C. albicans CHS genes showed that CHS2 mRNA levels were elevated in cells undergoing hyphal development, whereas the CHS1 mRNA was expressed only at low levels early in germ-tube formation (8).The role of each of the chitin synthase activities described in S. cerevisiae has been investigated by disrupting each of the relevant genes (for a review, see ref. 10). Gene disruptions are more difficult in C. albicans because the organism is constitutively diploid and because stable multiply marked strains are required for sequential gene disruptions. Sequential gene disruption of HEM3 of C. albicans has been achieved with two genetic markers (11), but host strains with sufficient markers for the disruption of more than one structural gene are not yet available. In this study, we employed the "ura-blaster" protocol originally described by Alani et al.(12) for use with S. cerevisiae, to disrupt all alleles of the hyphal-specific C. albicans CHS2 gene. This method allows the sequential disruption of target alleles. by using Ura3 auxotrophy as a single selectable marker. Because the selectable marker can be regenerated after the disruption of each allele, this method overcomes the need for multiply marked host strains and is, therefore, ideally suited for the analysis of families of genes such as the CHS genes in C. albicans. We show that the chs2 null mutant was still able to form germ tubes, albeit with a reduced chitin content compared with the isogenic wild-type parent. A prototrophic chs2-null mutant was still able to cause disease in normal and immunosuppressed mice and had a similar virulence to the parental CHS2 strain. A preliminary report of the construction of the Achs2::hisG null mutant has a...

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, andCryptococcus 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.

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.

SUMMARY. 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-DRl 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-DRl was more marked with the DR1-All and DR 1-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.

An N-acetyl-D-[14C]glucosamine radiolabel incorporation assay has been used to monitor chitin biosynthesis in whole cells of Candida albicans both in vitro and in vivo in two different mouse infection models, one using the peritoneal cavity as a chamber in which to add and retrieve cells and the other using infected kidneys. Specific labeling of chitin in alkali-insoluble material was confirmed by chitinase digestion, analysis of acid hydrolysates, and the use of nikkomycin Z as a probe. Nikkomycin Z was shown to strongly inhibit chitin biosynthesis in C. albicans grown in vitro and in vivo in both models. This demonstrates that nikkomycin Z-susceptible chitin synthase activity is present in C. albicans when the fungus is in its pathogenic state in vivo. The limited use of nikkomycin as a therapeutic agent is discussed.

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.As part of an antifungal screening programme we searched for compounds from microbial fermentation broths that inhibited fungal protein synthesis as determined by a Candida albicans cell free translation system using polyuridine as a synthetic template. This paper describes the isolation, chemical characterisation and biological properties of the compound designated GR135402 (Fig. 1) Seed cultures were inoculated with two agar plugs added to a 250ml Erlenmeyer flask containing 50ml of medium of the following composition: peptone (Oxoid L34) 10g, malt extract (Oxoid L39) 21g, glycerol 40g, Junlon PW 110 (Honeywell and Stein Ltd, Wallington, Surrey) 1g in 1 litre distilled water. The pH of the medium was adjusted to 6.5 by the addition of aq NaOH before autoclaving. The flasks of inoculated seed medium were at 250rpm with a 50mm throw, for 8 days.The seed cultures were used at 3% (v/v) to inoculate 50ml aliquots of fermentation medium in 250ml Erleneither statically or shaken at 250rpm. Fermenter cultures (5 liters working volume) were aerated at 2.5lpm and stirred at 700rpm. The fermentation medium SM2 contained arkasoy soy flour 15g, glycerol trioleate (Estol 1434) 10g and KH2PO4 1g in 1 litre of tap water. The ingredients were mixed in a Waring blender and the pH adjusted to 7.0 with KOH before autoclaving. Medium SM55 contained maltose 40g, maltodextrin MD30E 120g, peptone (Oxoid L37) 10g, cottonseed flour (Sigma) Fig. 1. Chemical structure of GR135402.

Summary:  Germination of Candida albicans was measured when blastospores were added to 10% rat serum and incubated for 2 h at 37°C. Sterols and steroids were tested for their ability to promote germination when added to serum. Oestriol, pregnanediol and pregnanetriol increased the percentage germination at concentrations of 0.001 — 1 μM. Luteinizing hormone similarly increased the percentage germination at concentrations of 0.001 — 1 μg/ml. 3‐isobutyl‐1‐methyl xanthine promoted germination at 1 — 100 μM. Zusammenfassung:  Es wurde die Keimschlauchbildung von Candida albicans‐ Blastosporen unter Zusatz von 10% Rattenserum und bei 2 h Inkubation bei 37°C beobachtet. Sterole and Steroide wurden auf ihre Fähigkeit hin untersucht, nach Zusatz zum Serum die Keimschlauchbildung zu fördern. Östriol, Pregnandiol and Pregnantriol erhöhten den Prozentsatz der Keimschlauchbildung bei Konzentrationen von 0.001 — 1 μM. Das Gelbkörperreifungshormon erhöhte den Keimschlauchprozent in Konzentrationen von 0.001 — 1 μg/ml. 3‐Isobutyl‐1‐methylxanthin begünstigte die Keimschlauchbildung im Bereich von 1 — 100 μM.

MANY ATTEMPTS have been made to set up experimental models to study staphylococcal infections. Often the route of challenge determines the disease produced. Mastitis results from inoculation into the mammary glands of mice and rabbits (Chandler, 1970;Adlam et al., 1976), osteomyelitis from injection into the bone marrow of rabbits (Norden, 1970), and endocarditis from intravenous injection in rabbits and rats (Nickerson et al,. 1969;Santoro and Levison, 1978). In respect of subcutaneous infection, except for a few important studies of experimental infection in man (Elek and Conen, 1957;Maibach, 1965; Marples and Kligman, 1976), most work has been performed on mice. As in most other experimental models of staphylococcal infection, large challenge doses are needed to produce subcutaneous lesions in adult mice, though these can be avoided if the organisms are injected with plugs of cotton dust (Noble, 1965). To assess the virulence of strains of staphylococci in terms of mortality, LD50 measurements have been made after intraperitoneal injections in mice (Chesbro, Wamola and Bartley, 1969), intracerebral injections in newborn mice (Namavar et al., 1976 and and intra-allantoic injections in chick embryos (McCabe, 1964).Recently, McKay and Arbuthnott (1979) showed that newborn mice are more susceptible than adult mice to subcutaneous challenge with staphylococci and compared the virulence of strains by assessing mortality and lesion formation. We have now made a more detailed investigation of this mouse model in an attempt to devise criteria other than mortality for assessing the virulence of staphylococci. MATERIALS AND METHODSBacterial strains. We studied 13 isolates of staphylococci (table I). These included six clinical isolates of Staphylococcus aureus from various infections, four multiple-antibiotic-resistant isolates of S. aureus from a study of hospital cross infection, two isolates of S. epidermidis and the reference "mouse-virulent" strain of S. aureus no. PS80 (NCTC9789).Mouse strain. The Sha Sha strain of mouse was used; this is homozygous from the gene "shaven". The mice never grow a first coat of hair and the adults have only a few short hairs. This "hairless" strain was chosen to study subcutaneous infection because it eliminated the necessity