MASTER'S THESIS

Assessing Molecular Heterogeneity in Clinical Isolates of Aspergillus fumigatus

Abstract

Invasive aspergillosis, caused primarily by Aspergillus fumigatus, has recently become a major concern in many tertiary care hospitals.  Even with aggressive antifungal treatment, it continues to have high mortality rates. 

Epidemiological studies have been hampered by the ubiquity of A. fumigatus and the lack of a reproducible strain-typing method.  A frequently-used method in many laboratories is the Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR).  In contrast, our studies suggest that the RAPD-based assays are problematic, and indicate that the microsatellite PCR assays are more reliable.

In addition to its strain-typing pattern, an isolate can be categorized by its gliotoxin (a mycotoxin) secretion level.  Our studies suggest that isolates can be separated into two groups: high gliotoxin producers (>230 mg/g) or low gliotoxin producers (<100 mg/g).  Moreover, our studies demonstrate gliotoxin secretion increases (~100%) in the presence of the antifungal agent amphotericin B.  Gliotoxin release may correlate with pathogenicity of the organism.

Introduction

Fungi are ubiquitous, eukaryotic microorganisms. Many are saprophytes, thriving on nonliving organic materials. Fungi can be differentiated into two types based on cell morphology and growth: yeasts and filamentous fungi (molds). Yeasts are small, unicellular microorganisms that produce daughter cells from the parent cell by budding. Molds are multicellular microorganisms whose cells are joined together, forming long filaments (hyphae), and elongate by apical extension. In higher fungi such as Ascomycota and Basidiomycota, adjacent hyphal cells are separated by a septum, which provide structural support and regulate diffusion of material between cells. The hyphae of more primitive fungi (e.g. Chytridiomycota and Zygomycota) are aseptate, although a vestigial septum may be present. The growing hyphae in higher fungi branch and anastomose, forming a complex intertwined colony, or mycelium.

As a fungal colony matures, reproductive structures develop and lead to the dispersal of asexual spores (e.g. conidia, sporangiospores) or sexual spores (e.g. ascospores, basidiospores). For instance, in an Aspergillus colony, a special hyphal cell gives rise to an erect, nonseptate cell (conidiophore). The tip of the conidiophore swells into a domelike vesicle, whose surface is then covered by a single layer of phialides, the flask-shaped, asexual conidiogenous cells. Conidial chains develop upward in a column, then radiate outward from the conidiophore. Once disturbed though, the conidia separate easily and become airborne in large numbers. Although it can withstand harsh conditions, the conidia will only germinate in advantageous, nutrient-rich environments, initiating a new colony (Lennette et al., 1985).

Pathogenesis of Aspergillus

Systemic Aspergillus infections are usually acquired by inhalation of airborne conidia or traumatic inoculation. There are many types of mycoses caused by the pathogenic strains of Aspergillus, including allergic reactions (allergic bronchopulmonary aspergillosis, ABPA) and the colonization of the external mucosal epithelia (aspergilloma), particularly in the respiratory tract. These infections often occur in patients with pre-existing pulmonary conditions. Approximately 2% of asthmatic patients and 10% of cystic fibrosis patients are found to have ABPA. Aspergillomas account for 0.02% of all US hospital patients, although they are complications in 15-20% of all patients internationally.

More serious, life-threatening illnesses arise when the hyphae invade tissue. The fungus can disseminate from the lung (the site of initial colonization) to the brain, heart, or kidneys. Even with early and aggressive antifungal treatment, invasive aspergillosis (IA) has a mortality rate of 80-95% (Bernardo et al., 2003; Bertout et al., 2001; Reeves et al., 2004a), with death often occurring 7-14 days post-diagnosis (Reeves et al., 2004a).

The severity of the infection depends on the immunologic state of the patient. Invasive aspergillosis primarily affects immunocompromised patients, in particular, people undergoing immunosuppressive therapy for the treatment of cancer or organ transplantation. Reports indicate that IA occurs in 3-9% of all renal transplant recipients, and as high as 5% of bone marrow, heart, lung, or liver transplant recipients (Singh and Paterson, 2005). Moreover, invasive aspergillosis has become much more prevalent recently because of the growing number of bone marrow transplant operations (Wald et al., 1997). A majority of invasive aspergillosis infections (76%) occur 40 to 180 days post-transplantation (Singh and Paterson, 2005). The incidence of invasive aspergillosis in AIDS patients is 4-5% in the United Kingdom and 1-12% worldwide (Khan, 2003).

Invasive aspergillosis is also a major problem for people with pre-existing pulmonary diseases (such as cystic fibrosis and tuberculosis) and has become the most frequent fungal pathogen found in tertiary care hospitals, overtaking candidiasis (i.e. infections with Candida spp.). About 4% of autopsy patients (irrespective of cause of death) are found to have IA, whereas only 2% of patients have invasive candidiasis (Groll et al., 1996). The Centers for Disease Control and Prevention (CDC) estimated the prevalence of aspergillosis in 2003 was 2 per 100,000 individuals.

Virulence factors

Although many fungi have been associated with human diseases, the majority of invasive infections are caused by a relatively small number of species. As an opportunistic fungal pathogen, Aspergillus fumigatus is responsible for 80% of all aspergillosis cases. This suggests that it has specific virulence factors that enable it to cause disease. A. fumigatus is known to secrete toxins (mycotoxins) such as gliotoxin, fumagillin, and helvolic acid (Tomee and Kauffman, 2000). One well-studied mycotoxin is gliotoxin (Fig. 1), which is an epipolythiodioxopoperazine. It is named after the organism from which gliotoxin was first isolated, Gliocladium fimbriatum. This lipid soluble thiol reactive reagent (molecular weight of 326.4 g/mol) blocks the sulfhydryl active site required for nucleotide binding and enzymatic activity of nucleotide binding proteins.

Gliotoxin has also antimicrobial and immunosuppressive properties, including the inhibition of phagocytosis by macrophages and mitogenic stimulation of lymphocytes, and the induction of apoptosis in a variety of cell types (Mullbacher et al., 1985). The apoptotic activity can be attributed to the reactive disulfide bridge in the molecule as this moiety undergoes redox cycling, generates oxygen radicals, and causes oxidative damage to DNA (Bernardo et al., 2003; Sutton et al., 1996). Such properties may enable A. fumigatus to suppress the innate immune response. In addition, gliotoxin has been implicated in the destruction of human respiratory epithelium, which may contribute to tissue invasion (Amitani et al., 1995a).

Aside from the production of secondary metabolites, the morphological characteristics of A. fumigatus may also help explain its pathogenicity to humans. The organism grows well at 37oC (body temperature). In addition, fungi such as A. fumigatus produce small-sized spores (2-3 m) which easily become airborne. The small size allows the spores to become trapped within the alveoli of the lungs.

Epidemiology

A. fumigatus is ubiquitous in the environment and its conidia can be found in ventilation systems, dust, carpeting, food, and soil (Fridkin and Jarvis, 1996). Thus, we are constantly exposed to the organism. However, primary host defense mechanisms, such as mucous membranes, mucociliary clearance, and local secretion of inflammatory mediators, usually provide sufficient protection (Tomee and Kauffman, 2000). Nevertheless, nosocomial aspergillosis still poses a serious problem to many hospitals and their patients (Leenders et al., 1996).

Studying the epidemiology of aspergillosis is important to improve infection control. Many emerging techniques involve molecular strain typing (Taylor et al., 1999). A common approach for strain typing bacteria is based on restriction fragment length polymorphism (RFLP) analysis, in which restriction endonucleases cut the DNA at specific sequences. Changes in the DNA may add or remove cut-sites and thus alter fragment patterns, thereby distinguishing isolates as different. However, the A. fumigatus genome (~28 Mbp in 8 chromosomes) is much larger than a bacterial genome (~4 Mbp) and RFLP analysis would be more complex.

PCR-based molecular typing methods

The randomly amplified polymorphic DNA-PCR (RAPD-PCR) is a method that is considered standard in many laboratories (Diaz-Guerra et al., 2000; Rath et al., 2002). Assays based on RAPD-PCR determine the DNA sequence variation by amplifying DNA fragments flanked by sequences complementary to short primers. DNA profiles will differ between strains by the presence or absence of primer sites, priming completeness, and the distance between priming sites. Although the RAPD-PCR has potentially great discriminatory power, it presents significant technical challenges and can be difficult to implement.

An alternative PCR-based approach is the microsatellite (or simple tandem repeats) assay. The genomic DNA of many organisms, including A. fumigatus, contains variable microsatellite regions, i.e. a stretch of polynucleotide repeats of various lengths. The length of a particular microsatellite may mark a particular lineage (i.e. strain). When the unique sequence flanking both ends of the microsatellite region is known, a PCR can be performed to determine the length of the repeat region (Groppe and Boller, 1997).

Antifungal therapy for Invasive Aspergillosis

Current antifungal treatment for aspergillosis remains unsatisfactory as IA continues to exhibit high mortality rates. In many healthcare institutions, amphotericin B preparations are the drugs of choice, though the development of new azoles and echinocandins are very promising (Singh and Paterson, 2005). Amphotericin B belongs to the polyene class of antifungals which directly targets ergosterol, the primary sterol found in fungal cytoplasmic membranes. This interaction increases membrane permeability, causing cell death through membrane leakage. Amphotericin B in some formulations (e.g. containing deoxycholate) interacts with cholesterol in human cell membranes, which may explain the nephrotoxicity in 80% of patients (Abuhammour and Habte-Gaber, 2004). To reduce the toxicity of amphotericin B, liposomal formulations have been developed. Furthermore, this formulation has the added benefit of improved pharmacokinetics.

Azoles, such as itraconazole and voriconazole, disrupt fungal cell integrity by inhibiting the cytochrome P450 (CYP-450) system involved in the conversion of lanosterol to ergosterol. Voriconazole is generally well tolerated. Both liposomal amphotericin B and voriconazole are currently considered first-line treatment options for IA (Abuhammour and Habte-Gaber, 2004). Echinocandins, such as Caspofungin, are a new class of antifugals. These agents act by inhibiting the synthesis of -1,3-glucan, a polysaccharide in the fungal cell wall (Abuhammour and Habte-Gaber, 2004). Such disruption of the fungal cell wall results in osmotic stress and eventual lysis.

Aside from the shortcomings of current antifungal therapies, the Division of Bacterial and Mycotic Diseases of the CDC has recently listed other challenges facing the treatment of aspergillosis. Chiefly among these: the ability to identify risk factors for disease in immunocompromised individuals, the improvement of understanding of the source and routes of transmission, and the development of sensitive and specific methods for earlier diagnosis (CDC, 2003). The purpose of this project is to optimize and compare PCR-based strain typing methods, detect and quantify gliotoxin production in clinical isolates, and test amphotericin B effects on A. fumigatus gliotoxin release.