The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the ␣1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the ␣1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1⌬ null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.Candida albicans is a commensal organism carried by a significant proportion of healthy individuals. It is the most common opportunistic fungal pathogen of humans causing superficial infections of the mucosa and in the immunocompromised host life-threatening systemic infections (1-4). The cell wall is the immediate point of contact between fungus and host and plays an important role in adherence, antigenicity, and the modulation of the host immune response (5-9). The outer layer of the cell wall is enriched in highly glycosylated mannoproteins (10), and both the protein and carbohydrate components have been implicated in the host-fungal interaction (5, 6, 11). The study of glycosylation in C. albicans therefore has its own relevance in identifying the carbohydrate epitopes involved in pathogenesis.Cell surface mannoproteins contain both O-and N-linked oligosaccharides. The O-linked oligosaccharides, attached to serine or threonine, consist of a linear chain of one to five ␣1,2-linked mannose residues (12)(13)(14) and are known to be required for full virulence (14). The process of N-glycosylation has been studied extensively in Saccharomyces cerevisiae. N-Linked glycosylation is initiated in the endoplasmic reticulum with the transfer of the Glc 3 Man 9 GlcNAc 2 oligosaccharide precursor to the protein target (15, 16). The oligosaccharide precursor is then processed by endoplasmic reticulum-resident glucosidases and a mannosidase to yield the mature triantennary Man 8 GlcNAc 2 core (17). Outer chain ...

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the ␦-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid ␦-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% ␦-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulationspecific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid ␦-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of ␦-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed. Dormant bacterial endospores formed by the genera Bacillus and Clostridium are the most resistant living structures known and are able to survive thousands if not millions of years (9). During the quiescent state, spores exhibit high-level resistance to many treatments, including heat, UV light, desiccation, and the action of deleterious chemicals. As a result of their resistance properties, spores are able to survive many food preservation and pasteurization procedures and thus cause huge problems to the food industry (11).Endospores are characterized by a relatively dehydrated protoplast encased in integument layers (20). The most prominent of the integuments are the spore coat layers which determine the physical properties of the spore surface and are responsible for resistance to enzymatic assault (56). However, the spore coats are not involved in the maintenance of dormancy and heat resistance (56). Between the spore coats and the protoplast membrane is a thick layer of bacterial peptidoglycan, consisting of two sublayers. Innermost is the ...

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Candida albicans is the major fungal pathogen of humans. This opportunistic pathogen can cause irritating superficial infections of the mucosa and serious life threatening systemic infections in the immunocompromised patient (1, 2). Invasive candidosis in hospitals now represents the third or fourth most common form of septicaemia (3, 4). The cell surface of C. albicans is the immediate point of contact between the fungus and host and plays vital roles in adhesion and immunomodulation of host responses, and it is a source of antigens (5-8). The outer cell wall layer is enriched in mannoproteins, which are embedded in a matrix of structural polysaccharides consisting of ␤-1,3-and ␤-1,6-linked glucan and chitin (9). This layer is important in adhesion to host surfaces and their subsequent colonization (10 -12). Both the protein and carbohydrate components of mannoproteins have been implicated in adhesion to the host (10, 13-15), although details of the nature of the ligands and receptors are still lacking. Hence, glycosylation of cell wall proteins is critical for host-fungal interactions and pathogenicity. Mnt2p also functions inKnowledge of glycosylation in Saccharomyces cerevisiae (16 -28) and information from the C. albicans genome data base has provided significant resources for the identification and analysis of glycosylation genes in C. albicans. Mannoproteins of S. cerevisiae and C. albicans contain both N-and O-linked oligosaccharides. The N-linked glycans, attached to asparagine residues of proteins, contain a conserved core structure and an elaborate, highly branched outer mannose chain that is specific to fungi and contains both acid-stable and acid-labile components (17,29,30). Glycosylation in C. albicans has its own relevance in investigations of the role of specific oligosaccharide moieties in host-fungal interactions. The acid-labile mannosylphosphate component, containing ␤-1,2-linked mannose, has been implicated in adhesion and recognition of phagocytic leukocytes, although mutants lacking this component have been shown to have normal interactions with macrophages (31). Both ␤-1,2-and ␣-1,2-linked mannan oligosaccharides have been implicated directly in adhesion functions (12,32).In C. albicans, O-glycans are linear oligosaccharides of one to five ␣-1,2-linked mannose residues (32-34). In S. cerevisiae an ␣-1,2-linked O-linked glycan is capped with one or two ␣-1,3-linked mannose residues (27). O-Glycosylation in S. cerevisiae is initiated in the endoplasmic reticulum where at least four of the seven-membered PMT gene family act to transfer mannose from dolichyl phosphate-activated mannose to serine or threonine (18,35,36). Evidently this step is essential, as certain combinations ...

The location and function of recognized cortex-lytic enzymes of Bacillus subtilis have been explored, and the involvement in germination of a number of related proteins tested. The SleB and CwlJ proteins are cortex-lytic enzymes, partially redundant in function, that are required together for effective cortex hydrolysis during B. subtilis spore germination. Spores were fractionated, and Western blotting of individual fractions suggests that the CwlJ protein is localized exclusively to the outer layers, or integument. The second spore-lytic enzyme, SleB, is localized both in the inner membrane of the spore and in the integument fraction. Neither protein changes location or size as the spore germinates. The ypeB gene is the second gene in a bicistronic operon with sleB. The SleB protein is absent from ypeB mutant spores, suggesting that YpeB is required for its localization or stabilization. In fractions of wild-type spores, the YpeB protein is found in the same locations as SleB -in both the inner membrane and the integument. As the absence of CwlJ protein does not affect the overall RP-HPLC profile of peptidoglycan fragments in germinating spores, this enzyme's hydrolytic specificity could not be defined. The effects of inactivation of several homologues of cortex-lytic enzymes of as yet undefined function were examined, by testing null mutants for their germination behaviour by OD 600 fall and by RP-HPLC of peptidoglycan fragments from dormant and germinating spores. The YaaH enzyme is responsible for a likely epimerase modification of peptidoglycan during spore germination, but the loss of this activity does not appear to affect the spore's ability to complete germination. Unlike the other cortex-lytic enzymes, the YaaH protein is present in large amounts in the spore germination exudate of B. subtilis. Mutants lacking either YdhD or YvbX, both homologues of YaaH, had no detectable alteration in either dormant or germinating spore peptidoglycan, and germinated normally. The ykvT gene, which encodes a protein of the SleB/CwlJ family, has no apparent association with germination : the gene is expressed in vegetative cells, and mutants lacking YkvT have no detectable phenotype.

The sigE gene of Streptomyces coelicolorA3(2) encodes an RNA polymerase sigma factor belonging to the extracytoplasmic function (ECF) subfamily. Constructed sigEdeletion and disruption mutants were more sensitive than the parent to muramidases such as hen egg white lysozyme and to the CwlA amidase fromBacillus subtilis. This correlated with an altered muropeptide profile, as determined by reverse-phase high-performance liquid chromatography analysis of lytic digests of purified peptidoglycan. The sigE mutants required high levels of magnesium for normal growth and sporulation, overproducing the antibiotic actinorhodin and forming crenellated colonies in its absence. Together, these data suggest that sigE is required for normal cell wall structure. The role of ςE was further investigated by analyzing the expression of hrdD, which is partially sigE dependent. The hrdDgene, which encodes the ςHrdD subunit of RNA polymerase, is transcribed from two promoters, hrdDp 1 andhrdDp 2, both similar to promoters recognized by other ECF sigma factors. The activities ofhrdDp 1 and hrdDp 2 were reduced 20- and 3-fold, respectively, in sigE mutants, although only hrdDp 1 was recognized by EςE in vitro. Growth on media deficient in magnesium caused the induction of both hrdD promoters in asigE-dependent manner.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilisvegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in thedacA mutant. This major low-molecular-weight PBP (dd-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

The role of the sleB gene of Bacillus subtilis, which encodes a putative sporecortex-lytic enzyme, and the downstream ypeB gene were investigated. Both SleB and YpeB were required for normal germination to occur. The corresponding mutants formed phase-bright, heat-resistant spores with no apparent defects in dormancy. However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the triggering of germination. Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked in the later stages of germination. The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore components can compensate for their function sufficiently to allow outgrowth. The expression and regulation of the operon was examined using a lacZ transcriptional fusion. Expression of the operon began 2 h after the onset of sporulation and was under the control of RNA polymerase containing the forespore-specific sigma factor, σ G . The application of reverse phase HPLC revealed that the mutants do not have any structural defect in the dormant spore cortex and therefore these genes are not required for normal sporecortex synthesis. The analysis of peptidoglycan dynamics during germination showed, however, that the cortex was only partially hydrolysed in both mutants. This analysis also revealed that the likely hydrolytic bond specificity of SleB is likely to be that of a lytic transglycosylase. Keywords :Bacillus subtilis, endospores, germination, peptidoglycan, cortex hydrolysis INTRODUCTIONThe formation of resistant dormant endospores is a survival strategy used by members of the genera Bacillus and Clostridium during nutrient deprivation. The spore cortex maintains heat resistance and dormancy (Ellar, 1978). The cortex consists of a thick layer of peptidoglycan with a unique spore-specific structure (Atrih et al., 1996 ;Popham et al., 1996a). Hydrolysis of the cortex peptidoglycan is essential for later germination events and outgrowth Atrih et al., 1998 ;Popham et al., 1996b).A number of spore-cortex-lytic enzymes have been isolated from spores of different organisms. Two lytic enzymes have been isolated from Clostridium perfringens S40 spores -a 31 kDa enzyme encoded by the gene sleC (Miyata et al., 1995) and a 38 kDa enzyme encoded by the gene sleM (Chen et al., 1997). The Bacillus cereus IFO 13597 gene sleB encodes a 24 kDa enzyme (Makino et al., 1994 ; Moriyama et al., 1996b), and its homologue has been identified and inactivated in Bacillus subtilis. The resulting mutant germinates slower than the wild-type (Moriyama et al., 1996a).Foster & Johnstone (1987) isolated a germinationspecific lytic enzyme (GSLE) that was capable of cortex hydrolysis from the spores of Bacillus megaterium KM. The enzyme was activated in vivo during germination and has a high specificity for intact spore cortex. Western blot analysis revealed cross-reactivity with proteins from spore fractions of...