1. Pathogenesis and Virulence
To understand how a given organism is able to infect its host, it is important to determine which aspect(s) of the biology of the fungus differs from nonpathogenic fungi. According to Bulmer and Fromtling, "Pathogenicity is the capacity of an organism to damage, ie, to produce disease in another animal or plant" (1). This property is the result of a direct interaction between the pathogen and the host. In many ways, pathogenicity and virulence are very broadly defined terms. If one considers all the possible gene products required for growth on or within a host, those that contribute to disease symptoms and host-range specificity, and those that govern their regulation, then we may be considering hundreds (or thousands) of potential components. Furthermore, the ability of an organism to adapt quickly to new environmental challenges is itself likely to be an effective virulence mechanism, in that it enables the pathogen to take advantage of new opportunities.
One of the most cited examples of virulence in the fungal world is the ability to switch morphological forms. While some fungi are pathogenic as yeast or conidial forms [eg, Histoplasma capsulatum, Coccidioides immitis, and Blastomyces dermatitidis, and Coccidioides immitis (2-4)], numerous correlations between hyphal growth and pathogenicity have been cited for many of the fungal pathogens. In fact, mutants incapable of switching to hyphal growth are often revealed as nonpathogenic cpg-1 deletions in C. parasitica (5), deletions offuz7 in U. maydis (6), or cph1/efg1 homozygous deletions in C. albicans (7). The ability to switch morphological forms is referred to as dimorphism and is governed by a number of external factors including nitrogen, pH, temperature, and intracellular biochemical pathways (eg, level of cAMP) (8). Similarly, correlations between a strain’s ability to myceliate and its virulence (defined by the LD50, lethal dose to obtain 50% killing of the host) have also been cited as strong virulence factors.
Pathogen-produced products which contribute in some way to either the establishment or continuation of the disease process have also been cited as virulence factors. Historically, secretion of degradative enzymes such as elastase and certain proteinases (9, 10) have been cited as potential virulence factors using a variety of methods to correlate disease progression with levels of expression. Paracoccidioides brasiliensis has been shown to alter its level of virulence with the production of a-1,3 glucan, suggesting that this polymer may act as a protective layer (11). Genes that contribute to adherence of the fungus to its host have also been regarded as virulence factors (12). Numerous genetic mutations or strain isolates have been analyzed in in vivo experiments to identify underlying gene products causing changes in pathogenicity. In fungus-plant interactions, the pathogen may produce a substance that specifically detoxifies plant-secreted antifungal compounds. For example, G. graminis deletion mutants have been generated that are relatively resistant to the production of avenacin by oat roots but retain full pathogenicity to wheat, which does not synthesize saponins (13); isolates of Nectria haematococca are similarly able to detoxify the pea phytoalexin, pisatin, using a cytochrome P450 mechanism (14). Furthermore, the biosynthetic pathway leading to melanin production in M. grisea correlates with altered virulence in this fungus; strains with reduced melanin content are less virulent (15). In some instances, these factors are reported to increase the pathogen’s ability to penetrate host tissue (as in the case of melanin or proteinase production), while in others they may simply contribute to the overall fitness of the organism in vivo. For example, some auxotrophies are known to decrease virulence in vivo but play little or no role in vitro. Mutations in the URA3 and ADE2 genes of C.albicans and C. neoformans attenuate virulence in vivo (16, 17).
Alternatively, pathogen-induced factors that result in direct attacks upon the host are also known. The best-characterized system of host-selective toxin production by a plant fungus is in Cochliobolus. Different species of this genus are known to produce plant-specific toxins: T-toxin, HC-toxin, and victorin, of which only HC-toxin production has been deleted and shown to be nonpathogenic on maize (reviewed in Ref. 18).
One area in which there is sparse molecular information, but a great deal of inferential information, is in the interactions between the host immune system and pathogen. Research directed toward understanding the role of the unique polysaccharide capsule of Cryptococcus neoformans pathogenicity has uncovered a large difference in ability to phagocytose capsulized versus acapsular C. neoformans mutants in vitro, although it is not yet clear whether the ability to engulf a cell correlates with killing (4). Additionally, macrophages appear to recognize and engulf wild-type, single, and double mutants of the MAPK pathway in C. albicans equally well; however, only wild-type strains were able to switch morphologies, adding further support that dimorphism plays a significant role in virulence in this organism (7). It is tempting to speculate that in the chs3 C. albicans cell wall mutant, prolonged survival in vivo (19) had more to do with a change in its interaction with the host immune system than with any other factor. If, in fact, the host defense system plays a more significant role in disease progression than has been realized, it will be interesting to determine whether other factors such as increased lipid content cited as a virulence factors in strains of Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatidtidis (1, 20) is a result of an alteration in the interaction rather than a simple defense on the part of the fungus.
2. Signal Transduction and Pathogenesis
Signal transduction pathways play an important role in cellular biology of all eukaryotic organisms, including fungi. The ability of a pathogen to receive signals from its environment and transmit those signals intracellularly to alter development or entire biochemical pathways so as best to take advantage of those changes has been recognized as a significant partner in pathogenesis of the organism. Some of the pathways have been carefully worked out in S. cerevisiae and S. pombe with regard to cell cycle, filamentation, stress, and the pheromone response. It appears that the molecules required to relay a signal from the external environment to the appropriate transcription factors within the nuclei of many of the filamentous fungi are highly homologous to those described in other organisms. Some of the molecules have been identified and isolated on the basis of sequence homology, and some by complementation or the use of similar screens. While the precise targets of many of these molecules have yet to be discovered, it is becoming clearer that many of the pathways in yeast are similarly conserved in more distantly related organisms.
Virulence and signal transduction pathways are inescapably intertwined in the biology of fungi and their pathogens. In the filamentous fungi, signal transduction pathways have been identified that regulate conidiation in A. nidulans, virulence in C. albicans, C. parasitica, U. maydis, and M. grisea, and mating. While only a few of the components have been identified in the filamentous fungi to date, there is much anticipation that most, if not all, of the players will eventually be isolated and characterized. Although their exact developmental pathways may be very different in the different fungi, it is important to keep in mind the similarities in order to appreciate the conservation of function across the biological world.
2.1. MAP Kinase Pathway
Signal transduction involves the activation of a receptor molecule at the surface of a cell, which transmits the signal inward toward associated proteins at the cytoplasmic interface of the plasma membrane. Changes in these proteins are relayed via a small number of known pathways (either by a cascade of kinases or by the release of a second messenger) which in turn activate a number of protein targets, some of which are themselves transcriptional activators. Subsequent alterations in transcription of whole sets of genes then reprogram the cell’s "transcriptome," resulting in a particular cellular response to the initial signal. That signal may be any of a number of ligands that tells the cell something about its external environment: for example, nutritional availability, osmolarity, presence of compatible mating partners, or light. In one common pathway observed in all eukaryotes, a heterotrimeric G protein activates a mitogen-activated protein (MAP) kinase module that results in the further activation of target proteins, among which is a transcription factor. In some cells, multiple MAPK signaling pathways can be operating simultaneously, altering the organisms’ transcriptome moment-by-moment. A MAP kinase pathway influence on pathogenicity has been shown for the corn smut, U. maydis, in which tumor production and completion of the sexual cycle is dependent upon successful compatible mating (21). Appressorium formation and pathogenic growth of M. grisea inplanta also require a functional MAP kinase pathway (22). Because the components of these pathways are so highly conserved, one of the central questions that remains unanswered is how specificity is achieved and how that affects the pathogenicity of the organism.
2.2. Cyclic AMP Pathway
A second common pathway that impinges upon pathogenicity is the cyclic AMP-mediated pathway, which begins with a plasma membrane-bound sensor molecule (none of which has been isolated for the filamentous fungi to date). Upon a functional receptor-ligand interaction, a signal is transmitted to a heterotrimeric GTP-binding protein, possibly through a conformational change in the membrane protein. Second messenger cyclic AMP (cAMP) is produced via adenylate cyclase, which then alters the activity of cAMP-dependent protein kinase A (PKA) by binding to the regulatory subunit of PKA. This releases the catalytic subunits, which continue to activate downstream targets (including transcriptional regulatory proteins). A few components of this pathway have been identified in filamentous fungi. Mutants in uac1 (adenylyl cyclase of U. maydis) have been shown to be defective in their ability to switch from budding to filamentous growth and, in addition, were unable to cause disease on corn (reviewed in (Ref. 23); disruption of adr1 (catalytic subunit of PKA) results in nonpathogenesis, whereas inactivation of uka1 (catalytic subunit of PKA) has apparently little or no effect on growth, morphology, or pathogenesis. Disruption offil1 (G a subunit of U. hordei) also prevented normal hyphal to bud switching. Interestingly, of the four G a proteins isolated from U. maydis, only one, gpa3, was shown to be required for pathogenic development, disruption of the other three displayed no obvious phenotypes. Furthermore, gpa3 mutants were unable to respond to pheromone signals strongly suggesting that a single G a subunit may play a role in multiple signaling pathways as has been observed in S. cerevisiae and C. albicans. Mutants disrupted in the catalytic subunit of M. grisea (CPKA) were shown to be unable to form appressoria on artificial surfaces, and they were also unable to infect rice unless exogenous cAMP was administered (23).
3. Strategies to Identify Virulence Factors
3.1. Gene Knockouts
There are a number of molecular genetic strategies that can be used to identify genes responsible for virulence in fungi (24). In a standard experiment, genetic manipulations are made such that the chromosomal copy of a gene in question is replaced by a marker gene. The growth rate of these strains is then compared to the normal wild-type strain and subsequently tested in vivo to determine the potential of the "knocked out" gene to cause disease. Proper controls wherein additional strains containing the chromosomal knockout, but also harboring a transformant carrying a reintroduced wild-type copy of the gene, or a heterozygote, is also compared in order to ensure that the effects observed are due to the presence or absence of a specific gene product and not some other underlying mechanism. It should be noted that due to the frequency of ectopic integration in filamentous fungi, these experiments may be difficult to interpret. They have been successfully accomplished for a wide variety of gene products in the dimorphic fungus, C. albicans. Using a modification of the original "ura-blaster" strategy (25), it is possible to knockout both copies of a given gene in this perpetually diploid organism. Genes in cell wall biosynthesis, signal transduction, and so on, have been identified as involved in virulence in such tests (7, 19, 26). This directed approach to virulence determination has also been successful in A. nidulans (27), Gaeumannomyces graminis (28), and C. neoformans (29), for example. Using this approach, there is the potential to knockout each gene in a given organism systematically and test its pathogenicity in vivo.
3.2. Gene Transfer
Virulence factors have also be identified by their ability to confer pathogenicity upon a related species or strain that was previously nonpathogenic. In this approach, a library is constructed and transformed into a nonpathogenic organism. Entire library pools are then tested for changes in related events that are suspected of having influences upon pathogenicity (adherence properties, toxin production, proteinase activity, etc.), and gene products are identified and then retested for altered virulence relative to the starting host strain. This approach has been successful in identifying a gene conferring adhesion properties from C. albicans to S. cerevisiae (30).
3.3. Gene Expression
This may be accomplished in a number of different ways. First is to identify all the genes expressed during an infection, by complementary DNA cloning, differential display analysis, or promoter fusions (all of which are technically feasible in the filamentous fungi). The idea is that crucial genes will be expressed sometime during or immediately prior to the establishment of an infection. These genes can then be further screened for their relative virulence, using a variety of knockout or deletion strategies. The MPG1 protein of M. grisea was identified using this approach and confirmed to be important in the development of fungal infection structures (31). Using a differential display approach, two genes from U. maydis (32) and six genes from C. albicans have been identified that are differentially induced during infection (33). As additional sequence information becomes available for other fungal genomes, the use of promoter fusions as an approach will probably become somewhat more popular. Following the initial identification of clones that express a viable fusion, sequence analysis of the upstream sequences directing the fusion will reveal the target gene. Conceivably, libraries in which the reporter proteins are cell surface-directed will aid in the initial identification of positive clones in this type of model.
One of the most fascinating stories concerning pathogenicity in fungal biology involves the interaction between Cryphonectriaparasitica (chestnut blight fungus) and a hypovirus. These fungi, which have wreaked havoc upon the chestnut forests of the northeastern United States, are themselves infection-susceptible to a hypovirus that causes reduced virulence of the fungus for the plant. In addition to causing reduced virulence, the virus causes altered colony morphology, reduced pigmentation, and attenuated asexual sporulation. Differential display analysis of infected and noninfected isolates identified a candidate gene cpg1 whose reduced expression in infected isolates appears to correlate with pathogenicity (5, 34).
3.4. Mutagenesis
Historically, a traditional genetic approach in which mutations are isolated and further characterized has proven beneficial in the understanding of pathogenesis of fungi. One of the more recent strategies to understanding and defining virulence factors has come from alterations in transposon-mediated mutagenesis screens. Transposons are relatively small fragments of DNA that are capable of recombining nearly randomly into different sites within a single genome. They have proven useful in creating large number of "insertional" mutant libraries that are easily screened for loss-of-function phenotypes. Unfortunately, filamentous fungi appear to lack such elements. However, restriction enzyme-mediated integration (REMI) has been be used to create insertional mutants in filamentous fungi. Pathogenicity genes were identified for Cochliobolus heterostrophus (35) and Ustilago maydis (36) using REMI.
A modification of this method, called signature-tagged mutagenesis, has been used successfully in bacterial pathogenesis to identify insertional mutants that fail to grow in vivo (37). In this method, every insertional mutant carries with it a unique oligomer tag that identifies it from all other mutants. After inoculating a mouse and recovering all survivors, DNA is prepared and hybridized to colony blots of the original input pool. Mutants not surviving are not recovered and are easily identified as not hybridizing. Identification of the gene into which the insertional tag is present is performed by sequencing. One of the only drawbacks to this approach is that only nonessential genes may be recovered, because insertions into essential genes are by definition lethal in culture.
3.5. Use of In Vivo Models
The use of in vivo models to confirm the role of individual genes in virulence is an important aspect of this field. However, it is important to recognize the difference between the establishment of infection and the maintenance of infection. Both are clearly critical factors in host-pathogen interactions, yet targets should be different, although somewhat overlapping. Second, the size of the inoculum is a well-established variable in disease models. It is not inconceivable that certain genes play a more significant role at low inocula in certain situations while other virulence genes are turned on under conditions of high inocula. Third, it is important to remember that virulence is multifactorial and results from a balance between the pathogen and the host immune system. Comparisons across different fungal backgrounds or different host models should proceed with caution.
4. Fungal Virulence Genes as Targets for Antifungal Therapy
It is not enough that we understand how an organism grows and develops or produces disease; it is our ability to use this information to circumvent those processes that will enable humanity to control and manage diseases produced by microorganisms. Compounds aimed toward inhibiting functions of known or suspected virulence factors have been suggested as likely to be effective in reducing disease even though many of the virulence genes identified to date have been shown to be nonessential for growth and reproduction of the organism in vitro. A compound directed toward such a nonessential target would likely prevent fungal growth until the compound were either removed or inactivated. In clinical situations where prophylaxis is appropriate, a reduction in anticipated fungal pathogen virulence could significantly influence patient outcome, because the disease will not likely gain entry initially. Unfortunately, the human patient population that often contracts fungal diseases is often immune-debilitated. From an agricultural view, inhibition of fungal growth will probably lead to increased production over a short season, making these attractive targets. Another consideration in choosing virulence factors as targets is the likelihood that parallel and/or redundant pathways may exist. With all of this, virulence genes are important to consider as potential drug targets.
Strategies for identifying fungal virulence genes as potential targets for drug development and/or vaccine development include many of the cloning strategies identified earlier. Furthermore, the use of bioinformatics and genome sequencing has made it possible to identify potential candidates based on previously known motifs, which may then be tested in vivo for specific disease.
