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In order to develop innovative tactics to combat the progressive infection of Aspergillus fumigatus in immunocompromised individuals, essential biochemical pathways have been observed as a method to determine integral components of virulence. David S. Askew of the department of Pathology and Laboratory Medicine of the University of Cincinnati, College of Medicine addresses how genes and gene products play a critical role in virulence of A. fumigates. In his journal article titled Aspergillus Fumigatus: virulence genes in street-smart mold specific genes and gene products have been highlighted in order to follow important biochemical pathways and determine how the fungus is able to sustain life within its host. Biochemical pathways of interest that contribute to virulence of A. fumigates are the genes and gene products that promote growth within a host environment (37oC), genes necessary for cell wall development and functionality, formation of secondary metabolites, signal transductions that promote an adaptive response to environmental stress, and mechanisms behind nutrient uptake. By obtaining a comprehensive understanding of the biochemical pathways of interest, novel solutions to this opportunistic pathogen can be discovered and can prevent further infections.
Studies conducted by Askew show that alterations in gene expression can lead to down regulation of gene products, leading to a decrease in virulence. Once inside its host, A. fumigatus experiences a tremendous difference in environmental pressures as compared to its natural habitat. For example, inside humans A. fumigatus must proliferate at 37oC, a feat that is controlled by biogenesis protein CgrA, O-mannoslytransferase Pmt1, and ThtA. After exposing A. fumigatus to conditions that promoted mutants (each with a disrupted form of the three genes of interest), Askew observed that without ribosome biogenesis protein CgrA the fungus was unable to grow efficiently at 37oC, thereby effecting its virulence as well.
The study also suggests that the biochemical pathway for the gene products of the cell wall also aid in virulence and the ability to sustain life within the host. Contributing factors to the integrity of A. fumigatus's cell wall are β-(1,3) and β-(1,4) glucan, α-(1,3) glucan, chitin, Glycophospatidyl-inositol (GPI)- linked proteins, and melanin. Mutants with a disrupted gene sequences for the gene products of interest were observed in order to determine which genes need to be disrupted to impair virulence. Each of these gene products experience redundancy in their respective genes, illustrating that they are essential components of the cell wall that must be able to withstand deleterious circumstances. Also, redundancy seems to also moderate the levels of gene products, deletion of certain genes seems to intensify its respective phenotypes. For example, mutants with a disrupted ags1 gene demonstrated a severe decrease in α-(1,3) glucan production but still were able to maintain homeostasis. However, deletion of the ags3 gene seemed to increase virulence without changing the production of α-(1,3) glucan in the cell wall. Chitin offers a flexibility and strength to the cell wall, controlled by at least seven synthase genes. Only mutants with disruption in both chsG and chsC genes demonstrate impaired virulence. The GPI protein serves as an anchor to the plasma membrane for the β-(1,3) glucan side chains and loss of function was only observed in a mutant with a deleted gene (Afpig-a) necessary in the biosynthesis of the GPI anchor. However, observations from the study indicate that deletion of Ecm33 increased virulence. Again, the Ecm33 gene can be considered as a modulating gene because the study does not indicate any change in the production of the gene product but virulence is increased. Pigmentation is also a critical factor in virulence of A. fumigatus by serving a similar function as observed in humans. A decrease in virulence was observed in mutants who did not have the enzyme polyketide synthase, resulting in a loss of melanin production. This is because polyketide synthase is an enzyme necessary for biosynthesis for the protein. Mutants with this phenotype were subjected to an increased rate of phagocytosis as well. Through these experiments, Askew is able to demonstrate how multiple genes that code for the same gene product serves as a common mechanism with which A. fumigatus uses preserve its cell wall within a human host.
The secondary metabolite observed by Askew is gliotoxin, a potent toxin that is hypothesized to attack neutrophils, further destroying the immune system in its immunosuppressed host. However, the model used by Askew could not accurately demonstrate the virulence of the secondary metabolite because the host of the fungus had depleted levels of neutrophils before infection. Although A. fumigatus is an opportunistic pathogen in immunosuppressed individuals, the gliotoxin seemed to have no effect. Once the host was introduced to the fungus, isolated mutants that lacked genes for gliotoxin production such as gliP had no effect on virulence. Based on the hypothesis that gliotoxins do target neutrophils, the results suggest that the potent secondary metabolite is only effective when the neutrophil concentration in A. fumigatus's environment is not suppressed. However, Askew was able to observe a decrease in gliotoxin in his model by deletion of a gene (laeA) that regulated the expression of the gene product. This is an essential gene in the production of gliotoxin, and thereby a gene of interest in prevention of progressive infections of immunosuppressed individuals with A. fumigatus infections (who are no neutropenic).
Response to environmental stress within its host is a manner in which A. fumigatus is able to adjust its internal environment so that it can continue its infection. This indirectly affects virulence and is a pathway of concern because if specific instances can be halted, infection can be stopped. The protein kinase and Calcineurin are two important signal transduction pathways which alert A. fumigatus of environmental fluctuations. The protein kinase pathway employs cAMP and as a messenger that alerts gene products downstream that eventually active a response. Mutants with loss of function of gpaB affected the expression of G-protein-α- subunit which produced cAMP. Also, loss of function of the pkaC1 gene also affects the product of catalytic subunit pkaC1 which triggers downstream adaptive responses. The Calcineurin pathway uses calcium as a signal for environmental changes. Mutants who do not have the gene cnaA (which coded for a catalytic subunit on Calcineurin) were unable to grow adequately. The ability to grow, however, is not related to virulence or appropriate response stress. This was proven by mutants that had disrupted genes that coded mitogen activated kinase and high osmolarity glycerol kinase (mpkA and sho1). Although the fungi had stunted growth, the virulence was not affected. Also, response to oxidative damage is also important to preserving the fungus inside the host as well. Mutants with disrupted genes for catalases (cat1 and cat1) and the pkaR gene showed an inability to sustain oxaditive damage.
As it is important for A. fumigatus to establish a defense against environmental stress within the host, establishing a method for nutrient acquisition is important as well. The study identifies several genes that are essential in nutrient acquisition that once disrupted, can cause serious damage to A. fumigatus. Along with the uptake of iron and zinc, A. fumigatus requires nitrogen as well. In response to the presence of nitrogen, signal transduction along a series of gene productions leads to the acquisition of nitrogen; for example, transcription factors and Ras-related proteins. Mutants that do not have the genes that code for these two factors (areA and rbhA) had impaired virulence within the host. The study has also proven that amino acid uptake is also essential. As A. fumigatus metabolizes amino acids, propionly-CoA is produced. This is a toxic byproduct which can cause potential damage to the fungus; however, methylcitrate disposes of the byproduct. Mutants who did not have the gene coding for methylcitrate demonstrated decreased virulence because they could not survive in the environment with propionly-CoA. It would seem halting the metabolic pathway of A. fumigatus would be detrimental to its ability to sustain life within its host.
The experiments conducted in the study outline possible steps that can be taken to eliminate progressive infection in immunosuppressed patients. Antifungal treatment which is specific to genes and gene products eliminates the repercussions the most may face because it will target specific products of A. fumigatus biochemical pathway. In order to determine which genes are necessary for virulence, specific genes of interest were deleted/disrupted. The phenotypes of the mutant fungi were observed and illustrated possible solutions to this problem. The study demonstrated how disruption cgrA gene at 37oC is necessary for other biochemical pathways to function. Redundancy is a feature that that preserves the integrity of the genes that code for the cell wall; therefore, mutants with multiple gene disruptions are necessary to cause interruptions in the biochemical pathway of the development the cell wall. Damaging secondary metabolites, signal transduction pathways that respond to environmental stress, and nutrient acquisition are important factors as well and interruption of genes in their respective biochemical pathways can decrease virulence as well.
It is efficient to consider a genetic approach because it would eliminate the phenotype that contributes to virulence altogether, limiting the need of taking repeated medication. This is because Askew's genetic approach to obtain antifungal treatment to A. fumigatus ensures that the fungus cannot carry out the proper steps it needs to sustain life and also does not seem to affect the host in a detrimental way, albeit this study did not consider host contribution to the virulence of A. fumigatus. Future work could identify a strain of A. fumigatus with interruption in biochemical pathways of interest in the study, as apposed to just one strains one mutation. It would be interesting to see how it would affect the host and will the completely avirulent and could it live alongside normal flora.