Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger.

Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis

Correlation of gene expression and protein production rate - a system wide study

<p>Abstract</p> <p>Background</p> <p>Growth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on <it>Saccharomyces cerevisiae </it>chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.</p> <p>Results</p> <p>We have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of <it>T. reesei</it>. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.</p> <p>Conclusions</p> <p>Based on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).</p

Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus

Operons

Operons (clusters of co-regulated genes with related functions) are common features of bacterial genomes. More recently, functional gene clustering has been reported in eukaryotes, from yeasts to filamentous fungi, plants, and animals. Gene clusters can consist of paralogous genes that have most likely arisen by gene duplication. However, there are now many examples of eukaryotic gene clusters that contain functionally related but non-homologous genes and that represent functional gene organizations with operon-like features (physical clustering and co-regulation). These include gene clusters for use of different carbon and nitrogen sources in yeasts, for production of antibiotics, toxins, and virulence determinants in filamentous fungi, for production of defense compounds in plants, and for innate and adaptive immunity in animals (the major histocompatibility locus). The aim of this article is to review features of functional gene clusters in prokaryotes and eukaryotes and the significance of clustering for effective function

Prevalence of Toxoplasma gondii in localized populations of Apodemus sylvaticus is linked to population genotype not to population location

Toxoplasma gondii is a globally distributed parasite infecting humans and warm-blooded animals. Although many surveys have been conducted for T. gondii infection in mammals, little is known about the detailed distribution in localized natural populations. In this study, host genotype and spatial location were investigated in relation to T. gondii infection. Wood mice (Apodemus sylvaticus) were collected from 4 sampling sites within a localized peri-aquatic woodland ecosystem. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii was detected using 4 specific PCR-based markers: SAG1, SAG2, SAG3 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples were positive giving an infection rate of 34.92% (95% CI: 27.14-43.59%). Juvenile, young adults and adults were infected at a similar prevalence, respectively, 7/17 (41.18%), 27/65 (41.54%) and 10/44 (22.72%) with no significant age-prevalence effect (P = 0.23). Results of genetic analysis of the mice showed that the collection consists of 4 genetically distinct populations. There was a significant difference in T. gondii prevalence in the different genotypically derived mouse populations (P = 0.035) but not between geographically defined populations (P = 0.29). These data point to either a host genetic/family influence on parasite infection or to parasite vertical transmission

Protein Kinase A Regulates Autophagy-Associated Proteins Impacting Growth and Virulence of Aspergillus fumigatus

Cellular recycling via autophagy-associated proteins is a key catabolic pathway critical to invasive fungal pathogen growth and virulence in the nutrient-limited host environment. Protein kinase A (PKA) is vital for the growth and virulence of numerous fungal pathogens. However, the underlying basis for its regulation of pathogenesis remains poorly understood in any species. Our Aspergillus fumigatus PKA-dependent whole proteome and phosphoproteome studies employing advanced mass spectroscopic approaches identified numerous previously undefined PKA-regulated proteins in catabolic pathways. Here, we demonstrate reciprocal inhibition of autophagy and PKA activity, and identify 16 autophagy-associated proteins as likely novel PKA-regulated effectors. We characterize the novel PKA-phosphoregulated sorting nexin Atg20, and demonstrate its importance for growth, cell wall stress response, and virulence of A. fumigatus in a murine infection model. Additionally, we identify physical and functional interaction of Atg20 with previously characterized sorting nexin Atg24. Furthermore, we demonstrate the importance of additional uncharacterized PKA-regulated putative autophagy-associated proteins to hyphal growth. Our data presented here indicate that PKA regulates the autophagy pathway much more extensively than previously known, including targeting of novel effector proteins with fungal-specific functions important for invasive disease