Gametocyte-specific and all-blood-stage transmission-blocking chemotypes discovered from high throughput screening on Plasmodium falciparum gametocytes.

Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.

Molecular epidemiology and characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans strains isolated in Italy during the 1990s.

Five cases of diphtheria were reported in Italy between January 1990 and June 2001. Three cases were confirmed microbiologically by the isolation of toxigenic Corynebacterium diphtheriae (two cases) and Corynebacterium ulcerans (one case). Over the same period, 11 cases of non-toxigenic C. diphtheriae infection were reported to the Italian Public Health Institute, from which the causative organism was isolated from a skin infection in one case and from the throat in the other ten. Seven of the throat isolates were associated with fever, severe pharyngitis and tonsillitis and were all biotype gravis. Because there are no standardized breakpoints, the antimicrobial sensitivities of C. diphtheriae were determined in accordance with the National Committee for Clinical Laboratory Standards guidelines for Streptococcus spp. other than Streptococcus pneumoniae. MICs for penicillin ranged between 0.125 and 0.250 mg l(-1) and 7 out of 11 strains had a minimal bactericidal concentration (MBC)/MIC ratio >or= 32. All strains were sensitive to clindamycin (MIC

A highly immunogenic recombinant and truncated protein of the secreted aspartic proteases family (rSap2t) of Candida albicans as a mucosal anticandidal vaccine.

Sap2 (secreted aspartyl proteinase2) is a member of the Sap family of Candida albicans, a human opportunistic pathogen, which acts as a virulence factor in experimental animal models of mucosal candidiasis. The C. albicans SAP2 was subcloned into vector pDS56-RBSII-6xhis, under the control of an inducible promoter to produce a truncated 6xhis-tagged, enzymatically inactive Sap2, lacking the N-terminus 76 amino acids (rSap2t). This recombinant protein was purified to homogeneity by one-step nickel-chelate affinity chromatography and used to immunize intravaginally oophorectomized estradiol-treated rats. These animals raised local anti-rSap2t immunoglobulin G (IgG) and IgA antibodies and were protected from the challenge of a highly vaginopathic strain of the fungus. Protection was possibly due to the specific antibodies as suggested by the passive transfer of immune vaginal fluid and the protective effects of passive vaccination with anti-rSap2t IgM and IgG monoclonal antibodies. Hence, this new recombinant proteinase constitutes a novel tool to investigate mechanisms of anti-Candida protection at the vaginal level and as vaccination against vaginal candidiasis, a common, frequently recurrent and sometimes antimycotic-refractory infection in women.

The 65 kDa mannoprotein gene of Candida albicans encodes a putative beta-glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity.

Mannoproteins are fungal cell wall components which play a main role in host-parasite relationship. Camp65p is a putative beta-glucanase mannoprotein of 65 kDa which has been characterized as a main target of human immune response against Candida albicans. However, nothing is known about its specific contribution to the biology and virulence of this fungus. We constructed CAMP65 knock-out mutants including null camp65/camp65 and CAMP65/camp65 heterozygous strains. The null strains had the same growth rate and morphology under yeast form as the wild-type strain but they were severely affected in hyphal morphogenesis both in vitro and in vivo. Hyphae formation was restored in revertant strains. The null mutants lost adherence to the plastic, and this was in keeping with the strong inhibition of fungal cell adherence to plastic exerted by anti-Camp65p antibodies. The null mutants were also significantly less virulent than the parental strains, and this loss of virulence was observed both in systemic and in mucosal C. albicans infection models. Nonetheless, the virulence in both infectious models was regained by the CAMP65 revertants. Thus, CAMP65 of C. albicans encodes a putative beta-glucanase, mannoprotein adhesin, which has a dual role (hyphal cell wall construction and virulence), accounting for the particular relevance of host immune response against this mannoprotein.

Human domain antibodies against virulence traits of Candida albicans inhibit fungus adherence to vaginal epithelium and protect against experimental vaginal candidiasis.

Antibody variable domains (domain antibodies [DAbs]) are genetically engineered antibody fragments that include individual heavy-chain (VH) or kappa-chain (Vkappa) variable domains and lack the Fc region. Human DAbs against the 65-kDa mannoprotein (MP65) or the secretory aspartyl proteinase (SAP)-2 of Candida albicans (monospecific DAbs) or against both fungal antigens (heterodimeric, bispecific DAbs) were generated from phage expression libraries. Both monospecific and bispecific DAbs inhibited fungus adherence to the epithelial cells of rat vagina and accelerated the clearance of vaginal infection with the fungus. When heterodimeric DAbs were used, the clearance of infection was at least equivalent to treatment with fluconazole. The in vivo protective effects of DAbs were demonstrated by both pre- and postchallenge schedules of DAb administration and with both fluconazole-susceptible and fluconazole-resistant strains of C. albicans. This is the first demonstration that human DAbs lacking the Fc constituent can efficiently control an infection and can act largely by inhibiting adherence.

Use of 65 kDa mannoprotein gene primers in Real Time PCR identification of Candida albicans in biological samples.

A method for the detection and quantification of Candida albicans in biological samples (blood, urine and serum) was developed with the use of Real-Time PCR utilizing CaMP65-specific primers. Two different systems were used for the detection in the LightCycler platform (Roche): the SYBR green fluorescent dye with melting peak analysis and the 5'nuclease fluorescent-probe detection. The amplification was highly specific for C. albicans, providing no cross-reaction on genomic DNA extracted from other Candida species or Aspergillus. The sensitivity in simulated biological samples was especially high (1 genome) when applied to sera and urine, and in blood samples the limit of detection was higher by ten-fold. Finally, the real-time PCR was employed in order to detect and quantify C. albicans in the sera from patients with invasive candidiasis.

Cell-cycle-regulatory elements and the control of cell differentiation in the budding yeast.

The stable differentiation of cells into other cell types typically involves dramatic reorganization of cellular structures and functions. This often includes remodeling of the cell cycle and the apparatus that controls it. Here we review our understanding of the role and regulation of cell cycle control elements during cell differentiation in the yeast, Saccharomyces cerevisiae. Although the process of differentiation may be more overtly obvious in metazoan organisms, those systems are by nature more difficult to study at a mechanistic level. We consider the relatively well-understood mechanisms by which mating-type switching and the pheromone-induced differentiation of gametes are coupled to the cell cycle as well as the more obscure mechanisms that govern the remodeling of the cell cycle during meiosis and filamentous growth. In some cases, the cell cycle is a primary stimulus for differentiation whereas, in other cases, the signals that promote differentiation alter the cell cycle. Thus, despite relative simplicity of these processes in yeast, the nature of the interplay between the cell cycle and differentiation is diverse.

Immunogenicity and protective effect of recombinant enolase of Candida albicans in a murine model of systemic candidiasis.

Enolase, a 46-kDa glycolytic enzyme, is an immunodominant antigen of the opportunistic pathogen Candida albicans. A recombinant 6 x His-tagged enolase was studied, in conjunction with interleukin-12 (IL-12), as an adjuvant for cytokine induction favouring protection in a murine model of haematogenous candidiasis. Mice immunized with enolase plus IL-12 showed increased antibody titres against enolase, as well as increased median survival time and decreased fungal burden in kidneys, in comparison to non-immunized or IL-12-treated mice. This increased survival was attributable to enolase-induced cell-mediated immunity as it also occurred in B-cell-deficient mice. Enolase immunization stimulated a predominant T-helper-1 (Th1) cytokine pattern in splenic cells and induced production of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by purified CD4+ T cells. However, despite the elevation of immunogenicity, recombinant enolase induced only a modest protection against disseminated candidiasis, suggesting a form of protection likely attributable to the induction of a Th1 cell-mediated immune response.