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Dr. Askew explains that Aspergillus fumigates is the number one fungal infection of immunosuppressed individuals. The fungus produces ubiquitous spores called conidia that have the ability to reach the smallest airways of the human respiratory system and germinate. Although in healthy individuals, the spores are of no trouble to the immune system, to individuals with compromised immune systems, the disease can germinate and cause rapid infections. A fungus like Aspergillus can reproduce by fragmenting pieces of its hyphae; in the lungs, these minute fragments are able to penetrate and spread throughout. Eventually the hyphae will penetrate the endothelial lining of the lungs and into the vessels; further fragmenting hyphae will disseminate and cause deadly infections. The method by which Aspergillus fumigatus propagates in the body was examined by researchers and found to be extremely complex, with reason, for A. fumigatus must adapt itself to the ever-changing environments of its complex hosts.
Temperature is the one environmental factor that can remain constant in a host, but can undergo wide fluctuations in A. fumigatus's natural habitat. Its ability to survive in such a wide range of temperature implies that Aspergillus fumigatus has a considerable amount of thermotolerance that is beneficial to its virulence. At 37o C there are three genes that have been observed to be essential to its thermotolerance: CgrA, which manufactures ribosomes, O-mannosyltransferase Pmt1, and ThtA. When any of these genes are manipulated, the effect is that the thermotolerance of A. fumigatus was affected, however at 37o C only the mutant with a deletion of cgrA had decreased growth and virulence. Interestingly, at 22o C the mutant is able to grow normally. A possibility exists that the metabolic requirements at a lower temperature is low enough to sustain growth. This experiment opens the opportunity to reduce the thermotolerance of A. fumigatus further by altering genes required for ribosome production, in effort to prevent growth at human body temperature.
The cell wall is perhaps the most important structural factor that gives Aspergillus fumigatus its success. The cell wall is the barrier that maintains homeostasis between the organism and its host environment, which is undoubtedly hostile. The cell wall composed of mostly chitin and polysaccharides such as glucans. Three genes have been studied that affect a-(1, 3) glucan synthesis, but deletions of ags1 showed decrease in glucan, even so, the fungus was able to retain its virulence. A mutant with deletion of ags3 actually increased virulence, and failed to decrease a-(1, 3) glucan content; suggesting that there is redundancy in the genes that promote synthesis, leading to up regulation of ags1. Ags3 also seems to have a profound effect on melanin production; increasing melanin will protect from higher levels of radiation. Also observed was a faster germination period for the mutant conidia.
Chitin is main structural component of the fungal cell wall of A. fumigatus, providing a tough outer coating. Researchers have found at least seven genes that are used in chitin synthesis; this redundancy is thought to have evolved because it is necessary to maintain cell wall integrity to ensure homeostasis. In experiments, only the deletion of both chsC and chsG showed decreased virulence.
Glycophosphatidyl-inositol linked proteins (GPI-linked) are a key component for maintenance and growth of the cell wall. Gel2 is a gene that is necessary for the GPI-anchored B-1, 3-glucanosyltransferases that elongate the B-(1, 3) glucan side chains. Disruption of gel2 has shown in experiments to reduce the virulence of A. fumigatus. However, not all deletions of GPI-linked proteins lead to a reduction in virulence; Ecm33 deletion mutants actually increased virulence by increasing the germination rate. Tests have shown that a deletion of the gene afpig-a, which produces an enzyme needed for the GPI anchor synthesis, will lead to a weakened cell wall and decreased virulence.
A unique feature of the Aspergillus fumigatus cell wall is the presence of melanin, a brown pigment that can reduce the levels of radioactive damage on the DNA. The synthesis of melanin is catalyzed by polyletide synthase. Interestingly, mutants that lack the enzyme have a white appearance in conidia and decreased virulence due to an increased susceptibility to phagocytosis than the wild type. Because cell walls are specific to A. fumigatus and not their hosts, and they are crucial for survival, strategies to disrupt synthesis are enticing to researchers.
Gliotoxin, a secondary metabolite secreted by Aspergillus fumigatus into its environment, is heavily studied because of its immune system suppression and cytocidal abilities. Studies have shown that disruption in the gliotoxin gene gliP has no effect on its virulence in mice, however, when neutrophils are present in the blood, the gliotoxin is thought to augment its pathogenicity; suggesting that gliotoxin targets the host neutrophils. laeA is a gene that codes for a methyltransferase that regulates the expression of A. fumigatus's secondary metabolites. A mutant with a deletion in laeA lacked gliotoxin and was found to have a decrease in virulence. However, no mechanism has been proposed yet because the laeA gene regulates the expression of around 10 percent of the Aspergillus genome.
A major contributor to Aspergillus fumigatus's virulence is its ability to adapt to the environment via cell signaling transduction pathways. cAMP dependant protein kinase (PKA) is a protein that is essential to many pathways, such as food sensing and response to environmental changes. cAMP is the central messenger of the pathway and regulate the downstream cascade. When cAMP builds up, it binds to PkaR, a subunit of PKA, which will then cleave off PkaC1 and PkaC2. Those two subunits are part of the cascade for activating a response to environmental stimuli. When deletions of gpaB or pkaC1, which disrupts the signaling cascade, are done, virulence is decreased in these mutants. Likewise, a deletion in the regulatory PkaR, allowing uninhibited activity, is examined, will also decrease virulence in A. fumigatus. This suggests that the PKA pathway is used to respond to the host environment, and any imbalance affects the virulence negatively.
Another pathway Aspergillus fumigatus uses to send signals to respond to host stressors is the calcium signaling pathway which requires calcineurin. Calcineurin is a calmodulin activated phosphatase used for response. The gene cnaA codes for the A subunit of calcineurin; in an experiment that deleted the cnaA, the fungus was completely unable to grow hyphae, which greatly reduced the virulence. CrzA codes for the calcineurin dependant transcription factor, and mutants lacking the gene were also noted to have reduced virulence. This suggests that reduced hyphal growth is not the sole factor in reducing A. fumigatus's virulence.
Mitogen-activated kinases, or MAP, in A. fumigatus have been researched to find if growth rate had any correlation to virulence. MpkA is a gene that codes for one of the four MAP's in A. fumigatus, was deleted in mutants and showed a weakened cell wall and growth defects. Interestingly, this mutant was just as virulent as the wild type phenotypes.
The human body uses oxidative damage to fight against pathogens via the white blood cells. In immunocompromised patients, the few immune cells left can hold up progression of the infection. Genes that code for the catalases, catA, cat1, and cat2 can help fight off the immune response with anti-oxidant responses. The PKA signaling cascade handles environmental oxidative stress, as does the MAPK pathway. Only deletions in pkaR and cat1/cat2 resulted in decreased virulence, which has put the notion that these genes play a major role in virulence under speculation.
As with any other parasitic organism, A. fumigatus must acquire nutrients from its hosts. Unfortunately, the host isn't always as co-operable as Aspergillus might want it to be, thus its ability to adjust its metabolism and actually prepare and store minerals is key to Aspergillus's survival. Stores of iron and zinc, which can be low in vivo, in siderophores allow the organism to survive when these minerals are of limited availability. ZafA, the transcriptional activator for zinc response, has shown that it is needed for virulence because it is responsible for zinc uptake mechanisms.
To obtain nutrients from its host, Aspergillus fumigatus uses a set of proteases secreted into host tissues. Although no deletion mutants of any of the genes that affect proteases has shown a significant decrease in virulence, proteases cannot be ruled out as an important virulence factor; to put into perspective, there are over 99 secreted proteases in the genome of A. fumigatus, and not everyone of them have been tested. Yet scientists know these enzymes are important because they degrade host tissue to provide amino acids, and this amino acid metabolism results in a toxic byproduct known as proprionyl-CoA. The toxic proprionyl-CoA is broken down by the fungal protein methylcitrate synthase (McsA) in the methylcitrate cycle. A deletion of McsA resulted in a great reduction in virulence, accounted by the buildup of proprionyl-CoA from amino acid metabolism. Targeting metabolic processes as a way of antifungal treatment is a very possible concept, since mammals and fungi handle proprionyl-CoA in separate methods.
As with all organisms, not all nutrients can be obtained from a host; this is when biosynthesis must account for certain compounds and amino acids. An experiment showed that auxotropic mutants of Aspergillus fumigatus that required p-aminobenzoic acid, uracil/uridine and lysine were avirulent, meaning that these compounds were not in sufficient numbers in the host cells. Transcription factors areA and rhbA are signals that alert the cell of a presence of nitrogen, a major element needed for growth. Deletions of those signaling molecules lead to reduced virulence in mice, therefore implicating that these proteins are needed for utilization of the nitrogen. Not all amino acid are able to be derived from the host, therefore there is a system of biosynthesis that makes most of them within A. fumigatus. The cpcA is a transcriptional activator that acts on the cross-pathway control system (CPC), making amino acids that contribute to virulence. The CPC system operates using an upstream sensor kinase called cpcC; this sensor upregulates the pathway during stress. In induced lab responses, virulence was unaltered in comparison to basic expression. The genes downstream in the CPC pathway are of interest because they control the responses.
In conclusion, Aspergillus fumigatus: virulence genes in a street-smart mold, by Dr. David Askew on Aspergillus fumigatus does well to highlight key concerns about the ability of the fungus to cause life threatening infections in immunosuppressed individuals, as well as providing sound scientific and experimental backing into methods that concern virulence in the species. Concerns about the ability to survive through tough environments were made apparent by the ability of A. fumigatus to maintain a rigid cell wall for support and adaptive responding to host stressors via signal cascades. Experiments with deletion mutants emphasize the intricate workings of the A. fumigatus genome and why and how they contribute to virulence. The paper and described research will pave a path for future research into how to better combat infections by reducing the virulence factors and rendering the fungus incapable of becoming systemic. However, Dr. Askew informs us that even though research has already pinpointed certain aspects of the genome to consider for further experimentation, many questions remain unanswered, such as why A. fumigatus does so well as an opportunistic infection, despite having the same virulence capabilities as many other environmental molds. I thought that this paper was very informative and lays the groundwork for increased awareness and future research; the paper was easy to understand and concise. The data provided only supports his thesis and enhances the quality content while informing me that A. fumigatus is quite the complex organism and truly a potent pathogen. The next step is to follow up on the new research and provide any answers to the ones posed towards the end of Dr. Askew's paper. Hopefully the new advances have curtailed the number of infections of Aspergillus fumigatus.
This paper by Dr. Askew was used throughout the whole paper:1. Askew, D.S., Aspergillus fumigatus: virulence genes in a street-smart mold. Current Opinion in Microbiology, 2008. 11(4): p. 331-337.