It is widely assumed that the ability of Candida albicans to switch between different morphologies is required for pathogenesis. However, most virulence studies have used mutants that are permanently locked into either the yeast or filamentous forms which are avirulent but unsuitable for discerning the role of morphogenetic conversions at the various stages of the infectious process. We have constructed a strain in which this developmental transition can be externally modulated both in vitro and in vivo. This was achieved by placing one copy of the NRG1 gene (a negative regulator of filamentation) under the control of a tetracyclineregulatable promoter. This modified strain was then tested in an animal model of hematogenously disseminated candidiasis. Mice injected with this strain under conditions permitting hyphal development succumbed to the infection, whereas all of the animals injected under conditions that inhibited this transition survived. Importantly, fungal burdens were almost identical in both sets of animals, indicating that, whereas filament formation appears to be required for the mortality resulting from a deep-seated infection, yeast cells play an important role early in the infectious process by extravasating and disseminating to the target organs. Moreover, these infecting Candida yeast cells still retained their pathogenic potential, as demonstrated by allowing this developmental transition to occur at various time points postinfection. We demonstrate here the importance of morphogenetic conversions in C. albicans pathogenesis. This engineered strain should provide a useful tool in unraveling the individual contributions of the yeast and filamentous forms at various stages of the infectious process.

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 M) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 M farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent. Candida albicans is the most frequently isolated human fungal pathogen (5). The most recent surveys have shown thatCandida is the third or fourth most commonly isolated bloodstream pathogen in United States hospitals, having surpassed gram-negative rods in frequency (2,13,20,21). Notably, yeasts (mainly C. albicans) are the third leading cause of catheterrelated infections, and they have the second highest colonization to infection rate and the highest crude mortality rate overall (8,22).Structured microbial communities attached to surfaces, commonly referred to as biofilms, have increasingly been found to be sources of infection by C. albicans, especially in view of the vast number of biomaterials that are now being used in the medical industry. Biomaterials, such as stents, catheters, and orthopedic joints, for example, serve as excellent substrates for microbial adhesion and subsequent biofilm formation (6, 10, 15). Biofilms are specific and organized communities of cells under the control of signaling molecules rather than random accumulations of cells resulting from cell division (9). Cell-cell signaling, particularly quorum sensing, has been the focus of much research over the past decade in the microbiological arena. It has been demonstrated that quorum-sensing molecules are essential for bacterial biofilm formation and that homoserine lactones act in a concentrationdependent manner; a threshold concentration triggers the formation of a biofilm (17,27). Rece...

No abstract

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.