Response to Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Aouacheria et al question the interpretation of contemporary assays to monitor programmed cell death with apoptosis-like features (A-PCD) in Aspergillus fumigatus Although our study focuses on fungal A-PCD for host immune surveillance and infectious outcomes, the experimental approach incorporates multiple independent A-PCD markers and genetic manipulations based on fungal rather than mammalian orthologs to circumvent the limitations associated with any single approach.

Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death.

Humans inhale mold conidia daily and typically experience lifelong asymptomatic clearance. Conidial germination into tissue-invasive hyphae can occur in individuals with defects in myeloid function, although the mechanism of myeloid cell-mediated immune surveillance remains unclear. By monitoring fungal physiology in vivo, we demonstrate that lung neutrophils trigger programmed cell death with apoptosis-like features in Aspergillus fumigatus conidia, the most prevalent human mold pathogen. An antiapoptotic protein, AfBIR1, opposes this process by inhibiting fungal caspase activation and DNA fragmentation in the murine lung. Genetic and pharmacologic studies indicate that AfBIR1 expression and activity underlie conidial susceptibility to NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-dependent killing and, in turn, host susceptibility to invasive aspergillosis. Immune surveillance exploits a fungal apoptosis-like programmed cell death pathway to maintain sterilizing immunity in the lung.

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior.

BACKGROUND: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. RESULTS: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. CONCLUSIONS: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development.

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.

The cysteine protease inhibitors EhICP1 and EhICP2 perform different tasks in the regulation of endogenous protease activity in trophozoites of Entamoeba histolytica.

Trophozoites of E. histolytica are equipped with two chagasin-like cysteine protease inhibitors, EhICP1 and EhICP2, also known as amoebiasin 1 and 2. Expression studies using E. invadens as model organism showed that corresponding mRNAs were detectable in both life stages of the parasite, cyst and trophozoite state. Unlike EhICP1 known to act in the cytosol, EhICP2 co-localized with cysteine protease EhCP-A1 in lysosome-like vesicles, as demonstrated by immunofluorescence microscopy. Silencing or overexpressing of the two inhibitors did not show any effect on morphology and viability of the trophozoites. Overexpression of the EhICPs, however, although dramatically dampening the proteolytic activity of cell extracts from the corresponding cell lines, did not influence expression rate or localization of the major amoebic cysteine proteases as well as phagocytosis and digestion of erythrocytes. Activity gels of cell extracts from strains overexpressing ehicp1 showed a drastically reduced activity of EhCP-A1 suggesting a high affinity of EhICP1 towards this protease. From these data, we propose that EhCP-A1 accidentally released into the cytosol is the main target of EhICP1, whereas EhICP2, beside its role in house-keeping processes, may control the proteolytic processing of other hydrolases or fulfils other tasks different from protease inhibition.

tRNA gene sequences are required for transcriptional silencing in Entamoeba histolytica.

Transcriptional silencing by trans inactivation can contribute to the regulation of gene expression in eukaryotic cells. In the human intestinal protozoan parasite Entamoeba histolytica, trans inactivation of the amoebapore-A gene (AP-A) was recently achieved by episomal transfection of E. histolytica trophozoites with the plasmid psAP1. The mechanism of AP-A trans inactivation is largely unknown, though it was suggested that a partial short interspersed transposable element (SINE) is required. By systematic assessment of various E. histolytica isolates transfected with psAP1 derivates, trans inactivation of AP-A was restricted to the strain HM-1:IMSS (2411) but could not be achieved in other standard laboratory strains. Importantly, sequences of an E. histolytica tRNA array that were located on psAP1 in close proximity to the AP-A upstream region and comprising the glutamic acid (TTC) (E) and tyrosine (GTA) (Y) tRNA genes were indispensable for AP-A silencing. In contrast to the case described in previous reports, SINE was not required for AP-A trans inactivation. AP-A expression could be regained in silenced cells by episomal transfection under the control of a heterologous E. histolytica promoter, opening a way toward future silencing of individual genes of interest in E. histolytica. Our results indicate that tRNA gene-mediated silencing is not restricted to Saccharomyces cerevisiae.

Major cysteine peptidases of Entamoeba histolytica are required for aggregation and digestion of erythrocytes but are dispensable for phagocytosis and cytopathogenicity.

Cysteine peptidases of Entamoeba histolytica (EhCPs) are considered to be important pathogenicity factors. It has been described that under standard axenic culture conditions, only three (ehcp-a1, ehcp-a2 and ehcp-a5) out of approximately 50 cysteine peptidase genes present in the E. histolytica genome are substantially expressed, thus representing the set of major EhCPs. In this study, transcriptional silencing of the major peptidase genes was used to characterize their physiological role in more detail. Analysing the transfectants a fourth major cysteine peptidase activity belonging to EhCP-A7 could be characterized. Neither cytopathic activity nor phagocytosis of erythrocytes was altered in CP-inactivated amoebae. However, a significant difference in haemolytic activity was observed. EhCP-A1 and EhCP-A7 apparently had no influence on haemolytic activity, whereas transfectants silenced for ehcp-a5 as well as those silenced for all major peptidases showed a significant reduction in their haemolytic activity. Furthermore, cells silenced for ehcp-a1 and ehcp-a7 and more effectively cells silenced in all major ehcps were impaired in digesting of phagocytosed erythrocytes. Moreover, amoebae silenced for all major peptidase genes lost the ability to form aggregates of erythrocytes prior to phagocytosis.

The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes.

BACKGROUND: A number of studies have shown that peptidases and in particular cysteine peptidases constitute major pathogenicity factors in Entamoeba histolytica. Recent studies have suggested that a considerable number of genes coding for proteolytic enzymes are present within the E. histolytica genome and questions remain about the mode of expression of the various molecules. RESULTS: By homology search within the recently published amoeba genome, we identified a total of 86 E. histolytica genes coding for putative peptidases, including 46 recently described peptidase genes. In total these comprise (i) 50 cysteine peptidases of different families but most of which belong to the C1 papain superfamily, (ii) 22 different metallo peptidases from at least 11 different families, (iii) 10 serine peptidases belonging to 3 different families, and (iv) 4 aspartic peptidases of only one family. Using an oligonucleotide microarray, peptidase gene expression patterns of 7 different E. histolytica isolates as well as of heat stressed cells were analysed. A total of 21 out of 79 amoeba peptidase genes analysed were found to be significantly expressed under standard axenic culture conditions whereas the remaining are not expressed or at very low levels only. In heat-stressed cells the expression of 2 and 3 peptidase genes, respectively, were either decreased or increased. Only minor differences were observed between the various isolates investigated, despite the fact that these isolates were originated from asymptomatic individuals or from patients with various forms of amoebic diseases. CONCLUSION: Entamoeba histolytica possesses a large number of genes coding for proteolytic enzymes. Under standard culture conditions or upon heat-stress only a relatively small number of these genes is significantly expressed and only very few variations become apparent between various clinical E. histolytica isolates, calling into question the importance of these enzymes in E. histolytica pathogenicity. Further studies are required to define the precise role of most of the proteolytic enzyme for amoeba cell biology but in particular for E. histolytica virulence.

Roles of a Trypanosoma brucei 5'->3' exoribonuclease homolog in mRNA degradation.

The genome of the kinetoplastid parasite Trypanosoma brucei encodes four homologs of the Saccharomyces cerevisiae 5'-->3' exoribonucleases Xrn1p and Xrn2p/Rat1p, XRNA, XRNB, XRNC, and XRND. In S. cerevisiae, Xrn1p is a cytosolic enzyme involved in degradation of mRNA, whereas Xrn2p is involved in RNA processing in the nucleus. Trypanosome XRND was found in the nucleus, XRNB and XRNC were found in the cytoplasm, and XRNA appeared to be in both compartments. XRND and XRNA were essential for parasite growth. Depletion of XRNA increased the abundances of highly unstable developmentally regulated mRNAs, perhaps by delaying a deadenylation-independent decay pathway. Degradation of more stable or unregulated mRNAs was not affected by XRNA depletion although a slight decrease in average poly(A) tail length was observed. We conclude that in trypanosomes 5'-->3' exonuclease activity is important in degradation of highly unstable, regulated mRNAs, but that for other mRNAs another step is more important in determining the decay rate.

Role of a 300-kilodalton nuclear complex in the maturation of Trypanosoma brucei initiator methionyl-tRNA.

tRNAs are transcribed as precursors containing 5' leader and 3' extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNA(Met) in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5' leader of initiator tRNA(Met) precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNA(Met) containing 5' extensions but processed 3' ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNA(Met) molecules with 5' and 3' extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNA(Met). Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNA(Met), 3' trimming precedes 5' processing.

Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements.

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian 'bloodstream form' trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.