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Skills to Develop
- Name at least three fungal virulence factors that promote fungal colonization.
- Name at least two fungal virulence factors that damage the host.
As with the bacteria, fungal virulence factors can be divided into two categories: virulence factors that promote fungal colonization of the host; and virulence factors that damage the host.
Virulence Factors that Promote Fungal Colonization
Virulence factors that promote fungal colonization of the host include the ability to:
1. adhere to host cells and resist physical removal;
2. invade host cells;
3. compete for nutrients;
4. resist innate immune defenses such as phagocytosis and complement; and
5. evade adaptive immune defenses.
Examples of virulence factors that promote fungal colonization include:
1. A compromised immune system is the primary predisposing factor for serious fungal infections. A person highly immunosuppressed, such as a person taking immunosuppressive drugs to suppress transplant rejection, or a person with advancing HIV infection, or a person with other immunosuppressive disorders, becomes very susceptible to infections by fungi generally considered not very harmful to a healthy person with normal defenses.
2. As with bacteria, the ability to adhere to host cells with cell wall adhesins seems to play a role in fungal virulence.
3. Some fungi produce capsules allowing them to resist phagocytic engulfment, such as the yeast Cryptococcus neoformans and the yeast form of Histoplasma capsulatum (Figure 1).
4. Candida albicans stimulates the production of a cytokine called GM-CSF and this cytokine can suppress the production of complement by monocytes and macrophages. This may decrease the production of the opsonin C3b as well as the complement proteins that enhance chemotaxis of phagocytes.
5. C. albicans also appears to be able to acquire iron from red blood cells.
6. albicans produces acid proteases and phospholipases that aid in the penetration and damage of host cell membranes.
7. Some fungi are more resistant to phagocytic destruction, e.g., Candida albicans, Histoplasma capsulatum, and Coccidioides immitis.
8. There is evidence that when the yeast form of Candida enters the blood it activates genes allowing it to switch from its budding form to its hyphal form. In addition, when engulfed by macrophages, it starts producing the tubular germ tubes which penetrate the membrane of the macrophage thus causing its death.
A movie of Candida killing a macrophage from within from the Theriot Lab Website at Stanford University Medical School: Candida albicans killing macrophages from inside out.
9. Factors such as body temperature, osmotic stress, oxidative stress, and certain human hormones activate a dimorphism-regulating histidine kinase enzyme in dimorphic molds, such as Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis, causing them to switch from their avirulent mold form to their virulent yeast form. It also triggers the yeast Candida albicans to switch from its yeast form to its more virulent hyphal form.
Virulence Factors that Damage the Host
Like bacteria, fungal PAMPs binding to PRRs can trigger excessive cytokine production leading to a harmful inflammatory response that damages tissues and organs. As fungi grow in the body, they can secrete enzymes to digest cells. These include proteases, phospholipases, and elastases. In response to both the fungus and to cell injury, cytokines are released. As seen earlier under Bacterial Pathogenesis, this leads to an inflammatory response and extracellular killing by phagocytes that leads to further destruction of host tissues.
Many molds secrete mycotoxins , especially when growing on grains, nuts and beans. These toxins may cause a variety of effects in humans and animals if ingested including loss of muscle coordination, weight loss, and tremors. Some mycotoxins are mutagenic and carcinogenic. Aflatoxins, produced by certain Aspergillus species, are especially carcinogenic. A mold called Stachybotrys chartarum is a mycotoxin producer that has been implicated as a potential serious problem in homes and buildings as one of the causes of "sick building syndrome." Mycotoxin symptoms in humans include dermatitis, inflammation of mucous membranes, , cough, fever, headache, and fatigue.
Medscape article on infections associated with organisms mentioned in this Learning Object. Registration to access this website is free.
- Candida albicans
- Cryptococcus neoformans
- Pneumocystis carinii
- Dermatophytic infections (tinea)
- Coccidioides immitis
- Histoplasma capsulatum
- Blastomyces dermatitidis
- Mold allergy
Many of the same factors that enable bacteria to colonize the body also enable fungi to colonize. Many of the same factors that enable bacteria to harm the body also enable fungi to cause harm.
Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)
Pathogenic fungi are fungi that cause disease in humans or other organisms. Approximately 300 fungi are known to be pathogenic to humans.  Markedly more fungi are known to be pathogenic to plant life than those of the animal kingdom.  The study of fungi pathogenic to humans is called "medical mycology". Although fungi are eukaryotic, many pathogenic fungi are microorganisms.  The study of fungi and other organisms pathogenic to plants is called plant pathology.
CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells
Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis.
Keywords: ALS genes Adhesion CRISPR/Cas9 Candida orthopsilosis Human buccal epithelial cells SAT1 flipper cassette Virulence factors.
Candida species are ubiquitous organisms (115). An increasing incidence of fungal infections with Candida species has been noted in immunocompromised patients such as intensive-care, postsurgical, and neutropenic patients (7, 11, 14, 67, 90, 175). Candida species are most frequently isolated from the oral cavity and are detected in approximately 31 to 55% of healthy individuals (115). Colonization rates increase with severity of illness and duration of hospitalization (115, 170, 175). Historically, C. albicans accounted for 70 to 80% of the isolates recovered from infected patients. C. glabrata and C. tropicalis each accounted for approximately 5 to 8% of isolates, while other non-albicans Candida species occur only rarely (3, 7). However, more recent epidemiological data reveal a mycological shift from C. albicans to the non-albicans Candida species such as C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei (7, 90, 107, 180, 183, 184).
The changing patterns and the increasing incidence of disseminated Candida infection are also evident in a large autopsy series (11). The high mortality rate associated with bacterial infections has declined with the early administration of empirical antibiotics, while systemic fungal infections have become increasingly important in causing morbidity and mortality in immunocompromised patients. Candida is now the fourth most common organism recovered from blood cultures in hospitalized patients (7). C. glabrata has recently emerged as an important nosocomial pathogen, yet little is known about its epidemiology. Although C. albicans is the most common fungal species isolated from blood, C. glabrata currently ranks fourth among Candida species (third in patients who have undergone surgery) and is associated with an equally high mortality rate (51, 90, 181, 184). C. glabrata is of special importance because of its innately increased resistance to antifungal agents, specifically the azoles (49, 61, 174, 181, 184). The current epidemiological data for C. glabrata is summarized in Table Table1. 1 .
Epidemiology of C. glabrata infection
|Predominantly nosocomial (except vaginal)|
|Immunocompromised or debilitated host|
|Specific risk factors:|
|Prior antibiotic use|
|Use of fluconazole|
|General use in hospital|
|Hand carriage by hospital personnel|
|Often mixed fungal infection|
A clear understanding of the epidemiology of Candida infection and colonization has been difficult because of a lack of reliable typing systems to evaluate strain homology. Previous typing systems have relied on phenotypic differences within a Candida species, which may not reflect true strain differences (26, 71, 106). However, recent advances in the use of molecular techniques have enabled investigators to develop a typing system with greater sensitivity (26, 34, 70, 71, 106, 169, 172). Molecular typing of Candida by DNA fingerprinting involving various molecular techniques (restriction fragment length polymorphism, CHEF, and randomly amplified polymorphic DNA), has the capability to differentiate closely related strains which may have phenotypic similarities (26, 70, 79, 161, 169, 172).
Based upon epidemiological studies, it is apparent that humans are exposed repeatedly to Candida in food and other sources. However, the natural history of this commensal “normal” colonization over weeks, months, and years is poorly understood. Nevertheless, one may reasonably conclude that Candida colonization is almost universal. A feature common to colonized individuals is that the most frequent species are still C. albicans, and so far no unique strains of C. albicans or any non-albicans Candida species with specific gastrointestinal tract tropism have been identified. DNA typing of Candida strains obtained from AIDS patients with oral and esophageal candidiasis indicate an identical distribution frequency to those of isolates present in healthy subjects (12). This suggests that AIDS-associated candidiasis is not caused by unique or particularly virulent strains but probably results from defects in host defense mechanisms.
Until recently, most reports describing the epidemiology of nosocomial C. glabrata have been retrospective, and few studies have evaluated independent risk factors associated with nosocomial C. glabrata acquisition and subsequent infection. Knowledge of the epidemiology of fungal nosocomial colonization and infection with C. glabrata is, however, essential for the prevention of further spread as well as of nosocomial infection. In a recent study by Vazquez and colleagues (170), multivariate prospective case-control analysis along with molecular analysis of C. glabrata demonstrated that patients with new acquisition of C. glabrata had a longer duration of hospitalization (18.8 and 7.6 days, respectively P < 0.001) and more frequent prior antimicrobial use (100 and 65%, respectively P < 0.001) compared to patients from whom Candida species were not recovered during the study. These results are similar to the findings noted in earlier epidemiological studies of C. albicans, C. lusitaniae, and C. parapsilosis (138, 139, 172). Little is known about the hospital reservoirs of C. glabrata, but, as with C. albicans, probable sources include a complex interaction of environmental and human reservoirs (72, 172). The unique role of the hospital environment as a potential reservoir for Candida species is further suggested by findings in a recent study in which identical strains of C. glabrata were isolated from the environment before being newly acquired by patients admitted into a Bone Marrow Transplant Unit (170). Fungal organisms isolated from the inanimate hospital environment were previously considered to contribute little to nosocomial fungal infection. Although infecting strains can be cultured from environmental surfaces, it is believed that the environment becomes passively contaminated by organisms from patients (170, 172). Two studies have implicated carriage on the hands of hospital personnel as a possible source of an outbreak (75, 172). Thus, C. glabrata may be similar to C. albicans and other nosocomial pathogens that are acquired directly or indirectly from contaminated environmental surfaces. Previous understanding of the pathogenesis of C. glabrata colonization and infection assumed that the organisms responsible for disease were endogenously acquired exclusively from the patients’ own flora.
The role of carriage by personnel in dissemination of C. glabrata remains to be clarified. Although C. glabrata is not frequently recovered from the hands of hospital personnel, transient carriage is suggested by its isolation on environmental surfaces in contact with hands (170). Perhaps more frequent culturing of the hands of personnel or the use of liquid media to recover yeasts may have improved the detection rates of C. glabrata. Proximity to a patient with infection or colonization increases the risk of nosocomial acquisition (170). As in earlier studies (124, 172), the results of longitudinal cultures showed that 75% of patients generally carried the same strain type of C. glabrata over time (170), with minimal strain diversity among individual patients. This finding is significantly different from the results described for the nosocomial acquisition of C. albicans, in which there was considerable strain diversity (172). Moreover, in this study, 71% of patients with positive C. glabrata cultures had more than one Candida species isolated. The most frequent combination was C. glabrata and C. albicans, which was found in approximately 70% of the patients. This again is in contrast to the findings previously described for C. albicans, which showed that only 39% of patients with C. albicans had more than one Candida species identified (175). Finally, unlike C. albicans, C. glabrata has not been recovered from the food provided to hospitalized patients, potentially contributing to the lack of identifiable C. glabrata strain diversity.
In conclusion, these studies suggest that nosocomial acquisition of C. glabrata is not uncommon and may be due to exogenous acquisition. In addition, two major risk factors associated with C. glabrata colonization are prolonged duration of hospitalization and prior antimicrobial use. Further prospective studies are sorely needed to define more clearly the reservoirs of infection, as well as the mode of transfer and measures for preventing the spread of infection.
5.2.4: Fungal Pathogenicity - Biology
Fungi cause a spectrum of diseases in humans ranging from comparatively innocuous superficial skin diseases caused by dermatophytes to invasive life-threatening infections caused by species such as Candida albicans, or Cryptococcus neoformans. Due to the opportunistic nature of most invasive mycoses, fungal pathogenicity has proven difficult to define. However the application of new genomic and other molecular techniques in recent years has revolutionized the field revealing fascinating new insights into the mechanisms of fungal pathogenesis.
In this book a panel of high profile authors critically reviews the most important research to provide a timely overview. The extensive reference sections in each chapter positively encourage readers to pursue the subject in greater detail. The book is divided into two sections: The first six chapters review the transformative effect of applying state-of-the-art tools and innovative approaches to research, particularly in the area of comparative biology. The second section consists of eight chapters, each dedicated to the molecular and cellular biology of a major fungal pathogen of humans: Candida, Aspergillus, Cryptococcus, dermatophytes, Histoplasma, Blastomyces, Pneumocystis and Paracoccidoides. These chapters provide a timely snapshot of the current state of research.
This volume is an essential reference for students, researchers and clinicians with an interest in fungal pathogenesis.
"The editors . have managed to bring together 34 of the most active and leading researchers in the field to produce such a review . the coloured diagrams and photographs are so informative and(or) stunning , that I can imagine that some of these will become much used in teaching medical mycology courses . This is a carefully edited and well-produced reference work that deserves to be widely available in laboratories exploring the molecular biology and pathogenicity mechanisms of human pathogenic fungi" from IMA Fungus (2014) 5: 56-57.
"this book is highly recommended . should be available in all college and university libraries where human pathology and biotechnology courses are offered. It is also a useful source of information for undergraduates as well as researchers." from Fungal Diversity
"The up-to-date, well-presented chapters serve to make this text a valuable reference for experts in the field as well as those new to fungal pathobiology . this well-presented work is a highly useful addition to the literature . The extremely readable nature of the well-edited text will certainly facilitate its use by graduate students entering into the fields of fundamental or medical mycology and established researchers will appreciate the crisp reviews and their thorough references. " from Frontiers in Microbiology
(EAN: 9781908230447 9781908230669 Subjects: [microbiology] [genomics] [mycology] )
Fungi are an increasing threat to human health, animals in the global ecosystem, and to agriculture and food security. Fungal Pathogenesis is committed to providing a broad, multidisciplinary platform for research that serves to cross-fertilize advances and contribute to thwart the fungal scourges of the planet.
The focus of this section spans the pathogenic microbes that cause life-threatening infections in humans, including Candida species, Aspergillus species, Cryptococcus species, dimorphic human fungal pathogens, Mucor and other zygomycetes, and Pneumocystis and the Microsporidia. In animals, the scope spans the chytrid pathogen of frogs, Batrachochytrium dendrobatidis and related species, as well as Pseudogymnoascus destructans the cause of bat white nose syndrome. In plants, submissions on fungal pathogens that cause important crop losses and studies focused on how plants respond and defend against fungal infections are invited.
We welcome novel heterologous model systems (Galleria, drosophila, zebrafish, amoeba, eggs, and others), genomic and population genetics studies, vaccine studies, novel drug and treatment approaches and strategies, mechanisms of drug resistance, diagnostics, and immunotherapy. Studies on the fungal components of the microbiome (GI, lung, skin, oropharynx) are invited.
We are particularly interested in studies that span disciplines and the emergence of new pathogens in clinical and agricultural settings, including fungal outbreaks with novel or rare pathogenic fungi. Studies contributing to the taxonomy and phylogeny of fungi will also be within the domain of this section
Fungal Pathogensis is led by Joseph Heitman, MD, PhD from Duke University and Anuradha Chowdhary, MD, PhD from the University of Delhi, and supported by an international editorial board of outstanding experts.
What is active host transmission?
Insects under the explicit control of parasitic fungi (entomopathogens) are sometimes characterized by colorful terms, even colloquially categorized as “zombies” [2,3], a moniker that draws comparison to both fictitious and factual elements of contemporary life. Though the effects of entomopathogenic fungi on their hosts are a far cry from behavior-modifying viruses such as rabies or the phantasmic world of brain-eating zombies that drag their way through our popular culture, both rabies and select entomopathogenic fungi are nevertheless archetypal examples of pathogens that actively enlist their living hosts for successful transmission, a phenomenon referred to hereafter as active host transmission (AHT) .
Victims of the rabies virus experience hydrophobia, refuse to swallow (allowing the virus to collect around their mouths), and are much more likely to aggressively bite and interact with others . This unsettling rewiring of animal behavior supplants the interests of the victim in favor of the interests of the virus within. The phenomenon of parasite-induced AHT in animal hosts has evolved numerous times across a variety of taxonomic groups. For example, Toxoplasma gondii, a protist parasite, suppresses the fear response of rodents and drives them to seek out feline foes to help complete the lifecycle of their protist partner . Horsehair worms (Nematomorpha) encourage their host crickets to drown themselves, which allows these parasites to complete their own lifecycle in water . Likewise, certain entomopathogenic fungi such as Massospora spp. manipulate their hosts’ sexual behaviors to increase their odds of transmission . Such engagements appear to serve the interests of the fungal pathogen over the interests of their hosts.
Manipulation of a host to focus on pathogen transmission is fascinating because it raises questions about the nature of autonomy and shines a light on the physical and behavioral manifestations of parasitism. AHT is a form of biological puppetry in which the pathogen manipulates the behavior of its powerless host. But, identifying clear behavioral manipulations and distinguishing AHT from other notable entomopathogen-induced behaviors such as summit disease, particularly when the infected insects are moribund or dead at the time of their discovery, is a challenge. In anamorphic fungi, including Metarhizium species , spores are dispersed on contact or passively through the environment. In summit diseases such as Entomopthora muscae  or Ophiocordyceps species , dissemination of spores is facilitated by the positioning of the host cadaver. In both of these modes of transmission, spores develop on the mummified host after death, and the deceased host does not actively disperse spores. In contrast, AHT requires 1) a living host and 2) host behavior that facilitates pathogen transmission, thereby increasing pathogen fitness at the expense of host fitness (Fig 1). To achieve these ends, AHT pathogens must produce transmissible reproductive structures while still allowing the host some level of functionality, which is a major distinction between AHT and most other entomopathogenic fungi, in which infectious spores (conidia) are not produced until after host death. Inconspicuous infectious stages also present a challenge for the pathogen itself: developing complex reproductive structures while still inside the living host could result in physical disruption from insect organs, muscles, and exoskeleton that would be static on an insect cadaver. Even when infections are conspicuous, such as when the abdomens of Massospora-infected cicadas swell and are eventually shed (Fig 2), the remaining internal organs must retain some functionality to keep the cicada alive. AHT parasites also modify host behaviors so that parasite reproductive structures appear when hosts are manipulated to increase their interactions with uninfected potential hosts. This synchronization could either exploit natural host behaviors or induce behaviors that increase the frequency of interaction between host insects.
DprA (Afu4g00860) and DprB (Afu6g12180) encode fungal dehydrin-like proteins
The gene Afu4g00860 was retrieved from an expression profiling study conducted in A. fumigatus aiming at the identification of germination-regulated genes (Lamarre et al., 2008). The deduced sequence corresponded to a protein containing 246 amino acids. Scanning of the sequence by prediction software neither revealed known localization signatures nor gave indications of protein function. However, blast results gave a hit with a 435-residue protein from A. fumigatus, encoded by Afu6g12180. Remarkably, blast results aligned only on small portions of the proteins, with very poor homology in between. The homologous sequences were repeated five and nine times, respectively, and corresponded to the signature pattern of fungal dehydrins (Abba et al., 2006). The repeated domain consisted of a stretch of 23 amino acids, containing a conserved dehydrin-like protein (DPR) motif (Figure 1). For this reason, the genes were called Dpr. Dehydrins were described in plants, where they are involved in the protection against dehydration-related stresses (Rorat, 2006). However, they have not been studied in fungi. In silico analysis confirmed that DprA and DprB were dehydrin-like proteins, by virtue of their physicochemical properties (Wise, 2003 Abba et al., 2006 Supplemental Figure S1). The presence of hydrophobic residues, predicted phosphorylation sites, and proline residues within the DPR domains (Figures 1 and S1B) suggest that DPR domains could form a hydrophobic core, within which protein–protein interactions would take place.
FIGURE 1: Alignment of the DPR domains from DprA and DprB. Conserved amino acids are boxed in black (identical) or gray (similar). D1A-D5A designate the five domains from DprA and D1B-D9B refer to the nine domains from DprB, numbered from N- to C-terminal end. Numbers indicate the amino acid positions. The DPR motif is indicated with a red border. Asterisks designate a predicted phosphorylation site conserved in all DPR domains.
DprA and DprB are down-regulated upon conidial germination
Expression of DprA and DprB was assessed during conidial germination (Figure 2A). In dormant conidia (time 0), DprA transcripts were ∼120 times more abundant than DprB. Both transcript types underwent significant down-regulation with a 1000- and a 350-fold decrease in expression level from 0 to 30 min, respectively. Very weak expression of DprA was detected beyond 30 min, at least up to 24 h. In contrast, DprB transcripts became abundant again after 8 h and reached a twofold increase at 24 h.
FIGURE 2: Expression of DprA and DprB. (A) Evaluation of the expression levels of DprA and DprB by real-time PCR. RNA was extracted from dormant conidia of the AkuB strain (0 h) and conidia that were incubated for 0.5, 2, 4, 8, 16, or 24 h in liquid YPD medium at 37°C, 150 rpm. An arbitrary value of 1.0 was attributed to the expression level of DprB at time 0. Data are from three independent experiments ± SE. Developmental stages corresponding to the selected time points are shown. (B) Assessment of DprA expression by eGfp fusion in hyphae undergoing conidiation (7 d at 25°C). Note that DprA-eGfp is present only in the conidia. (C) Same as (B), with DprB expression. Scale bars: 5 μm.
To monitor the expression during development, the coding sequences of DprA and DprB were fused to eGfp under the control of their own promoters. DprA-eGfp fluorescence was detected only in dormant conidia (Figure 2B). In agreement with the real-time PCR data, DprB-eGfp fluorescence was observed in dormant conidia. However, in contrast to DprA-eGfp, DprB-eGfp was also observed in the conidiophores (Figure 2C) and in hyphae (unpublished data), indicating DprB was associated with late stages of development, unlike DprA.
DprA is involved in the oxidative stress response of conidia
Growth of DprAΔ (but not DprBΔ) mutants was inhibited by oxidative stress generated by hydrogen peroxide or paraquat at concentrations > 2 mM (Figure 3A). Consistent with this, DprA expression was up-regulated upon treatment with 2 mM H2O2 or 2 mM paraquat (Figure 3B). In the absence of stress, no difference was observed between the germination curves of the mutant and control strains (Figure 3C). However, when 2 mM H2O2 was added to the medium, germination of the DprAΔ (and DprAΔ DprBΔ) mutants was impaired (Figure 3D). After 13 h, germination had reached its maximum stage. At that time point, only 30% of the mutant conidia had undergone swelling, compared to 70% for the control strains. This defect in swelling, and subsequently in germination, could be explained by the higher susceptibility of DprAΔ conidia to the fungicidal effect of H2O2. To check the behavior of conidia challenged with host-derived reactive oxidant species, conidial survival was assessed after 36 h in the lungs of immunocompetent mice. Accordingly, the conidia of the DprAΔ and DprAΔ DprBΔ mutants were hypersensitive to killing by the lung phagocytes (Figure 3E). However, no difference was observed in the virulence of the strains in two experimental models of invasive aspergillosis (Figure S2).
FIGURE 3: Oxidative stress-induced phenotype of DprAΔ mutants. (A) Growth of DprΔ mutants (AΔ, BΔ and AΔBΔ) in the presence or absence of oxidative stress induced by H2O2 or paraquat, compared with the control strains (AkuB, or the revertant strains RevA and RevB). Conidia (10 5 ) were spotted on Sabouraud medium supplemented with 2 mM H2O2 or 2 mM paraquat (P). The plates were incubated at 37°C for 30 h. (B) Expression levels of DprA under oxidative stress. Overnight cultures of the AkuB strain were treated for 1 h with 2 mM H2O2 or 2 mM paraquat at 37°C 150 rpm, prior to RNA extraction. An arbitrary value of 1.0 was attributed to the expression level corresponding to the control without stress. Data are from three independent experiments ± SE. (C) Germination rates of the control (AkuB) and the DprΔ mutant conidia on Sabouraud medium at 37°C. Conidia undergoing germination were scored on a total of 100 conidia at 1 h intervals, in three independent experiments ± SE. (D) Same as (C), but in the presence of 2 mM H2O2. (E) Conidial killing in immunocompetent mice 36 h after intranasal infection. Conidial survival was evaluated by plating serial dilutions of bronchoalveolar lavages. Data are the mean value obtained with 3 mice ± SE.
DprB is involved in the osmotic stress response
When grown in the presence of sorbitol, DprBΔ (in contrast to DprAΔ mutants), displayed an abnormal colony morphology, which could be observed as from 0.5 M sorbitol (Figure 4A). DprBΔ colonies were restricted to forming a “central zone,” in which conidiation would take place, with no “peripheral zone,” in which the colony would normally expand by apical extension of the hyphae. With 1.5 M sorbitol, the DprBΔ colonies had one-half the diameter of the control strain colonies. Microscopic observation of the colony edges showed extensive branching (Figure 4B). The results were similar irrespective of the osmoticum (sorbitol, glycerol, mannitol, NaCl, KCl) or medium (Sabouraud, YPD, minimal medium, malt extract unpublished data) used. Addition of 1.5 M sorbitol to the medium did not affect the germ tube emergence nor the germination kinetics of the mutant conidia compared with the control strains (unpublished data), indicating that osmostress did not affect the DprBΔ mutants at the conidial stage, but rather at later stages of development. When the wild-type strain was subjected to sorbitol concentrations ranging from 0 to 1 M, DprB transcript levels were up-regulated (Figure 4C). Regulation was dose-dependent, with an expression peak with 0.75 M sorbitol.
FIGURE 4: Osmotic stress-induced phenotype of DprBΔ mutants. (A) Growth of DprΔ mutants (AΔ, BΔ, and AΔBΔ) in the presence or absence of osmotic stress induced by sorbitol, compared with the control strains (AkuB, or the revertant strains RevA and RevB). Conidia (10 5 ) were spotted on Sabouraud medium supplemented with 0, 0.5, 1.0, or 1.5 M sorbitol. The plates were incubated at 37°C for 30 h. (B) Microscopic observation of the colonies grown on solid Sabouraud supplemented with 0.5 M sorbitol. Note the hyperbranched mycelium of the DprBΔ and DprAΔ DprBΔ mutants, compared to the parental and revertant strains. (C) Expression levels of DprB under osmotic stress assessed by real-time PCR. Extracts are from overnight liquid cultures of the wild-type strain subjected to a range of sorbitol concentrations (0, 0.25, 0.5, 0.75, 1 M) for 30 min at 37°C. An arbitrary value of 1.0 was attributed to the expression level without addition of sorbitol. Data are from three independent experiments ± SE.
The DprBΔ mutant displays a pH-dependent phenotype that is PacC-related
Growth of the DprBΔ (but not of the DprAΔ) mutant was impaired at pH 7 and pH 9, but not at pH 5 (Figure 5A). At pH 9, after 72 h of growth, the DprBΔ colonies had one-half the diameter of the control strain colonies (unpublished data). Real-time PCR indicated DprB was expressed preferentially at neutral or alkaline pH (Figure 5B), consistent with the phenotype of the DprBΔ mutant. As seen in the assays under osmostress, germination of the conidia was not affected by pH stress (unpublished data). The PacC transcription factor controls pH-regulated genes in Aspergillus spp. (Tilburn et al., 1995). A putative binding site of PacC (GCCAGG) was detected in the promoter of DprB at position −264. The binding of recombinant PacC to the DNA sequence was confirmed (Figures 5C and S3). In Aspergillus spp., PacC acts as both an activator of alkaline-expressed genes and a repressor of acid-expressed genes. Loss-of-function mutations of PacC (PacC +/− ) cause an acidity-mimicking phenotype and result in an increased expression of acid-expressed genes, and a reduced expression of alkaline-expressed genes. Gain-of-function, alkalinity-mimicking mutations of PacC (PacC c ) result in a phenotype opposite that of acidity-mimicking mutations. DprB expression was checked in the alkalinity-mimicking and in the acidity-mimicking strains (Amich et al., 2009). In agreement with a positive regulation of DprB by PacC, the expression of DprB in the acidity-mimicking strain was similar to that in the wild-type, whereas it was overexpressed in the alkalinity-mimicking strain (Figure 5D).
FIGURE 5: pH stress-induced phenotype of DprBΔ mutants. (A) Growth of DprΔ mutants (AΔ, BΔ, and AΔBΔ) under pH stress, compared with the control strains (AkuB, or the revertant strains RevA and RevB). Conidia (10 5 ) were spotted on Sabouraud medium supplemented with 0.1 M MES, MOPS, or Tris and adjusted to pH 5, 7, or 9. The plates were incubated at 37°C for 30 h. (B) DprB expression according to extracellular pH. Real-time PCR was performed on extracts from overnight cultures shifted for 1 h to medium at pH 5, 7, or 9 at 37°C, 150 rpm. An arbitrary value of 1.0 was attributed to the expression level corresponding to pH 5. Data are from three independent experiments ± SE. (C) Gel-mobility shift assay using the GST::PacC (30–195*) protein produced in Escherichia coli and a 37 bp fragment of the DprB promoter. The 32 P-labeled DNA fragment (40 fmol) was incubated in the absence (−) or presence (+) of the purified GST::PacC protein (40 fmol). Unlabeled probe (4 pmol) was used as competitor (c) to check the specificity of the binding. (D) DprB expression in PacC mutants. Real-time PCR was performed on extracts from overnight cultures at pH 7 of the reference strain AkuB, the acid-mimicking strain PacC +/− , and the basic-mimicking strain PacC c . An arbitrary value of 1.0 was attributed to the expression level obtained with the wild-type strain (WT). Data are from three independent experiments ± SE.
DprA and DprB act downstream of the SakA MAPK
To gain insight into the signaling cascades involved in the recruitment of Dpr proteins, the expression of Dpr genes was assessed in signal transduction mutants from the mitogen-activated protein kinase (MAPK) pathways (SakAΔ, MpkAΔ, MpkBΔ, and MpkCΔ Du et al., 2006 Reyes et al., 2006 Valiante et al., 2008), the cyclic AMP (cAMP) signaling pathway (AcyAΔ, PkaC1Δ, and PkaRΔ Liebmann et al., 2003, 2004 Grosse et al., 2008), and the calcium/calcineurin transduction pathway (calAΔ da Silva Ferreira et al., 2007). In contrast with other signaling mutants, DprA and DprB transcripts were not detected in the SakAΔ mutant (Figure 6, A and B), indicating DprA and DprB acted downstream of the stress-activated kinase (SAK) SakA MAPK cascade. In A. fumigatus, the SakA signaling pathway regulates the response to hyperosmotic and oxidative stress (Du et al., 2006 Reyes et al., 2006). An in silico comparative analysis was undertaken to identify targets of SakA that could link the MAPK to Dpr genes. In Saccharomyces cerevisiae, the SakA homologue Hog1 interacts with 4 transcription factors, Msn2, Msn4, Sko1, and Hot1 (Hohmann et al., 2007). However, A. fumigatus lacked clear orthologues of these proteins (Bahn, 2008), indicating that other sets of transcription factors achieved stress-response regulation downstream of SakA. In Schizosaccharomyces pombe, two bZIP-type transcription factors, Atf1 and Pap1, intervene downstream of the SakA-related MAPK cascade in response to environmental stress signals (Toda et al., 1991 Takeda et al., 1995 Shiozaki and Russell, 1996 Gaits et al., 1998 Toone et al., 1998). Aspergillus spp. possess an Atf1 and a Pap1 homologue, and SakA was shown to interact with AtfA in A. nidulans (Lara-Rojas et al., 2011). Mutant strains for AtfA (Afu3g11330) and Yap1 (Afu6g09930) were constructed, and real-time PCR analysis showed that DprA expression was impaired in the AtfAΔ but not in the Yap1Δ mutant, indicating that DprA acted downstream of AtfA (Figure 6, A and B).
FIGURE 6: Signal transduction pathways involved in the expression of Dpr genes. (A) Expression of Dpr genes in A. fumigatus mutants under 0.5 M sorbitol, pH 9. Transcript levels of DprA and DprB were estimated by real-time PCR in the reference strain AkuB the Dpr mutants DprAΔ, DprBΔ, DprAΔ DprBΔ the MAPK mutant SakAΔ and the transcription factor mutants AtfAΔ and Yap1Δ. An arbitrary value of 1.0 was attributed to the expression levels in the AkuB strain. (B) Same as (A), under 2 mM H2O2. (C) Expression of Dpr genes in the adenylyl cyclase mutant AcyAΔ, and the effects of adding 50 mM exogenous cAMP as determined by real-time PCR. An arbitrary value of 1.0 was attributed to the expression levels in the AkuB strain without treatment with cAMP. (D) Gel-mobility shift assay using the AtfA::6His recombinant protein and 37–base pair fragments from the DprA promoter (A-142 or A-23). The 32 P DNA fragments (40 fmol) were incubated in the absence (−) or presence (+) of the recombinant AtfA::6His protein (40 fmol). Unlabeled probe (4 pmol) was used as competitor (c) to check the binding specificity.
The expression of DprA was also affected in the adenylyl cyclase mutant AcyAΔ (Figure 6C). When the AcyAΔ strain was grown on medium supplemented with 25 mM cAMP, the wild-type expression level of DprA was restored, indicating regulation by the cAMP-related pathway. Consistent with this finding, putative cAMP-responsive element (CRE) sequences were found in the promoter of DprA at positions −142 (TGACGTAA) and −23 (GAACGTCA), to which a recombinant AtfA protein was able to bind (Figures 6D and S3).
DprA and DprB are induced by DTT
A major class of stress-protective molecules is represented by molecular chaperones. These molecules are essential for cells to prevent the aggregation of partially unfolded proteins. This requirement is increased when cells experience protein unfolding stresses. Upon treatment with dithiothreitol (DTT), an inducer of the unfolded-protein response, significant up-regulation of DprA and DprB expression was observed (Figure 7), suggesting a potent role of the corresponding proteins as molecular chaperones.
FIGURE 7: Expression of DprA and DprB upon treatment with DTT. Real-time PCR was performed on RNA extracted from overnight cultures of the AkuB strain treated for 1 h with 0, 1, or 5 mM DTT at 37°C, 150 rpm. An arbitrary value of 1.0 was attributed to the expression level of DprA in the absence of DTT. Data are from three independent experiments ± SE.
DprA and DprB fused to eGfp are associated with the cytosol and the peroxisomes
To check their subcellular localization, DprA and DprB were fused at their carboxy-terminal end to eGfp, under the control of their native promoters, in the respective mutant strains. The functionality of the constructs was checked by the restoration of the wild-type phenotype (unpublished data). Both DprA-eGfp and DprB-eGfp fusion proteins accumulated in the cytoplasm and in punctuate organelles. The labeled organelles did not stain with the membrane- and endocytosis-selective dye FM4–64 (Supplemental Movie S1 Fischer-Parton et al., 2000). To test the hypothesis that the organelles might be peroxisomes, the DprA- and DprB-eGfp strains were transformed with a plasmid bearing a DsRed-serine-lysine-leucine (DsRed-SKL) fusion typical of the type 1 peroxisomal targeting sequence PTS1 (Ruprich-Robert et al., 2002 Elleuche and Pöggeler, 2008). Colocalization of eGfp and DsRed showed that DprA and DprB were associated with peroxisomes (Figure 8 and Movie S2).
FIGURE 8: Subcellular localization of DprA and DprB using eGfp fusions. DprA and DprB were fused at their 3′ end to the coding sequence of eGfp. Expression was driven by the gene's own promoter in the corresponding DprAΔ and DprBΔ strains. Localization of DprA in dormant conidia (A), and localization of DprB in dormant conidia (B), in germinating conidia (C), and in hyphae (D). Scale bars: 5 μm
DprΔ mutants have altered catalase and β-oxidation activities
Peroxisomes contain a large battery of enzymes that are important notably for oxygen species detoxification and β-oxidation of fatty acids. Catalases have a protective role against H2O2 and have been shown to be localized in peroxisomes (Schrader and Fahimi, 2006). A. fumigatus possesses three catalase activities: CatA, which is produced exclusively in conidia, and Cat1 and Cat2, which are produced in the mycelium (Paris et al., 2003). In an attempt to explain the higher sensitivity of DprAΔ conidia to H2O2, catalase activity was assessed in conidial protein extracts. As shown by in-gel detection, activity due to the conidial catalase CatA was reduced in the DprAΔ mutant (Figure 9A). This was not the case for the mycelial catalases Cat1 and Cat2 when mycelial extracts were assessed (unpublished data).
FIGURE 9: Effect of DprΔ mutation on peroxisomal functions. (A) In-gel detection of catalase activity. Each line was loaded with 30 μg total protein. (B) Growth on different carbon sources. The following carbon sources were added to modified minimal medium: 1% glucose, 10 mM butyrate, 10 mM valerate, 5 mM hexanoate. Growth was for 3 d at 37°C.
Growth of the DprBΔ mutant was impaired when short-chain fatty acids (butyrate [C4], valerate [C5], hexanoate [C6]) were used as sole carbon sources (Figure 9B), but not with longer-chain fatty acids such as oleic acid (C18), or Tween 20, whose major component is lauric acid (C12 unpublished data). In Aspergillus spp., β-oxidation of short-chain fatty acids with odd-numbered carbons (notably valerate) requires the peroxisomal β-oxidation pathway (Hynes et al., 2008). The two results are in agreement with a peroxisomal association of Dpr proteins.
Adaptive Pathogenicity Strategies
The immune system possesses elegant strategies to cope with potential harmful invading microorganisms. Nevertheless, pathogens themselves have evolved mechanisms to deal with the threats imposed by the immune system. Yeasts like Candida species are common commensals of the human microbiota, yet also major opportunistic fungal pathogens that frequently cause superficial and even fatal infections. The commensal co-existence with the human host allows the co-evolution of fungal adaptation strategies in line with the threats imposed by the host.
While the competing microbiota is potentially the major challenge for host-associated Candida species during commensalism, the immune system is the major threat that can compromise the survival during infection. The fungus, therefore, employs strategies to evade immune recognition or even escape the immune cells after it has been attacked. A constant expression of these pathogenicity strategies is not efficient and may also jeopardize the commensal lifestyle of these pathogens. An adaptive regulation is essential to only engage these pathogenicity strategies when needed. We investigate the adaptations induced by host conditions (like temperature) and host molecules. In particular, we are interested in the underlying molecular mechanisms inducing these adaptations and the host proteins the fungus may sense to do so.
This research group is funded by the Deutsche Forschungsgemeinschaft (DFG) Emmy Noether Program (project no. 434385622 / GR 5617/1-1) and an ESCMID research grant 2019.
Further related topics dealing with immunotherapy and Interaction with the microbiota are investigated in close collaboration with the Department of Microbial Pathogenicity Mechanisms.
Significant advances have been made in our understanding of stress adaptation in C. albicans, and progress is being made towards the elaboration of specific stress signalling pathways. This is important because stress adaptation contributes to the virulence of this major fungal pathogen of humans. However, host niches are complex and dynamic, and the impact of this complexity and dynamism upon stress adaptation remains largely unexplored. In particular, how are stress responses regulated temporally during host colonisation and disease progression? The elegant microarray studies performed by Bernie Hube's group go some of the way to addressing this question (Fradin et al., 2005 Thewes et al., 2007 Zakikhany et al., 2007 Wilson et al., 2009). However, microarray studies average the molecular behaviour of the fungal population as a whole, and fungal populations display heterogeneous behaviours in host niches (Barelle et al., 2006). This is because the microenvironments of individual cells vary even within specific host niches. Therefore, the spatial regulation of stress adaptation must also be examined during infection. This must either be done by examining the responses of individual cells in vivo, for example using GFP-based single-cell profiling methods (Barelle et al., 2006 Enjalbert et al., 2007 Miramón et al., 2012), or by increasing the sensitivity of RNA sequencing technologies and increasing their spatial resolution, for example by exploiting laser capture microscopy. These approaches are being pursued by the Aberdeen Fungal Group (J.P., S.S. and A.J.P.B., unpublished).
In addition, at least three aspects of stress adaptation that are of direct relevance in vivo need further dissection in vitro. First, which anticipatory responses in C. albicans influence host colonisation and disease progression, and how are these anticipatory responses controlled at the molecular level? Second, which combinatorial stress responses in C. albicans influence host–fungus interactions, and how are they regulated? Third, how does metabolic adaptation influence stress resistance within host niches? Despite the limited exploration of these issues, it is already clear that they involve non-additive behaviours that reflect unexpected signalling, transcriptional, biochemical and chemical cross-talk. Furthermore many of these responses are dynamic and dose dependent. Given their complexity, a combination of experimental approaches and predictive mathematical modelling seems especially important for the development of a true understanding of these adaptive processes. Such studies will provide important insights into the forces that have driven the recent evolution of this pathogen in its host.
In closing, it is worth emphasising that studies of stress adaptation are revealing points of fragility in C. albicans that could potentially provide targets for translational research directed towards the development of novel antifungal therapies. Indeed, the therapeutic potential of Hsp90 inhibitors is being pursued by a number of laboratories (Dolgin and Motluk, 2011). Therefore, observations such as the acute sensitivity of C. albicans towards combinatorial cationic plus oxidative stress could, in principle, be exploited therapeutically.