Tor-dependent post-transcriptional regulation of autophagy: Implications for cancer therapeutics.

Paradoxically, both anticancer immunosurveillance and tumor progression have been associated with intact autophagy, which is regulated by the target of rapamycin (Tor1). Here, we describe the potential impact on the design of cancer therapeutics of a newly described highly conserved post-transcriptional mechanism whereby Tor regulates autophagy.

TOR-dependent post-transcriptional regulation of autophagy.

Regulation of autophagy is required to maintain cellular equilibrium and prevent disease. While extensive study of post-translational mechanisms has yielded important insights into autophagy induction, less is known about post-transcriptional mechanisms that could potentiate homeostatic control. In our study, we showed that the RNA-binding protein, Dhh1 in Saccharomyces cerevisiae and Vad1 in the pathogenic yeast Cryptococcus neoformans is involved in recruitment and degradation of key autophagy mRNAs. In addition, phosphorylation of the decapping protein Dcp2 by the target of rapamycin (TOR), facilitates decapping and degradation of autophagy-related mRNAs, resulting in repression of autophagy under nutrient-replete conditions. The post-transcriptional regulatory process is conserved in both mouse and human cells and plays a role in autophagy-related modulation of the inflammasome product IL1B. These results were then applied to provide mechanistic insight into autoimmunity of a patient with a PIK3CD/p110δ gain-of-function mutation. These results thus identify an important new post-transcriptional mechanism of autophagy regulation that is highly conserved between yeast and mammals.

A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy.

Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110δ gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.

The role of transcriptional 'futile cycles' in autophagy and microbial pathogenesis.

Eukaryotic cells utilize macroautophagy (hereafter autophagy) to recycle cellular materials during nutrient stress. Target of rapamycin (Tor) is a central regulator of this process, acting by post-translational mechanisms, phosphorylating preformed autophagy-related (Atg) proteins to repress autophagy during log-phase growth. We recently reported an additional role for post-transcriptional regulation of autophagy, whereby the mRNA decapping protein, Dcp2, undergoes Tor-dependent phosphorylation, resulting in increased ATG mRNA decapping and degradation under nutrient-rich, repressing conditions. Dephosphorylation of Dcp2 during starvation is associated with dissociation of the decapping-ATG mRNA complex, with resultant stabilization of, and accumulation of, ATG transcripts, leading to induction of autophagy. Regulation of mRNA degradation occurs in concert with known mRNA synthetic inductive mechanisms to potentiate overall transcriptional regulation. This mRNA degradative pathway thus constitutes a type of transcriptional 'futile cycle' where under nutrient-rich conditions transcript is constantly being generated and degraded. As nutrient levels decline, steady state mRNA levels are increased by both inhibition of degradation as well as increased de novo synthesis. A role for this regulatory process in fungal virulence was further demonstrated by showing that overexpression of the Dcp2-associated mRNA-binding protein Vad1 in the AIDS-associated pathogen Cryptococcus neoformans results in constitutive repression of autophagy even under starvation conditions as well as attenuated virulence in a mouse model. In summary, Tor-dependent post-transcriptional regulation of autophagy plays a key role in the facilitation of microbial pathogenesis.

A copper hyperaccumulation phenotype correlates with pathogenesis in Cryptococcus neoformans.

Cryptococcus neoformans is a major human pathogen and a cause of meningoencephalitis in immunocompromised patients. Many factors contribute to the extraordinary survivability and pathogenicity of this fungus in humans, including copper homeostasis pathways. Previous work has shown that deletion of the copper-dependent regulator Cuf1 results in decreased virulence and dissemination in brain infection, suggesting that copper acquisition is important to the persistence of this pathogen. Here, we show that the minimal copper quota of C. neoformans is maintained at a high level even when grown under conditions of stringent copper limitation. Intriguingly, when this fungal pathogen is grown in standard and copper-enriched media, it sequesters even higher levels of this essential metal, achieving levels that are far higher than non-pathogenic S. cerevisiae. The hypothesis that copper acquisition plays an essential role in virulence is further corroborated by the findings that a hypovirulent CUF1-deletant strain of C. neoformans retrieved from infected mice contains almost a 6-fold lower concentration of intracellular copper than the pathogenic wild-type strain. The concentration difference arises in part from larger-sized cuf1Δ cell. Under in vitro growth conditions, the size of the cuf1Δ cells is normal and the hypertrophy phenotype is readily induced in vitro under conditions of copper starvation. Taken together, these data suggest that acquisition of extraordinary levels of copper is an important factor in the survivability of the pathogen in the copper-deplete environment of infection, and effective copper concentration may play an important role in the pathogenesis of C. neoformans.

Role of CTR4 in the Virulence of Cryptococcus neoformans.

UNLABELLED: While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-length CTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function of CTR4 in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role for CTR4 in copper-related homeostasis and subsequently in fungal virulence. IMPORTANCE: Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a Δctr4 mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4 during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.

PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans.

Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Delta), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8-labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Delta cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Delta C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.

Role of a CUF1/CTR4 copper regulatory axis in the virulence of Cryptococcus neoformans.

The study of regulatory networks in human pathogens such as Cryptococcus neoformans provides insights into host-pathogen interactions that may allow for correlation of gene expression patterns with clinical outcomes. In the present study, deletion of the cryptococcal copper-dependent transcription factor 1 (Cuf1) led to defects in growth and virulence factor expression in low copper conditions. In mouse models, cuf1Delta strains exhibited reduced dissemination to the brain, but no change in lung growth, suggesting copper is limiting in neurologic infections. To examine this further, a biologic probe of available copper was constructed using the cryptococcal CUF1-dependent copper transporter, CTR4. Fungal cells demonstrated high CTR4 expression levels after phagocytosis by macrophage-like J774.16 cells and during infection of mouse brains, but not lungs, consistent with limited copper availability during neurologic infection. This was extended to human brain infections by demonstrating CTR4 expression during C. neoformans infection of an AIDS patient. Moreover, high CTR4 expression by cryptococcal strains from 24 solid organ transplant patients was associated with dissemination to the CNS. Our results suggest that copper acquisition plays a central role in fungal pathogenesis during neurologic infection and that measurement of stable traits such as CTR4 expression may be useful for risk stratification of individuals with cryptococcosis.

Cell wall targeting of laccase of Cryptococcus neoformans during infection of mice.

Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Delta cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

The translocated Salmonella effector proteins SseF and SseG interact and are required to establish an intracellular replication niche.

The facultative intracellular pathogen Salmonella enterica causes a variety of diseases, including gastroenteritis and typhoid fever. Inside epithelial cells, Salmonella replicates in vacuoles, which localize in the perinuclear area in close proximity to the Golgi apparatus. Among the effector proteins translocated by the Salmonella pathogenicity island 2-encoded type III secretion system, SifA and SseG have been shown necessary but not sufficient to ensure the intracellular positioning of Salmonella vacuoles. Hence, we have investigated the involvement of other secreted effector proteins in this process. Here we show that SseF interacts functionally and physically with SseG but not SifA and is also required for the perinuclear localization of Salmonella vacuoles. The observations show that the intracellular positioning of Salmonella vacuoles is a complex phenomenon resulting from the combined action of several effector proteins.

Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling.

Mechanisms controlling the balance between proliferation and self-renewal versus growth suppression and differentiation during normal and leukemic myelopoiesis are not understood. We have used the bi-potent FDB1 myeloid cell line model, which is responsive to myelopoietic cytokines and activated mutants of the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor, having differential signaling and leukemogenic activity. This model is suited to large-scale gene-profiling, and we have used a factorial time-course design to generate a substantial and powerful data set. Linear modeling was used to identify gene-expression changes associated with continued proliferation, differentiation, or leukemic receptor signaling. We focused on the changing transcription factor profile, defined a set of novel genes with potential to regulate myeloid growth and differentiation, and demonstrated that the FDB1 cell line model is responsive to forced expression of oncogenes identified in this study. We also identified gene-expression changes associated specifically with the leukemic GM-CSF receptor mutant, V449E. Signaling from this receptor mutant down-regulates CCAAT/enhancer-binding protein alpha (C/EBPalpha) target genes and generates changes characteristic of a specific acute myeloid leukemia signature, defined previously by gene-expression profiling and associated with C/EBPalpha mutations.

Identification of the promoter regions and sigma(s)-dependent regulation of the gadA and gadBC genes associated with glutamate-dependent acid resistance in Shigella flexneri.

Resistance to killing by low pH is a common feature of both Escherichia coli and Shigella flexneri. The most effective E. coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB, and a putative glutamate/gamma-amino butyric acid antiporter encoded by gadC. Expression of the gad system is dependent upon the alternate sigma factor, sigma(s). We confirm that gadA, gadB, and gadC are also all dependent upon sigma(s) for their expression in S. flexneri. -10 sequences similar to the sigma(s)-10 consensus sequence were identified by primer extension in the upstream promoters of all three genes and the transcriptional start points were identical in both E. coli and S. flexneri.

Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system.

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella-containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid.

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.

Transcriptional expression of Escherichia coli glutamate-dependent acid resistance genes gadA and gadBC in an hns rpoS mutant.

Resistance to being killed by acidic environments with pH values lower than 3 is an important feature of both pathogenic and nonpathogenic Escherichia coli. The most potent E. coli acid resistance system utilizes two isoforms of glutamate decarboxylase encoded by gadA and gadB and a putative glutamate:gamma-aminobutyric acid antiporter encoded by gadC. The gad system is controlled by two repressors (H-NS and CRP), one activator (GadX), one repressor-activator (GadW), and two sigma factors (sigma(S) and sigma(70)). In contrast to results of previous reports, we demonstrate that gad transcription can be detected in an hns rpoS mutant strain of E. coli K-12, indicating that gad promoters can be initiated by sigma(70) in the absence of H-NS.