Aspergilli are saprophytes that commonly grow on dead or decaying organic material, recycling carbon and nitrogen sources. Are the second most frequent cause of nosocomial fungal infections after Candida species. Invasive aspergillosis is mostly caused by A. fumigatus, the most important airborne human pathogenic fungus. A. fumigatus is a thermotolerant species able to grow at up to 55 ◦C and surviving even up to 70 ◦C. They are able to utilize a wide range of organic substrates and adapt well to a broad range of environmental conditions. In contact with air the mycelium forms specialized structures, so-called conidiophores. These produce large numbers of conidia (asexual spores) that are sufficiently dispersed through the air and inhaled by humans. The virulence of A. fumigatus is a multifactorial process. Putative virulence determinants include conidial, pigments or intermediates, surface proteins, toxins, allergens, enzymes, cell wall components, nutrient sensing, and adaptation to hypoxia and iron depletion.
Different stages of conidiophore and conidium development in Aspergillus fumigatus
Imaging living cells of Aspergillus in vitro.2009
Characteristic of the pathogen
The cell wall is the main line of defense of the fungus against a hostile environment providing structural integrity and physical protection to the cell. The fungal cell wall is also the structure responsible for the interaction with the host and their components are often the targets of the host immune system during fungal infections. In A. fumigatus, the cell wall is mainly composed of polysaccharides (at least 90%) and proteins. Thus this thick protective cell wall protects the fungus from the host residual immune system.
Mycotoxins can be described as a chemically diverse group of low molecular weight organic substances produced by fungi. These substances are formed in the hyphae during growth, and may be actively expelled into the environment, or released after the death of the hyphae.The presence of preformed mycotoxins in conidia means that the toxins must be incorporated during conidiogenesis. However, these substances might be also produced during germination. Many of these toxins are secondary metabolites of these fungi. Depending on the mycotoxin, they can affect the synthesis of proteins, DNA and RNA, or alter the cell membrane, the consequences of which may be death or impairment of cellular functions. Gliotoxin is the major and the most potent toxin produced by A. fumigatus. It belongs to the family of epipolythiodioxopiperazines, which are characterized by a disulfide bridge across a piperazinering which is essential for their toxicity. Gliotoxin has several immune suppressive roles including inhibition of macrophage phagocytosis, mitogen-activated T cell proliferation, mast cell activation, cytotoxicT-cell response, and monocyte apoptosis. It also inhibits the NADPH of neutrophils, suppresses ROS production and impairs neutrophil phagocytic capacity, reduces the ciliary movement of epithelial cells and leads to epithelial cells damage.
What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis.2010
A. fumigatus produces a significant number of allergenic molecules. In immunocompetent patients Aspergillus can produce several hyper- sensitivity diseases due to these allergens, such as ABPA (allergic bronchhopulmonary aspergillosis), allergic rhinosinusitis, asthma, and aspergilloma. All Aspergillus allergens appear to activate a TypeI hypersensitivity response in sensitized patients with production of high affinity IgG and IgE antibodies. In immunocompromised patients with debilitated innate immuneresponses, these allergenic compounds can increase the risk associated with aspergillosis because they may redirect the immuneresponse to the fungus by the activation of Th2 lymphocytes, a response that does not seem to be efficient in eliminating this fungus.
Biofilms are by definition highly structured communities of microorganisms that are surfaceassociated, and/or attached to one another, enclosed within a self-produced protective extracellular matrix (ECM). These can form in the natural environment as well as inside the human host, and can be considered as complex cities of microbes that cooperatively interact in an altruistic manner. The advantages to an organism of forming a biofilm include protection from the environment, resistance of physical and chemical removal of cells, metabolic cooperation and a community based regulation of gene expression, including increased resistance to antimicrobial agents and protection from host defenses. The phases are: initial adhesion, filamentation, hyphal proliferation, with maturation and associate ECM production. The overall morphology of sessile A. fumigatus populations is highly dependent on the initial conidial seeding concentration. The architecture of biofilms is highly ordered to enable the perfusion of nutrients and expulsion of waste products. Mature biofilms exhibit spatial heterogeneity with microcolonies and water channels being present. This complexity is governed by defined genetic pathways.
Aspergillus fumigatus biofilms in the clinical setting.2011
Biofilm produced by Aspergillus Fumigatus
Reproductive cycle
Sexual development and cryptic sexuality in fungi: insights from Aspergillus species.2012
Aspergillus species have traditionally been recognized by the presence of a common morphological feature, the ‘aspergillum’, an asexual reproductive structure consisting of a characteristic conidiophore culminating in an expanded bulbous region from where originate chains of conidia. Sexual and asexual spore production can be viewed as two competing forms of reproduction with one developmental process inhibiting the other and vice versa. The type of reproduction that occurs is largely governed by environmental factors. A first key factor influencing sexual development is the absence or presence (including wavelength) of light. Given that many Aspergillus species are natural soil dwellers, the need to determine whether they are above or below ground can be seen as essential because this can favour the production of airborne conidia. A series of genes that are responsive to light have been identified. A second major factor determining whether sex can occur is the composition of the growth medium such as nutrient content and pH. In general, a balanced carbon/nitrogen ratio is most favourable for sex, and when suitable nitrogen sources are available, sexual reproduction is preferred over asexual sporulation. Another aspect of well-nourished conditions is the presence of a suitable amino acid source(s) and low phosphorus concentrations suppress sexual development, presumably due to a requirement for phosphorus in the generation of ATP. A third factor influencing the extent of sexual reproduction is the presence of various atmospheric gases. There are many potential reasons why a number of aspergilla have retained the ability to undergo sexual reproduction despite the increased metabolic costs compared to purely asexual reproduction. Sexual reproduction involving outcrossing can generate large amounts of genetic variation and produce novel genotypes much faster than by asexual means. This can increase the mean fitness of the next generation, quickening adaptation to changes in the environment and improving their chances for long-term survival. However, it is important to note that there are also many benefits to asexual reproduction in the aspergilli. These include the ability to produce prolific numbers of conidia for dispersal of the species in a shorter time than is required for ascospore production, the lower metabolic costs and the ability to produce asexual propagules on a wider range of substrates and under a broader set of environmental conditions than the often more fastidious requirements for sexual reproduction.
Acquisition of nutrients
Nutrient acquisition by pathogenic fungi: nutrient availability, pathway regulation, and differences in substrate utilization.2011
These fungi are trained to cope with competitors for nutrients, and they are well adapted to rapidly changing environmental conditions. Additionally, predators frequently attack fungi in their natural environment, and strategies to escape these predators have been assumed to represent ‘virulence schools’ for human infections. Different metabolic adaptations are required at different host sites, and it remains questionable, whether targeting of a single metabolic pathway can prohibit fungal growth at all host niches. To understand the acquisition of nutrients by the pathogen, it is necessary to estimate the variation of nutrients in different body site.
• Skin surface: the dry environment and the extremely limited nutrient supply in combination with immune effector cells in subcutaneous regions prohibit the establishment of an infection unless the skin is harmed by trauma, irritation, or maceration.
• Mucosal surface: as found in the oral region or the gut, are rich in nutrients from food uptake, but reproduction is limited by competing microorganisms, and tissue invasion is restricted by the immune system. The mucosal surface of the lung, in contrast, may provide a more nutrient-limited condition, since it is not in direct contact with nutrients from food intake. Additionally, the surface is covered with a mucus layer, which contains large amounts of antimicrobial factors. This allows the inactivation of inhaled microorganisms and their embedment for removal by ciliated cells and subsequent coughing.
• Bloodstream: Since blood is the major carrier of nutrients, glucose, proteins, amino acids, and vitamins are present in larger quantities. Therefore, it is essential for the host prohibiting the entry of pathogens to the blood, which is realized by large quantities of immune effector cells keeping the bloodstream virtually pathogen-free. In case of a systemic infection, pathogens can reach different internal organs such as the liver, which is the main storage compartment of glucose in the form of glycogen.
Glucose
Glucose as carbon source does not require gluconeogenesis for its synthesis and is easily degraded via glycolysis or the pentose phosphate pathway. The latter not only provides building blocks for nucleic acids and cofactors, but also generates NADPH for biosynthetic processes and for combating oxidative stress. Therefore they prefer to use glucose instead gluconeogenic nutrients. Additionally, the use of glucose allows fermentation even under aerobic conditions by gaining energy from glycolysis.
The interaction of microrganism with immune effector cells, such as macrophages and neutrophils, seems to suppress high glycolytic activity, but, in turn, favors induction of gluconeogenesis. Because these phagocytes may form extremely nutrient-limited conditions causing severe starvation. But given than within the phagocytes is a large amount of lipids and phospholipids the main pathway is represented by glyoxilate cycle, that allows to produce glucose from lipid substrates. In contrast, once penetrating a tissue and establishing an infection, significant proportions of cells seem to use, at least temporarily, the glycolytic path. Therefore, glycolysis may be of temporal importance during growth within host tissues, but may not be essential for the general ability of fungi to cause an infection at all host niches.
A schematic view of the above
Nitrogen
Besides the importance of an adequate carbon source, uptake of nitrogen is an essential prerequisite for biomass formation. An elegant way combining the simultaneous uptake of suitable carbon and nitrogen sources is the intake of amino acids. However, the largest proportion of amino acids is fixed in host proteins, requiring the production of proteolytic enzymes, farther from hyphae. The main secreted protease produced by A. fumigatus is the alkaline serine protease Alp1, which was found to be highly abundant in infected lung tissues. But also metalloprotease Mep1, that shows elastinolytic activity. As a third protease, probably important for virulence, a secreted aspartic protease was identified. Additionally, A. fumigatus produces several dipeptidyl-peptidases and sedolisins.
Lipid signaling
Lipid signalling in pathogenic fungi.2011
Microbial sphingolipids and their metabolizing enzymes play a key role in the regulation of fungal pathogenicity, through the modulation of different microbial pathways and virulence factors.
• The quorum sensing molecule (QSM) farnesol is involved not only in mycelial growth and biofilm formation but also in many stress related response. In Aspergillus fumigatus, QSM and sphingolipids are important for maintaining cell wall integrity and virulence.
• Oxylipins are the collective term used for all oxygenated lipids. Fungal oxylipins are derived from oleic acid (18:1), linoleic (18:2) and linolenic acid (18:3), after the addition of an O2 molecule to polyunsaturated fatty acids. Three genes (PPOA, PPOB and PPOC) have been implicated. Alterations in the PPO genes lead to a malfunctional signalling system in fungal cells, resulting in the inability to regulate a myriad of processes required for pathogenicity. Oxylipins increase the virulence of A. Fumigatus and counteract the host immune defences.
Physiopathology
Aspergillus fumigatus: contours of an opportunistic human pathogen.2010
Invasive Aspergillus infections usually start in the non-inflamed lung, hence at normal body temperature. Conidia of numerous Aspergillus and other fungal species are constantly inhaled by humans, but A. fumigatus is responsible for the vast majority of infections. What makes the difference? Potential criteria that may decide the success of infection are: spore size, thermoreistance, metabolic trait and secondary metabolites.
Mechanism of infection
Most cases of invasive aspergillosis are associated with haematological malignancies, particularly haematopoietic stem cell transplantation, leukaemia or lymphoma. The risk of invasive Aspergillus infection is particularly high for patients with persistent neutropenia, graft-versus-host disease (especially with concomitant steroid therapy) and certain types of allogeneic transplantation.
Healthy humans are generally not affected by severe invasive fungal infections, because the interplay of different immune effectors, especially from the innate immune system, successfully combats fungal invaders. A.fumigatus conidia bind efficiently to the surface of cells and receptors for matrix proteins may reside in the surface layer of resting conidia and mediate the primary adhesion to host tissue in the lung. Resident alveolar macrophages engulf conidia and respond to this encounter by producing cytokines and chemokines. This triggers a massive recruitment of neutrophils, which is the hallmark that distinguishes a substantial inflammation from a daily skirmish. Conidia are also internalized by cells and travel to an acidic compartment comprising lysosomal markers. But only 3% of these asexual spores survive.
Interaction of phagocytes with filamentous fungi.2010 The few conidia that survived in this hostile environment formed germ tubes, breached host membranes and escaped from the infected cell, destroying cell membrane of macrophages by mechanical forces. However, elongation of hyphae and growth are energy consuming reactions and without nutrition an infection could not establish. NETs represent an anti-microbial effector mechanism that mediates killing of a diverse range of pathogens as well as C. albicans, but in this case are unable to eliminate A. fumigatus, but reduce hyphal growth by depleting zinc ions. PTX3 is an opsonin produced by macrophages that mutually binds to the complement protein C1q and ficolin-2, a recognition molecule of the lectin complement pathway. Thus, PTX3, C1q and ficolin-2 might form complexes on the conidial surface and thereby amplify the innate immune response. After penetration of the epithelial layer of the alveoli, the fungus immediately comes in direct contact with the underlying blood vessels. Here, A. fumigatus requires no sophisticated adhesion and invasion mechanisms to breach epithelial or endothelial barriers. Instead it can rely on the robust architecture of its cell wall and the enormous driving force of the polarized hyphal growth following gradients.
Hyphae of A. fumigatus
Mitochondria stained with Rhodamine-123. Note the gradient in mitochondrial fluorescence observed towards the growing hyphal tip
Role of hypoxia during infection
Sterol regulatory element binding proteins in fungi: hypoxic transcription factors linked to pathogenesis.2010
Local obstruction of the airways may induce oedema, alveolar flooding and completely shut down the oxygen supply. Consequently, the fungus has to adapt to a hypoxic environment and the adaptation to hypoxia is a prerequisite for the survival of A. fumigatus in the inflamed tissue and its ability to spread to different organs. Moreover hyphae activate platelets and this host–pathogen interaction probably promotes thrombosis and contributes to inflammation. Viable fungal cells are rarely found in the peripheral blood, a fact that severely hampers diagnosis of disseminated Aspergillus infections. This is a consequence of the hyphal architecture that establishes tight cellular cohesion by a common cell wall and prevents the release of single cells or fragments. However, a detachment of short hyphal segments may occasionally occur and drive the systemic spread of infection. Thrombosis is a common feature of these lesions and instrumental to generate hypoxic conditions. Aspergillus furthermore inhibits angiogenesis through production of secondary metabolites, like gliotoxin, and thus enforces the formation of hypoxic conditions.
Gene expression of A. fumigatus under hypoxic conditions reveal a substantial change in the overall gene expression. The transcription factors called sterol regulatory element binding proteins (SREBPs), act as principal regulators of both cellular cholesterol uptake and de novo synthesis in mammalian cells. SREBP-like proteins have been identified in Aspergillus fumigatus. In addition to their role in hypoxic adaptation, these proteins are important for pathogenesis and resistance to antifungal drugs. Mutants lacking the SREBP homolog SrbA grow normally in atmospheric oxygen at 21% but fail to grow at 1% oxygen and SrbA is required for the hypoxic induction of genes involved in ergosterol biosynthesis. SrbA mutants show abnormal hyphal branching, indicating a defect in the establishment of cell polarity, a process which requires cell wall synthesis and restructuring. A. fumigatus cells lacking SrbA were dramatically defective for virulence. The function of these transcription factors in regulating sterol homeostasis makes the SREBP pathway an attractive target for antifungal therapy. These drugs cause fungal cell growth arrest by blocking ergosterol biosynthesis. However, azole drugs are fungistatic and not fungicidal agents, in that they do not directly kill fungal cells. This distinction is thought to contribute to the rising incidence of azole resistant fungal strains, particularly in immunocompromised patients who are unable to rapidly clear nondividing fungal cells.
INSIGHTS:
1. Regulation of sterol synthesis in eukaryotes : classical role of SREBP in regulation of sterol synthesis especially in mammalian cells.
2. Fatty Acid Synthase Gene Is Up-regulated by Hypoxia via Activation of Akt and Sterol Regulatory Element Binding Protein-1 : role of SREBP during hypoxia in humans.
Model of invasive aspergillosis development
1. First step of colonization and invasion of pulmonary epithelium.
2. Invasion of blood capillaries and haematogenous dissemination of hyphal fragments, galactomannan and other molecules.
3. Dissemination and first step of invasion of deep organs.
Have been identified some genetic susceptibility to A. fumigatus infections. For example some SNPs of TLR or MBL. MBL (the serum protein mannose-binding lectin) is a key regulator during fungal infections that enhances innate immune mechanisms by binding to cell wall components of A. fumigatus. Attachment to fungal cells is a strong signal for phagocytosis by immune effector cells and for release of proinflammatory cytokines.
Genetic susceptibility to Aspergillus fumigatus infections.2011
Some cases of disease caused by A. Fumigatus
1. Endocarditis
Aspergillus fumigatus endocarditis of the mitral valve in a heart transplant recipient: a case report.2008 : aspergillus endocarditis has a poor prognosis, requiring surgical valve replacement and aggressive antifungal therapy. This case raises the question of the contamination source. Although a pulmonary portal of entry of Aspergillus cannot be excluded, but rather has been demonstrated, the huge number of Aspergillus hyphae seen in cardiac specimens in contrast to respiratory specimens suggests that the infection probably started in the graft.
Transesophageal echocardiogram showing the large vegetation on the mitral valve and microscopic examination of the mitral valve biopsy showing several hyphae typical of a hyaline septated filamentous fungi.
Another example of endocartitis caused by A.fumigatus involves a large fungus ball attached to the right coronary cusp of the aortic valve with near complete obliteration of the left ventricular outflow tract.
Aspergillus endocarditis: a case of near complete left ventricular outflow obstruction.2012
Successful treatment of endocarditis requires the combination of antifungal therapy and surgical debridement. In general the prognosis is poor. This may be in part because of the immunocompromised status of the hosts, delay in diagnosis, and rapidity of embolization. Mortality approaches 100% among those who receive medical therapy alone.
2. Osteomyelitis
Aspergillus osteomyelitis has been recognized as an emerging extrapulmonary manifestation of invasive aspergillosis. Vertebral aspergillosis can be classified into 3 major categories, depending on the mode of acquisition. Direct inoculation related to trauma, spinal surgery, or epidural injection occurs rarely and manifests generally within months after the procedure. Contiguous spread from pleuropulmonary disease generally affects the thoracic spine. Hematogenous infection arising from a pulmonary focus occurs mainly in immunosuppressed patients.
Aspergillus vertebral osteomyelitis in immunocompetent hosts: role of triazole antifungal therapy. 2011
3. Central nervous system
One of the major complications during Aspergillus infection is its dissemination into the central nervous system (CNS), surmounting the blood–brain barrier. Although the blood–brain barrier consists of tight junctions between all endothelial cells in capillaries supplying brain cells, A. fumigatus is able to overcome this barrier and to penetrate into the cerebrospinal fluid. Infiltration of A. fumigatus into the CNS is often fatal because of reduced penetration capacities of most antifungal agents and impaired numbers of immune cells present in the CSF.
4. This infection is also often present in people with HIV , expecially in the last phase advanced AIDS, and with Falciparum malaria.
Renal abscess due to Aspergillus fumigatus as the only sign of disseminated aspergillosis in a patient with AIDS.2010
Invasive Aspergillus fumigatus infection after Plasmodium falciparum malaria in an immuno-competent host: Case report and review of literature.2009
Pharmacological treatment
The numbers of antifungals available for combating lifethreatening fungal infections are rather limited, and they mainly act on the integrity of the fungal cell membrane and cell wall. Species-specific resistance has been observed leading to ineffective therapy. A basic problem is the incomplete knowledge on the importance of metabolic pathways during pathogenesis. Unlike virus and bacteria, there are few specific drugs for fungi and non-toxic to the host, because the fungal cells are eukaryotic cells such as those of mammals. The main drugs used against the aspergillosis, in addition to one described before (azole), are:
1. Amphotericin B: induces cell death by loss of intracellular ions and macromolecules, by binding to ergosterol;
2. 5-fluorocytosine: an antimetabolite drug that inhibits the synthesis of DNA and RNA at level of thymidylate synthase.
