Genetic manipulation to enhance siderophore production in Talaromyces marneffei

Abstract

Siderophores are low molecular weight organic compounds with a high affinity and specificity for iron (III), which are produced by microorganisms, especially bacteria and fungi. Siderophores have received much attention because of their potential roles in the medical field. Siderophores can be used as iron chelator drugs for the treatment of iron-overload conditions. Another medical application is using the "Trojan horse strategy", to generate complexes between siderophores and antimicrobials to facilitate the delivery of antibiotics to pathogens. Some siderophores possess antimalarial or anticancer activity. Moreover, siderophores can be used as the diagnostic markers for the detection of infectious diseases. Fungal siderophores have more advantages than those produced from bacteria because molecules are more diverse and have a higher iron binding affinity. Currently fungal siderophores have not been widely used, even though several types have been previously characterized with studies demonstrating their feasibility for clinical applications. It is possible that fungi are limited by slow growth and low siderophore production. Hence, the purpose of this study is to generate a genetically modified strain (mutant) that enhances siderophore production, as well as to conduct preliminary testing of the characteristics of siderophore, to guide its medical application. Talaromyces marneffei is chosen in conducting research since there is currently established methodology for genetic manipulation in this fungus. Additionally, the siderophore biosynthesis pathway is previously described. Our goal for this study is to generate the strain of T. marneffei which possesses the ability to produce high amounts or a specific type of siderophore. Then the produced siderophores are purified and tested for possibilities to be used in the medical field. First, several mutants of genes involved in siderophore biosynthesis in T. marneffei were generated. The subjected genes are hapX and sreA which encode transcription factors in control of siderophore biosynthesis, and sidC and sidF which encode enzymes involved in intracellular and extracellular siderophore production. Additionally, two genes encoding transcription factors acuK and acuM, that are presumed to be involved in iron metabolism from previous studies were included. Phenotypic analysis revealed that ∆hapX and ∆sidF mutant strains exhibited limited growth and decreased percentage of germination, whereas ∆sreA and ∆sidC did not show growth defects when cultured in normal medium. The ∆acuK and ∆acuM, despite showed normal growth on normal medium; however, they showed no growth in iron-depleted conditions. The siderophore level was measured by using Chrome Azurol S (CAS) assay. Siderophore production in ∆hapX and ∆sidF significantly decreased while ∆sreA increased. The level of siderophore production in ∆acuK and ∆acuM did not differ from the wild type. Transcriptome analysis indicated that genes encoding iron-containing proteins, required for general metabolism and energy production in ∆acuK and ∆acuM, were downregulated. The failure of ∆acuK and ∆acuM to grow on iron-depleted conditions could be explained by the combination of lack of these proteins and iron element, not by lack of siderophores. Among all mutants, the ∆sreA resulted in markedly elevated siderophore production. From phenotypic studies, we therefore selected the ∆sreA strain for further experiments from its ability to produce highest siderophores among all mutants. Then, the siderophores produced from ∆sreA strain were purified from the culture filtrate and analyzed. Chemical characterization by FTIR and NMR identified the siderophore as a coprogen B extracellular type of siderophore. The ∆sreA strain could produce high amounts of coprogen B, and it could be purified with high purity. Cytotoxicity testing with Huh7 human hepatocellular carcinoma found that the coprogen B was nontoxic. Importantly, the compound showed powerful iron-binding activity, and it could significantly reduce the labile iron pool (LIP) levels in an iron-loaded cell model. Furthermore, antimicrobial activity of the coprogen B was tested with representative microorganisms, Escherichia coli, Staphylococcus aureus and Candida albicans. The coprogen B did not support the growth of all microorganisms tested. In contrast, it could inhibit growth of C. albicans and E. coli in a dose-dependent manner. Collectively, these results suggested that coprogen B from T. marneffei ∆sreA siderophore-enhancing strain could be used as a new iron chelating agent, from its ability to withdraw irons from the iron-loaded cells, without disturbing viability of human cells. The coprogen B was found not to support the growth of microbial pathogens; therefore, it be safely used as the iron chelator.