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A vaccine capable of protecting at-risk persons against infections due to Cryptococcus neoformans and Cryptococcus gattii could reduce the substantial global burden of human cryptococcosis. Vaccine development has been hampered though, by lack of knowledge as to which antigens are immunoprotective and the need for an effective vaccine delivery system. We made alkaline extracts from mutant cryptococcal strains that lacked capsule or chitosan. The extracts were then packaged into glucan particles (GPs), which are purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Subcutaneous vaccination with the GP-based vaccines provided significant protection against subsequent pulmonary infection with highly virulent strains of C. neoformans and C. gattii. The alkaline extract derived from the acapsular strain was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the most abundant proteins were identified. Separation of the alkaline extract by size exclusion chromatography revealed fractions that conferred protection when loaded in GP-based vaccines. Robust Th1- and Th17-biased CD4+ T cell recall responses were observed in the lungs of vaccinated and infected mice. Thus, our preclinical studies have indicated promising cryptococcal vaccine candidates in alkaline extracts delivered in GPs. Ongoing studies are directed at identifying the individual components of the extracts that confer protection and thus would be promising candidates for a human vaccine., IMPORTANCE The encapsulated yeast Cryptococcus neoformans and its closely related sister species, Cryptococcus gattii, are major causes of morbidity and mortality, particularly in immunocompromised persons. This study reports on the preclinical development of vaccines to protect at-risk populations from cryptococcosis. Antigens were extracted from Cryptococcus by treatment with an alkaline solution. The extracted antigens were then packaged into glucan particles, which are hollow yeast cell walls composed mainly of β-glucans. The glucan particle-based vaccines elicited robust T cell immune responses and protected mice from otherwise-lethal challenge with virulent strains of C. neoformans and C. gattii. The technology used for antigen extraction and subsequent loading into the glucan particle delivery system is relatively simple and can be applied to vaccine development against other pathogens.

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.

Theplasmid determinants ofkiller phenotypes intypeK1andK2killer yeast cells arethe1.9-kilobase (kb) Mland1.7-kb M2double-stranded RNAs(dsRNAs), respectively. Thesearedependent fortheir maintenance andencapsidation, inSaccharomyces cerevisiae virusScV-M, orScV-M2virus-like particles, onthecapsid provided byoneofagroupofmoderately related 4.7-kb dsRNAscalled LA.TheLIAandL2AdsRNAsfound innaturally isolated K1andK2killers encode 88-kilodalton VLlA-P1 and86-kilodalton VL2A-Pl capsids, respectively. Thesearecompetent forencapsidating homologous LAdsRNAsaswellasM dsRNAs.Most strains ofS.cerevisiae, including killers, contain oneofasecond groupofclosely related 4.7-kb dsRNAscalied LBC.Theseencode their own82-kilodalton capsid protein, VLBC-P1, which, atleast instrains containing only LBc, encapsidates homologous dsRNAinScV-Lnc virus-like particles. InaKlkiller strain containing bothLlA andLBC,ScV-M, particles contain onlyVLIA-Pl. Insuchstrains itisprobable thateachvirus-like particle contains asingle capsid typeandthateachLdsRNAisencapsidated byahomologous capsid. Moststrains oftheyeast Saccharomyces cerevisiae contain species ofdouble-stranded RNA (dsRNA) called L(ca. 4.7kilobases), encapsidated incytoplasmic virus-like parti

Synthesis of enzymes functional in the synthesis of nucleotide precursors of peptidoglycan ceases upon initiation of sporulation in Bacillus sphaericus. During sporulation, two periods of synthesis of these enzymes occur. The first starts at spore septum formation and is conincident with forespore engulfment; it involves the synthesis of those enzymes required for making the precursor of vegetative-type peptidoglycan, including L-lysyl ligase but no mesodiaminopimelyl ligase. The second period occurs shortly before the appearance of cortex. It involves the synthesis of diaminopimelyl ligase and the other enzymes needed for making the precursor of cortical peptidoglycan, but not lysyl ligase. Both events are a consequence of derepression at the level of transcription. Neither period of synthesis occurs in asporogenous mutants whose morphological block is at the point of spore septum formation.

In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae. Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species. Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs. 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated. Other L-dsRNA gene products and their transcripts may exist. For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA. Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m. A functional model for M1-dsRNA killer plasmid structure is presented.

Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.