Background:The molecular mechanism underlying the regulation of cellulase production by T. reesei is unclear. Results: The absence of sugar transporter Stp1 enhanced cellulase gene induction whereas the absence of Crt1 abolished cellulase gene expression. Conclusion: Crt1 is essential in cellulase gene induction independent of intracellular sugar delivery. Significance: These data shed light on the mechanism by which T. reesei senses and transmits cellulose signal.

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular ␤-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple ␤-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular ␤-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular ␤-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three ␤-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three ␤-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei.

bLactose (1,4-O-␤-D-galacto-pyranosyl-D-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular ␤-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular ␤-glucosidases abolished cbh1 gene expression on lactose. D-Galactose restored the efficient cellulase gene induction in the ⌬cel1a strain independently of its reductive metabolism, but not in the ⌬cel1a ⌬cel1b strain. A further comparison of the transcriptional responses of the ⌬cel1a ⌬cel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the ⌬cel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis ␤-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular ␤-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.C ost-effective conversion of plant cell wall-derived polysaccharides holds the potential for production of an environmentally clean and renewable source of energy and platform chemicals (1). Trichoderma reesei (teleomorph Hypocrea jecorina) is well known for its high capacity to secrete large amounts of lignocellulosic enzymes that release fermentable sugars and has thus been developed into one of the most prolific industrial cellulase producers. High-yield production of the bulk of the plant cell walldegrading machinery in T. reesei is, however, dependent on induction by insoluble substrates that include cellulose, hemicellulose, and mixtures of plant polymers. Considering the ease of manipulation and the complication of separating enzymes from insoluble plant cell wall materials, soluble inducing substrates are usually preferred or required (2). Among others, the disaccharide lactose (1,4-O-␤-D-galacto-pyranosyl-D-glucose) is an important and economic soluble carbon source for cellulase production by T. reesei. However, the induced cellulase yields on lactose are usually lower than those on cellulose (3, 4). Understanding the differences in the inducing efficiency and the mode by which lactose triggers cellulase formation would be helpful for improving the performance of industrial strains.In fungi, catabolism of lactose is thought to proceed either by extracellular hydrolysis and subsequent uptake of the resulting sugar monomers or by uptake of the disaccharide followed by intracellular hydrolysis (4). For T. reesei, it has been assumed that lactose metabolism relies on the first strategy, based on several findings, including the absence of appar...

Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans. We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4+ T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance.