S., Reck-Peterson S. ; Raetz and Whitfield, 2002 ; Whitfield, 2006 ). In fungi, most of the cell wall is composed of polysaccharides, including – and/or -glucans, chitin, and some mannans (Bose is an environmental basidiomycetous yeast that causes IITZ-01 fatal infections in mammals. The two most widespread serotypes, A and D, are a leading cause of fungal meningoencephalitis in immunocompromised individuals, with the majority of cases caused by serotype A strains. The characteristic of that is unique among pathogenic fungi is the polysaccharide capsule that surrounds its cell wall (Bose Strains lacking GXM are avirulent in animal models (Chang and Kwon-Chung, 1994 , 1998 , 1999 ; Chang (1967) observed a halo between the cell wall and the capsule using conventional transmission electron microscopy (TEM) and suggested it was a zone of capsule synthesis. Several years later, Takeo (1973a , 1973b) demonstrated that this halo Rabbit Polyclonal to BMX corresponded to a layer of 20-nm granules, which they speculated were involved in polymerization of capsule precursors. These investigators used a freeze-etching technique to compare grown in vitro to cells grown in a mouse model of infection, which have more extensive capsule. They noted that the number of intracellular vesicles in the latter cells was greater and hypothesized that the capsule was synthesized inside these vesicles. In both cell populations they also observed unusual plasma membrane invaginations containing small vesicles, which they termed the baglike paramural bodies. This observation led them to alternatively speculate that capsule was made in the vesicles of the paramural body. Twenty years later, Sakaguchi (1993) used the quick-freeze, deep-etch technique to improve image resolution in the comparison IITZ-01 of in vivo- and in vitro-grown cells and observed that a particle-accumulating layer between the IITZ-01 cell wall and the capsule was thicker in cells grown in vivo. They proposed that the accumulated particles represented capsule precursors that were then polymerized into fibrils, but did not indicate a model for overall capsule synthesis. More recently, two other reports touched on the location of capsule synthesis in (2001) performed immunoelectron microscopy (immuno-EM) using anti-GXM antibodies, biotin-conjugated secondary antibodies, and gold-conjugated streptavidin and observed 200C300-nm aggregations of label within the cytosol, which they termed vacuolar structures. In another report, this group used the same immuno-EM method and noted clusters of gold particles in the cytoplasm and cell wall of (Garcia-Rivera exocytosis mutants, vesicles of 100 nm in diameter accumulate upon shift to a restrictive temperature, and these post-Golgi vesicles have been shown to contain secreted proteins (Novick Sec4p homolog to address whether GXM uses the secretory pathway to exit the cell. MATERIALS AND METHODS Strains and Growth Conditions strains stored in 25% glycerol at ?80C were streaked onto yeast extract peptone dextrose (YPD) plates for growth at room temperature (RT) or at 30C. Where IITZ-01 indicated, plates were supplemented with nourseothricin (100 g/ml) or G418 (100 g/ml). Liquid cultures were grown in YPD medium with shaking (230 rpm) at RT or at 30C. Serotype A strain H99 was provided by G. Cox (Duke University) and serotype D strain JEC21 was from J. Lodge (St. Louis University). Generation of other strains is described below. Identification of a Cryptococcal Homolog of the Sec4/Rab8 Subfamily of Rab GTPases BLAST was used to query the Institute of Genome Research (TIGR) annotations of the serotype D strain JEC21 (http://www.tigr.org/tdb/e2k1/cna1/) with the amino acid sequence of Sec4p (“type”:”entrez-protein”,”attrs”:”text”:”NP_116650″,”term_id”:”14318517″,”term_text”:”NP_116650″NP_116650). The closest homolog (E-value of 6 10?61) IITZ-01 was “type”:”entrez-protein”,”attrs”:”text”:”CNC04340″,”term_id”:”814290416″,”term_text”:”CNC04340″CNC04340 (GenBank “type”:”entrez-protein”,”attrs”:”text”:”XP_569689″,”term_id”:”58265066″,”term_text”:”XP_569689″XP_569689), which showed 53% identity and 67% similarity to Sec4p when whole sequences were compared using AlignX (VectorNTI Suite, Invitrogen, Carlsbad, CA). The genomic sequence of “type”:”entrez-protein”,”attrs”:”text”:”XP_569689″,”term_id”:”58265066″,”term_text”:”XP_569689″XP_569689, termed database (http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans/Home.html); this was identified as Supercontig 5: 681180C681966. The amino acid sequence encoded by this serotype A protein predicted by TWINSCAN.