MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5'-GNG/C-3'). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production. IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila, which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila. Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.

The endocytosis of foot-and mouth disease virus requires clathrin and caveolin and is dependent on the existence of Rab5 and Rab7 in CHO-677 cells.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes severe vesicular disease of cloven-hoofed animals. Various endocytosis mechanisms are involved in the entry of FMDV after binding to the integrin and heparan sulfate (HS) receptors. However, the mechanism of FMDV using other unknown receptors to enter the cells remains unclear. Here, we reported that the endocytosis and endosomal pathways are employed by FMDV to invade the Chinese hamster ovary cell line (CHO-677) without the integrin and HS receptors. We demonstrated that the internalization of FMDV into CHO-677 cells was abrogated by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Knockdown of the clathrin heavy chain decreased the viral protein abundance. Incubation of the CHO-677 cells with the inhibitors of caveolae-mediated endocytosis or transfection by caveolin-1 siRNA also limited FMDV replication. In addition, we determined that the acidic environment and the existence of dynamin were essential for FMDV infection in CHO-677 cells. The endosomal proteins Rab5 (early endosome) and Rab7 (late endosome), but not Rab11 (recycling endosome), were utilized by FMDV during infection. These data provide a new entry model of FMDV by unknown receptors which will help to better understand the pathogenesis mediated by FMDV.

Establishment and evaluation of a murine ανß3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus.

Integrin ανß3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of ανß3 integrin, a stable CHO-677 cell line expressing the murine ανß3 heterodimer (designated as "CHO-677-mανß3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits αν and ß3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-mανß3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-mανß3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable ανß3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of ανß3 integrin, and as a cell model for FMDV research.