Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response

Mycobacterium tuberculosis causes tuberculosis, which kills more people than any other infection. M. tuberculosis grows in macrophages, cells that specialize in engulfing and degrading microorganisms. Like many intracellular pathogens, in order to cause disease, M. tuberculosis damages the membrane-bound compartment (phagosome) in which it is enclosed after macrophage uptake. Recent work showed that when chemicals damage this type of intracellular compartment, cells rapidly detect and repair the damage, using machinery called the endosomal sorting complex required for transport (ESCRT). Therefore, we hypothesized that ESCRT might also respond to pathogen-induced damage. At the same time, our previous work showed that the EsxG-EsxH heterodimer of M. tuberculosis can inhibit ESCRT, raising the possibility that M. tuberculosis impairs this host response. Here, we show that ESCRT is recruited to damaged M. tuberculosis phagosomes and that EsxG-EsxH undermines ESCRT-mediated endomembrane repair. Thus, our studies demonstrate a battle between host and pathogen over endomembrane integrity.Intracellular pathogens have varied strategies to breach the endolysosomal barrier so that they can deliver effectors to the host cytosol, access nutrients, replicate in the cytoplasm, and avoid degradation in the lysosome. In the case of Mycobacterium tuberculosis, the bacterium perforates the phagosomal membrane shortly after being taken up by macrophages. Phagosomal damage depends upon the mycobacterial ESX-1 type VII secretion system (T7SS). Sterile insults, such as silica crystals or membranolytic peptides, can also disrupt phagosomal and endolysosomal membranes. Recent work revealed that the host endosomal sorting complex required for transport (ESCRT) machinery rapidly responds to sterile endolysosomal damage and promotes membrane repair. We hypothesized that ESCRTs might also respond to pathogen-induced phagosomal damage and that M. tuberculosis could impair this host response. Indeed, we found that ESCRT-III proteins were recruited to M. tuberculosis phagosomes in an ESX-1-dependent manner. We previously demonstrated that the mycobacterial effectors EsxG/TB9.8 and EsxH/TB10.4, both secreted by the ESX-3 T7SS, can inhibit ESCRT-dependent trafficking of receptors to the lysosome. Here, we additionally show that ESCRT-III recruitment to sites of endolysosomal damage is antagonized by EsxG and EsxH, both within the context of M. tuberculosis infection and sterile injury. Moreover, EsxG and EsxH themselves respond within minutes to membrane damage in a manner that is independent of calcium and ESCRT-III recruitment. Thus, our study reveals that T7SS effectors and ESCRT participate in a series of measures and countermeasures for control of phagosome integrity

Toward an Integrated Model of Capsule Regulation in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation

Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide

The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdo

Model-driven mapping of transcriptional networks reveals the circuitry and dynamics of virulence regulation

Key steps in understanding a biological process include identifying genes that are involved and determining how they are regulated. We developed a novel method for identifying transcription factors (TFs) involved in a specific process and used it to map regulation of the key virulence factor of a deadly fungus—its capsule. The map, built from expression profiles of 41 TF mutants, includes 20 TFs not previously known to regulate virulence attributes. It also reveals a hierarchy comprising executive, midlevel, and “foreman” TFs. When grouped by temporal expression pattern, these TFs explain much of the transcriptional dynamics of capsule induction. Phenotypic analysis of TF deletion mutants revealed complex relationships among virulence factors and virulence in mice. These resources and analyses provide the first integrated, systems-level view of capsule regulation and biosynthesis. Our methods dramatically improve the efficiency with which transcriptional networks can be analyzed, making genomic approaches accessible to laboratories focused on specific physiological processes

Cryptococcus neoformans Dual GDP-mannose transporters and their role in biology and virulence

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis

Yeast IME2 Functions Early in Meiosis Upstream of Cell Cycle-Regulated SBF and MBF Targets

BACKGROUND: In Saccharomyces cerevisiae, the G1 cyclin/cyclin-dependent kinase (CDK) complexes Cln1,-2,-3/Cdk1 promote S phase entry during the mitotic cell cycle but do not function during meiosis. It has been proposed that the meiosis-specific protein kinase Ime2, which is required for normal timing of pre-meiotic DNA replication, is equivalent to Cln1,-2/Cdk1. These two CDK complexes directly catalyze phosphorylation of the B-type cyclin/CDK inhibitor Sic1 during the cell cycle to enable its destruction. As a result, Clb5,-6/Cdk1 become activated and facilitate initiation of DNA replication. While Ime2 is required for Sic1 destruction during meiosis, evidence now suggests that Ime2 does not directly catalyze Sic1 phosphorylation to target it for destabilization as Cln1,-2/Cdk1 do during the cell cycle. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that Sic1 is eventually degraded in meiotic cells lacking the IME2 gene (ime2Δ), supporting an indirect role of Ime2 in Sic1 destruction. We further examined global RNA expression comparing wild type and ime2Δ cells. Analysis of these expression data has provided evidence that Ime2 is required early in meiosis for normal transcription of many genes that are also periodically expressed during late G1 of the cell cycle. CONCLUSIONS/SIGNIFICANCE: Our results place Ime2 at a position in the early meiotic pathway that lies upstream of the position occupied by Cln1,-2/Cdk1 in the analogous cell cycle pathway. Thus, Ime2 may functionally resemble Cln3/Cdk1 in promoting S phase entry, or it could play a role even further upstream in the corresponding meiotic cascade

Grid Portal Application

This project concerns EMMIL (E-Marketplace Model Integrated with Logistics) and its viability as a grid application. The model was evaluated on a single processor and on the SEEGRID network using MTA-SZTAKI's P-GRADE Portal. A granularity heuristic was developed to guide the mapping of EMMIL datasets to processes. A portlet for P-GRADE Portal was also created to aid in data entry. Finally, pre-processing filters were added. These were designed to discard useless combinations and reduce overall computing time