antifungal – Drug Discovery Opinion

Control of ROS in Fungal Virulence

Aspergillus fumigatus Image: Wikipedia — *Aspergillus fumigatus*

Image: Wikipedia

Although saprophytic fungi play an essential role in recycling dead and decaying organic material, they can also cause disease in both plants and animals. Plants and animals have evolved defence mechanisms against infection; one of the most important of these is the production of reactive oxygen species (ROS). This means that pathogenic fungi, in turn, need strategies to deal with oxidative stress and researchers at the Virginia Bioinformatics Institute (VBI) at Virginia Tech and Montana State University have discovered a fungal protein that plays a key role in causing disease in plants and animals and which also shields the pathogen from oxidative stress.

The team looked at two different fungal pathogens; Alternaria brassicicola, which causes widespread damage in cultivated brassicas such as cabbage, broccoli and oilseed rape, and Aspergillus fumigatus, which causes severe, and usually fatal, disease in immunocompromised individuals. With increases in the number of immunosuppressed patients – as a result of transplants, cancer treatment or HIV infection – aspergillosis has become the most common mould infection worldwide.

The researchers found that the fungal protein TmpL is essential for infection of host tissues and helps the fungi to regulate oxidative stress responses caused by the presence of ROS produced both by the host in response to infection and by the fungi themselves as signalling molecules involved in physiological responses, development and virulence. The study suggests that TmpL consists of an AMP-binding domain, six transmembrane domains, and a FAD/NAD(P)-binding domain and is localised in the Woronin body, a specialized peroxisomal organelle found in the cells of hyphae in filamentous fungi. Although TmpL-deficient mutants were more sensitive to external oxidative stress and less virulent than wild-type fungi, experiments with mice that are deficient in generating an oxidative burst suggest that the intracellular regulation of reactive oxygen species in the fungus is most likely more important for pathogenesis than resistance to host-derived oxidative stress. The authors hope that their work, which is published in PLoS Pathogens, could lead to the development of efficient and novel therapeutics for both plant and animal fungal disease.

More ‘Moonlighting’ Proteins – This Time in Yeast

Coenzyme A (CoA) is an indispensable cofactor in all living organisms, operating as an acyl carrier and carbonyl-activating group in a variety of biochemical transformations, including fatty acid metabolism. Many bacteria as well as plants and yeast are capable of de novo CoA biosynthesis from aspartate and ketovalerate via pantothenic acid. In contrast, animals and some pathogenic microbes lack a de novo route, and they are totally dependent on scavenging exogenous pantothenic acid (vitamin B5). Biosynthesis of CoA from pantothenic acid is an essential, universal pathway in prokaryotes and eukaryotes, comprising five steps. The third step is the decarboxylation of phosphopantothenoylcysteine to 4′-phosphopantetheine by phosphopantothenoylcysteine decarboxylase (PPCDC).

The gene involved in the formation of PPCDC has previously been identified in plants and humans, where the functional enzyme is a homotrimeric complex. Until now, however, the nature of the enzyme in the yeast Saccharomyces cerevisiae has been unclear. Researchers at Universitat Autònoma de Barcelona (UAB), Spain, in collaboration with the University of Stellenbosch, South Africa, have now unravelled the mystery.

S. cerevisiae appears to contain three genes capable of coding a PPCDC (HAL3, VHS3 and YKL088w), but none of them have been associated with this function. In recent years the UAB group has discovered that the genes HAL3 and VHS3 regulate the activity of a protein phosphatase involved in saline tolerance and in the cell cycle, but the new research has demonstrated that the proteins encoded by these genes have additional functionality. Unlike the plant and human counterparts, the S. cerevisiae PPCDC exists as a heterotrimer. One of these proteins is necessarily coded by the YKL088w gene and the others can be two molecules coded by either HAL3 or VHS3, or one of each. The active site in this case is made up of amino acids from two different proteins: the one coded by YKL088w, which provides a catalytic cysteine, and the one coded by HAL3 or VHS3, which provides a histidine, also essential for the catalysis. So, in S. cerevisiae, HAL3 and VHS3 have apparent multiple functions.

The research, published in Nature Chemical Biology, demonstrates that the heterotrimeric structure of PPCDC can exist in a wide group of yeasts from the Ascomycetes family. This group not only includes yeasts which are used in biotechnology and industry, such as S. cerevisiae and Pichia pastoris, but also potential pathogens such as Candida albicans. The difference between the PPCDC structure in these organisms and that of the human enzyme, together with its essential nature, makes it a potential target for antifungal therapy.

Protease Inhibitors Could Be Effective For Fungal Disease

Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissue most commonly caused by infection with Fonsecaea pedrosoi. The disease occurs most often in rural areas in tropical and subtropical countries and is caused when fungus is introduced by minor injury such as that caused by a splinter or thorn. Chromoblastomycosis is very difficult to cure; antifungal chemotherapy, surgical excision and/or cryosurgery have traditionally been used but with varying degrees of success.

Recently, HIV protease inhibitors have been shown to have a direct effect on AIDS-related opportunistic pathogens such as Candida albicans by inhibiting production of C. albicans secreted aspartyl proteases. The proteases assist the fungus to colonize tissues and to evade the host’s antimicrobial defense mechanisms. F. Pedrosoi also secretes aspartyl proteases and a study published in PloS ONE describes the effect of selected HIV protease inhibitors on secreted protease activity and survival of F. Pedrosoi. At high concentration (100µM), saquinavir and nelfinavir robustly inhibited growth of F. Pedrosoi in vitro. The high concentration needed possibly reflects a much lower affinity for the fungal protease than for HIV protease, or may suggest alternative mechanisms of control. The authors also studied the in vitro effect of combining sub-inhibitory concentrations of the aspartyl protease inhibitors with sub-inhibitory concentrations of antifungal drugs and found good synergistic actions.

The results suggest that combination therapy with protease inhibitors and antimycotic drugs may be effective for treatment of chromoblastomycosis, especially if more potent inhibitors of the F. Pedrosoi aspartyl protease were developed.