Investigation Of The Functions Of Aspergillus Fumigatus ChsC Gene By Agrobacterium Tumefaciens-Mediated Transformation

Backgrounds and ObjectivesAspergillus fumigatus, an airborne fungal pathogen, is the major cause of allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Of particular importance is the invasive aspergillosis that starts with inhalation of conidia and progresses to life-threatening infection in immunocompromised patients. Due to the rising prevalence of cancer, organ transplantation, and other causes of immunosuppression, the number of patients at risk of invasive aspergillosis is on the rise. Despite aggressive antifungal therapy, the overall mortality of invasive aspergillosis remains high, and new strategies to prevent and treat this disease are urgently needed. Many molecular and genes involved the virulence of Aspergillus fumigatus. But, at present, it is not known what molecular or gene play a predominant role in invasive aspergillosis (IA). Chitin is the third polysaccharide component of the wall,several chitin synthases (chs) have been detected in Aspergillus fumigatus. The family of chs genes in Aspergillus fumigatus include at least seven different genes, called chsA (class I), chsB (class II), chsC (class III), chsD (class VI), chsE (class V), chsF (class IV) and chsG (clase III). Disruption of these genes may leads to an altered phenotype in Aspergillus fumigatus. The chsE- mutant has reduced levels of chitin in the mycelium, swellings throughout the length of the hypha and conidiation. The strains with chsG- mutations showed a reduced activity of the chitin synthetase enzyme, reduced radial growth and highly branched hyphae, suggesting that the function of the enzyme is in the apex of the hypha. The chsE-/chsG- double mutantion is not lethal for this fungus, it has reduced chitin synthase activity and colony radial growth rate, and produced highly branched hyphae. But whether or not chitin synthase gene chsC plays a role in lifestyle, appearance and sensitivity for antifungus drugs in Aspergillus fumigatus is unknown.As genomic sequencing of Aspergillus fumigatus has been succeeded, investigation of the functions of such genes can be accomplished via mutational analysis either by reverse or forward genetic approaches. To disrupt a gene by homologous integration in Aspergillus fumigatus, transformation via the spheroplast method has been most widely used; this is a laborious and time-consuming method, and the homologous recombination frequency can be relatively low. Electroporation and biolistic methods were also used to transform Aspergillus fumigatus. these methods, however, resulted in a high frequency of multiple integrations and a low frequency of homologous recombination. As an alternative method, Agrobacterium tumefaciens-mediated transformation (ATMT) has been used to transform several Aspergillus species. Here we report transformation of Aspergillus fumigatus mediated by Agrobacterium tumefaciens as a tool for insertional mutagenesis to construct chsC- mutant of Aspergillus fumigatus, and investgate the function of chsC gene in lifestyle, appearance and sensitivity for antifungus drugs in Aspergillus fumigatus. For ATMT is a new method of transformation of Aspergillus fumigatus, we investgate the optimize condition of ATMT as well.Methods and Results1. The construction and identification of pDHt/chsC-::hph in ATMT. To construct the plasmid used for ectopic mutagenesis, , we amplificate two approximately 500 bp fragments templeted two side of chsC gene. Following, insert the two fragments into pPK2, and made the hygromycin resistance gene (hph) in pPK2 flanked by two chsC gene fragment sequence, this plasmid named as pPK2/ chsC-::hph. The pDHt/chsC-::hph was constructed by insertion of a 5.5-kb HindIII/SacI fragment from pPK2/ chsC-::hph containing the hygromycin resistance gene flanked by two chsC gene fragment sequence into the HindIII/SacI restricted pDHt/SK plasmid. The plasmid used for ectopic mutagenesis named as pDHt/chsC-::hph. To confirm the pDHt/chsC-::hph been constructed successfully, we digested the plasmid by HindIII/SacI restriction enzyme and found the plasmid be digested into two fragment, 5.5-kb fragment and 7.45-kb fragment. All constructs were sequenced and blasted for the further confirmation.2. Tageted disruption of chsC of Aspergillus fumigatus by ATMT. Agrobacterium tumefaciens strain EHA105 was transformed by Calcium chloride with pDHt/chsC-::hph plasmids. Strain of Agrobacterium tumefaciens harboring pDHt/chsC-::hph was named EHA105/chsC-. To investget the tansformation efficience, the Agrobacterium tumefaciens-mediated transformation was conducted at different co-cultivation temperature (23,24,25,26,27,28℃),different transformation media(nylon,nitrocellulose filters,liquid phase cultivation),different co-cultivation period(12,24,36,48h),AS in preincubation or not,different concentration of AS in co-cultivation(100,200,300,400μg/ml),co-cultivated at different ratios of conidia to bacteria(1:1,1:10,1:100,1:1000),hygromycin resistance cassette was used to select the positive transformants. Tansformants were inoculated to LB broth with high concentration hygromycin( 200,400μg/ml) to identify the positive transformant, reverse transcription PCR (RT-PCR) was carried out for further identification.Agrobacterium tumefaciens strain EHA105 could be transformed by Calcium chloride with pDHt/chsC-::hph plasmids. Nylon and liquid phase cultivation have the same tansformation efficience. Compared with the two method, nitrocellulose filters had lower tansformation efficience. Co-cultivation at 25℃during ATMT could gain the most transformants, Co-cultivation at lower or higher than this temperature during ATMT usually got lower tansformation efficience. With the elongation of co-cultivationt period, we ould gain more transformants. Co-cultivated for 72 hours, it is difficult to pick the monoclone for too many transformants. From 1:1 to 1:100, with the increasing of co-cultivationt ratios, the transformants were increasing as well. But if the ratio of conidia to bacteria reached to 1:1000, the transformants do not increase any more. The tansformation efficience of ATMT is not changed significantly while the media contain AS or not. Higher concentration of AS in inducing medium usually got higher tansformation efficience. The positive transformant growed well in the media with high concentration hygromycin. RT-PCR showed that ATMT could transfor T-DNA to Aspergillus fumigatus and made the chsC gene tageted disruption.3. Investegation of basical character of chsC- mutant. Conidia were inoculated into Czapek medium and grown at 37℃for 3 days to observe the growth velocity. Aspergillus fumigatus chsC- mutant and wild type Aspergillus fumigatus were microcultured to observe the difference of morphous and the sensitivity to antifungus drugs of the chsC- mutant. NCCLS M38-A protocol was employed to investigate the activities of amphotericin B (AmB), itraconazole (ICZ), terbinafine (TBF), fluconazole (FCZ) and 5-flucytosine (5-FC) against Aspergillus fumigatus.On growth velocity chsC- mutant is obviously slower than wild type Aspergillus fumigatus, but there have no difference on figure and color in following observation. Under light microscope , the conidium chains of wild type Aspergillus fumigatus MIDA1 are short, and the hypha are uniformity, conidial head are round and full. The conidium chains of chsC- mutant are long, and the hypha are not uniformity, intumescence can be seen in some section. Under high vacuum of electron microscope,the hypha of wild type Aspergillus fumigatus MIDA1 are collapse because of anhydration, but the conidium are round and full. Under high vacuum of electron microscope,The hypha of chsC- mutant are collapse because of anhydration, and the conidium are collapse as well. Under black vacuum of electron microscope,the hypha and conidium of wild type Aspergillus fumigatus MIDA1 are full, the conidium chains are short. The hypha of chsC- mutant are collapse because of anhydration, the conidium chains are long. ChsC- mutant and wild type Aspergillus fumigatus have the same sensitivity to 5-Fc,AmB,FCZ,ICZ,but to TBF the value of MIC chsC- mutant obviously lower than wild type Aspergillus fumigatusConclusions1. The transformat system composed of Agrobacterium tumefaciens species EHA105 and pDHt/SK plasmid Agrobacterium tumefaciens-mediated transformation can transformat T-DNA to Aspergillus fumigatus and targeted disrupt the destinating gene.2. During ATMT,co-cultivation temperature, transformat media, co-cultivation period, concentration of AS in co-cultivation,co-cultivated ratios of conidia to bacteria play important roles in the transformation efficience,but AS in preincubation or not does not affect the tansformation efficience seriously.3. ChsC gene of Aspergillus fumigatus play an important role in its lifestyle, appearance and sensitivity for antifungus drugs.