Study On Salt Induced Drug Resistance Mechanism Of Trichoderma Harzianum Towards Hygromycin B

With the widespread application of antibiotics,the phenomenon of fungal resistance has also emerged.At the same time,the development of antifungal has slowed down,making the situation even worse.The key to overwhlem the clinical drug resistance of fungal is the analysis of its molecular mechanism.In this paper,we studied the effect and possible factors that affect the resistance of Trichoderma harzianum to hygromycin B induced by sea salt.Combined with the transcription analyze of T.harzianum under different culture conditions,the drug resistance mechanisms and related genes were also proposed.In this paper,the influence of sea salt to the hygromycin B tolerance of T.harzianum was studied by concentration gradient control.The results showed that the presence of sea salt could significantly increase the tolerance of marine-derived T.harzianum to hygromycin B from 40 to 500 ?g/mL.The hygromycin tolerance test of six strains of fungi showed that the phenomenon of increased tolerance of T.harzianum to hygromycin B induced by sea salt is non-specific,it also works on five other marine or terrestrial fungi?two strains of Aspergillus tubingensis,two strains of Aspergillus niger and one strain of Mucor circinelloides?.At the same time,sea salt could also improve the tolerance of T.harzianum to aminoglycoside antibiotic G418 and another kind of antifungal drug carbendazim.However,it was not caused by the osmotic pressure or chelation of hygromycin B with ions.Some metal cations like K+,Na+,Mg2+ and Ca2+,played a key role in this phenomenon,and their effect to the tolerance of T.harzianum to hygromycin B was Ca2+>K+>Mg2+>Na+.Among the tested anions,SO42-may have some effect on drug resistance,while Cl-and Br-almost not.Three potential drug resistance mechanisms were proposed by analysing the transcription of T.harzianum in the presence of sea salt and hygromycin B.Firstly,in the presence of sea salt,the expression of fungal cell walls and related genes changed,and the organic layer on the cell surface was thicker,which could decrease the uptake of hygromycin B.Secondly,sea salt and hygromycin affected the expression of genes coding ion transporters and multidrug resistance related transporters,promoted the efflux of hygromycin B.Finally,sea salt and hygromycin might induced the expression of acetyltransferases and resulted in its inactivation,increasing the tolerance of T.harzianum to hygromycin B.The CRISPR Cas9 technology was used to knock out the related significantly differentially expressed genes.The resistance test results of the mutant strains showed that M431DRAFT₅12552 gene which encoding Na+-ATP plays a key role in the development of sea salt induced resistance to hygromycin B in T.harzianum.The inhibition rate of mutant strain ?512552 was 19.06%lower than that of wild type when cultivated on PDA with sea salt and 600 ?g/mL hygromycin B;The tolerance of the hydroxysteroid 17-? dehydrogenase mutant strain ?504126 to hygromycin B was slightly improved.The inhibition rate of mutant strain?504126 was 8.51%and 7.50%lower than wild type when cultivated on PDA and PDA with sea salt;In addition,the tolerance to hygromycin B of the amino acid permease mutant strain ?506686 was alos slightly changed.Compared with wild type,the maximum tolerance concentration to hygromycin B of T.harzianum on PDA increased from 50 ?g/mL to 70?g/mL.This study could enrich the research on the mechanism of fungal drug resistance and shed light on new targets for the development of new antibiotics or drugs.It also has great significance on the rational use of antifungal drugs and the reduction of drug-resistant strains in clinics.