An Omics Perspective on Candida Infections: Toward Next-Generation Diagnosis and Therapy.

Candida species can cause severe infections associated with high morbidity and mortality. Therefore, it is essential to gain more insight into the anti-fungal host defense response. The advent of omics technology and development of advanced systems biology tools has permitted to approach this in an unbiased and quantitative manner. This review summarizes the insights gained on anti-Candida immunity from genetic-, transcriptome-, proteome-, metabolome-, microbiome-, mycobiome-, and computational systems biology studies and discusses practical aspects and future perspectives.

Pattern recognition pathways leading to a Th2 cytokine bias in allergic bronchopulmonary aspergillosis patients.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterised by an exaggerated Th2 response to Aspergillus fumigatus, but the immunological pathways responsible for this effect are unknown. OBJECTIVE: The aim of this study was to decipher the pattern recognition receptors (PRRs) and cytokines involved in the Aspergillus-specific Th2 response and to study Aspergillus-induced responses in healthy controls and ABPA patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were stimulated with heat-killed Aspergillus conidia, various other pathogens, or PRR ligands. PRRs and cytokine pathways were blocked with PRR-blocking reagents, anti-TNF (Etanercept or Adalimumab), IL-1Ra (Anakinra) or IFNγ (IFN-gamma). ELISA and FACS were used to analyse cytokine responses. RESULTS: Aspergillus was the only pathogen that stimulated the Th2 cytokines IL-5 and IL-13, while Gram-negative bacteria, Gram-positive bacteria, Candida albicans, chitin, ß-glucan or Toll-like receptor (TLR) ligands did not. Depletion of CD4(+) cells abolished IL-13 production. Blocking complement receptor 3 (CR3) significantly reduced IL-5 and IL-13, while blocking TLR2, TLR4 or dectin-1 had no effect. ABPA patients displayed increased Aspergillus-induced IL-5 and IL-13 and decreased IFNγ production compared with healthy controls. All biological agents tested showed the capability to inhibit Th2 responses, but also decreased Aspergillus-induced IFNγ. CONCLUSIONS AND CLINICAL RELEVANCE: Aspergillus conidia are unique in triggering Th2 responses in human PBMCs, through a CR3-dependent pathway. ABPA patients display a significantly increased Aspergillus-induced Th2/Th1 ratio that can be modulated by biologicals. These data provide a rationale to explore IFNγ therapy in ABPA as a corticosteroid-sparing treatment option, by dampening Th2 responses and supplementing the IFNγ deficiency at the same time.

Autophagy is redundant for the host defense against systemic Candida albicans infections.

Autophagy has been demonstrated to play an important role in the immunity against intracellular pathogens, but very little is known about its role in the host defense against fungal pathogens such as Candida albicans. Therefore, the role of autophagy for the host defense against C. albicans was assessed by complementary approaches using mice defective in autophagy, as well as immunological and genetic studies in humans. Although C. albicans induced LC3-II formation in macrophages, myeloid cell-specific ATG7(-/-) mice with defects in autophagy did not display an increased susceptibility to disseminated candidiasis. In in vitro experiments in human blood mononuclear cells, blocking autophagy modulated cytokine production induced by lipopolysaccharide, but not by C. albicans. Furthermore, autophagy modulation in human monocytes did not influence the phagocytosis and killing of C. albicans. Finally, 18 single-nucleotide polymorphisms in 13 autophagy genes were not associated with susceptibility to candidemia or clinical outcome of disease in a large cohort of patients, and there was no correlation between these genetic variants and cytokine production in either candidemia patients or healthy controls. Based on these complementary in vitro and in vivo studies, it can be concluded that autophagy is redundant for the host response against systemic infections with C. albicans.

Analysis of high density expression microarrays with signed-rank call algorithms.

MOTIVATION: We consider the detection of expressed genes and the comparison of them in different experiments with the high-density oligonucleotide microarrays. The results are summarized as the detection calls and comparison calls, and they should be robust against data outliers over a wide target concentration range. It is also helpful to provide parameters that can be adjusted by the user to balance specificity and sensitivity under various experimental conditions. RESULTS: We present rank-based algorithms for making detection and comparison calls on expression microarrays. The detection call algorithm utilizes the discrimination scores. The comparison call algorithm utilizes intensity differences. Both algorithms are based on Wilcoxon's signed-rank test. Several parameters in the algorithms can be adjusted by the user to alter levels of specificity and sensitivity. The algorithms were developed and analyzed using spiked-in genes arrayed in a Latin square format. In the call process, p-values are calculated to give a confidence level for the pertinent hypotheses. For comparison calls made between two arrays, two primary normalization factors are defined. To overcome the difficulty that constant normalization factors do not fit all probe sets, we perturb these primary normalization factors and make increasing or decreasing calls only if all resulting p-values fall within a defined critical region. Our algorithms also automatically handle scanner saturation.

In vivo progression of LAPC-9 and LNCaP prostate cancer models to androgen independence is associated with increased expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR).

Androgen deprivation therapies for metastatic prostate cancer are useful initially, but progression to androgen independence usually results in relapse within 2 years. The molecular mechanisms underlying the clinically important transition from androgen dependence to androgen independence are poorly described. Several lines of investigation have suggested that insulin-like growth factors (IGFs) are involved in the biology of prostate cancer, but little is known about their relevance to progression to androgen independence. We used three in vivo models of androgen-dependent (AD) human prostate cancer to study this issue. Progression to androgen-independent (AI) growth was associated with a 60-fold increase in expression of IGF-I mRNA in LAPC-9 xenografts and a 28-fold increase in IGF-I expression in LNCAP xenografts, relative to the initial AD neoplasms. IGF type I receptor (IGF-IR) mRNA levels were approximately 2.5-fold and approximately 5-fold higher, respectively, in AI LAPC-9 and LNCaP tumors compared with the original AD neoplasms. AI growth of these xenografts was also associated with significant reductions in IGF binding protein-3 expression. LAPC-4 xenografts, which previously have been shown to exhibit molecular pathology related to HER-2/neu expression with progression to AI, showed relatively minor changes in expression of the genes investigated, but we nevertheless found evidence of increased IGF-IR phosphorylation with progression to androgen independence in this model. Taken together with prior observations, our results suggest that deregulation of expression of genes related to any one of several critical receptor tyrosine kinase regulatory systems, including IGF signaling, may confer androgen independence.

Genomic structure, chromosomal localization, and expression of human cathepsin W.

A 12 kb genomic fragment containing the entire open reading frame of the human cathepsin W was isolated and the genomic organization of this papain-like protease gene was determined. The 3.8 kb gene was mapped by fluorescence in situ hybridization to chromosome 11q13.1. The gene contained ten exons with introns ranging from 81 to 1119 bp. Four of the nine introns and a 5' untranslated exon were conserved when compared to related genes such as cathepsins L, K and S, whereas there was no similarity to the genomic organization of cathepsins B or C. In contrast to conserved splice site locations in other cysteine protease family members, the cathepsin W gene contained five unique locations. Furthermore, human cathepsin W cDNA was expressed, and was found to be localized within the rough endoplasmic reticulum in transiently transfected Cos-7 and Hela cells.

Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T-lymphocytes.

A 750-bp fragment of a novel human cysteine protease has been identified from the dbEST databank. PCR cloning and DNA sequencing yielded a 1.38-kb full-length cDNA which encodes a polypeptide of 376 amino acids. The protein consists of a putative 21-residue signal peptide, a 106-residue propeptide and a 252-residue mature protein. The deduced amino acid sequence contains the highly conserved residues of the catalytic triad of papain-like cysteine proteases: cysteine, histidine, and asparagine. Furthermore, the protein sequence possesses two potential N-glycosylation sites: one in the propeptide and one in the mature protein. Comparison of the amino acid sequence of human cathepsin W with other human thiol-dependent cathepsins revealed a relatively low degree of similarity (21-31%). In contrast to cathepsins L, S, K, B, H and O, cathepsin W contains a 21-amino acid peptide insertion between the putative active site histidine and asparagine residues and an 8-amino acid C-terminal extension. This unique sequence may indicate that cathepsin W belongs in a novel subgroup of papain-like proteases distinct from that of cathepsin L- and B-like proteases. Northern blot analysis indicates a specific expression of cathepsin W in lymphatic tissues. Further analysis revealed predominant levels of expression in T-lymphocytes, and more specifically in CD8+ cells. The expression of the protease in cytotoxic T-lymphocytes may suggest a specific function in the mechanism or regulation of T-cell cytolytic activity.

Identification of potential tyrosine-containing endocytic motifs in the carboxyl-tail and seventh transmembrane domain of the neurokinin 1 receptor.

Although agonist-induced endocytosis of G-protein-coupled receptors is critical for receptor desensitization and resensitization, receptor motifs that interact with the endocytic apparatus have not been adequately characterized. We examined the effects of mutating the rat neurokinin-1 receptor on endocytosis using 125I-substance P, fluorescent substance P, and receptor antibodies. Substance P induced rapid internalization of wild-type receptors that were targeted to perinuclear endosomes. Truncation of the C-tail at residues 324, 342, and 354 reduced internalization up to 60% and caused retention of receptors at the cell surface and in superficial endosomes. Mutation of Tyr-341 and Tyr-349 in potential tyrosine-containing endocytic motifs of the C-tail also impaired internalization. A Y305A mutant within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain showed impaired signaling and was minimally expressed at the plasma membrane but was found in cytoplasmic vesicles. In contrast, a Y305F mutant signaled normally and was normally expressed at the plasma membrane but showed impaired internalization. Thus, endocytosis of the neurokinin 1 receptor relies on several tyrosine-containing sequences in the C-tail and seventh transmembrane domain.

Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization.

We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin. The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein. The protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (Cys, His, Asn) characteristic for cysteine proteases of the papain superfamily. Human bleomycin hydrolase has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. The 52 kDa recombinant protein forms a hexamer of 310 kDa and acts strictly as an aminopeptidase with a broad substrate specificity. The lack of a leader sequence and its pH optimum at 7.2 suggest a cytosolic/nuclear localization. Human bleomycin hydrolase was detected at low to moderate expression levels in most of the human organs tested. Significantly higher RNA levels have been observed in a variety of tumor cell lines. The human enzyme effectively degrades both forms of bleomycin (A2 and B2) in vitro and could indeed be responsible for the resistance of various tumors to this widely used anticancer drug.

Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2.

We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.

Characterization of multiple prohormone convertase PC1/3 transcripts in porcine ovary.

Overlapping cDNAs encoding porcine prohormone convertase, PC1/3, have been isolated from a pregnant sow ovary cDNA library using a mouse PC1/3 cDNA as a probe. Nucleotide sequence analysis of these cDNAs predicts a PC1/3 precursor protein of 753 amino acid residues, which shares an overall sequence homology of 96, 92, and 92% with the human, rat, and mouse counterparts, respectively. Furthermore, five different polyadenylation sites have been observed. The utilization of these polyadenylation sites results in a length difference of 40-440 bp in the 3' untranslated regions of the transcripts.

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.

Processing of protein precursors by a novel family of subtilisin-related mammalian endoproteases.

The recent identification of a novel family of mammalian endoproteases that carry out intracellular processing of protein precursors at dibasic sites has ended a search that began twenty-five years ago with the discovery of the first such precursor, proinsulin. The five proteases found thus far are all related to the yeast dibasic-specific endoprotease kex2, and include PC2, PC3/PC1, PC4, furin/PACE, and PACE4. All are Ca(2+)-dependent serine proteases with catalytic domains organized similarly to the bacterial subtilisins. The emerging characteristics of these endoproteases, including their tissue-specific expression, subcellular localization, and cleavage site selectivity, indicates that members of this family arose during evolution to process a diverse group of functionally distinct precursors in a highly specific, compartmentalized and regulated fashion.

Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3.

Experiments using recombinant vaccinia viruses expressing rat proinsulin I coinfected into COS-7 cells with recombinant vaccinia virus expressing human furin, human PC2, mouse PC3 (subtilisin-related proprotein convertases 1-3, respectively), or yeast Kex2 indicate that in this system both Kex2 and furin produce mature insulin, whereas PC2 selectively cleaves proinsulin at the C-peptide-A-chain junction. This is a property consistent with its probable identity with the rat insulinoma granule type II proinsulin processing activity as described by Davidson et al. [Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, 93-96]. PC3 generates mature insulin but cleaves preferentially at the proinsulin B-chain-C-peptide junction. This pattern of cleavage by PC3 is similar, but not identical, to that of the highly B-chain-C-peptide junction-selective type I activity as described by Davidson et al., perhaps due to the presence of a P4 arginine residue near the C-peptide-A-chain junction unique to the rat proinsulins. These results along with data presented on the expression of both PC2 and PC3 in islet beta cells strongly support the conclusion that these proteases are involved in the conversion of proinsulin to insulin in vivo.

A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase.

PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.

Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

Isolation and characterization of a rat ventricular cDNA expressed specifically in cardiac and skeletal muscles.

We describe the isolation of a novel cDNA named Myomy and show that its transcripts are present in skeletal and cardiac muscles as well as in differentiated Sol 8 skeletal muscle cell line. Sequence analysis revealed that neither nucleotides nor deduced protein product have any significant homology to those previously described. The encoded protein of Myomy cDNA consists of 76 amino acids and has a molecular weight of 8,000 dalton. Based on its muscle specific expression, low abundance and a higher occurrence of SP(T)XX, S(T)S(T)XX motifs, we suggest that Myomy encodes a new muscle specific transcription factor.