Animal models in the analysis of Candida host–pathogen interactions Louis de Repentigny An increasingly diverse array of clinically relevant animal models of candidiasis have been established that mimic both the immune perturbations of the host and tissue-specific features of candidiasis in humans. Cause-and-effect analysis of Candida host–pathogen interactions using these animal models has made a quantum leap forward in the genomic era, with the concurrent construction of C. albicans mutants with targeted mutations of putative virulence factors, the application of microarrays and other emerging technologies to comprehensively assess C. albicans gene expression in vivo, and construction of transgenic and knockout mice to simulate specific host immunodeficiencies. The opportunity to combine these powerful tools will yield an unprecedented wealth of new information on the molecular and cellular pathogenesis of candidiasis. Addresses Department of Microbiology and Immunology, Sainte-Justine Hospital and University of Montreal, 3175 Coˆte Ste-Catherine, Montreal, Quebec, Canada, H3T 1C5 e-mail:
[email protected]
Current Opinion in Microbiology 2004, 7:324–329 This review comes from a themed issue on Host–microbe interactions: fungi Edited by Ken Haynes Available online 1st July 2004 1369-5274/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2004.06.001 Abbreviations OMP orotidine 50 -monophosphate
Introduction Candida species are the most frequent cause of mucosal and invasive fungal infections [1]. The leading cause of candidiasis, Candida albicans, is a dimorphic fungus that resides as a commensal of the human mucosae and the gastrointestinal tract. In immunocompromised hosts, however, saprophytic colonization often leads to opportunistic mucosal or life-threatening deep organ infection. Invasion of the human gastrointestinal mucosa by C. albicans and its passage across the bowel wall into the bloodstream is an important portal of entry for this opportunistic pathogen in the neutropenic host, leading to systemic or disseminated candidiasis [2]. Hematogenous candidiasis is a frequent complication in the treatment of patients with acute leukemia [3]. Oropharyngeal Current Opinion in Microbiology 2004, 7:324–329
candidiasis, a common mucosal infection, occurs in the majority of human immunodeficiency virus (HIV)infected patients, although decreases have been observed with highly active antiretroviral therapy [4]. Although vaginal candidiasis is not associated with HIV-infection [5], it has been estimated to occur in approximately 75% of women at least once [6]. The ability of C. albicans to colonize, penetrate and damage host tissues depends on imbalances between Candida virulence attributes and specific defects in host immune defenses. However, the exact mechanisms leading to this imbalance are still unclear. As a highly evolved pathogen capable of colonizing and infecting a multiplicity of body sites, each representing a distinct ecological niche, C. albicans must have the ability to selectively express the required specific sets of virulence attributes at each step of host invasion, while simultaneously exploiting weakened host defense mechanisms which in the normal host limit C. albicans proliferation. In this review, I discuss the development and use of animal models for studying these Candida host–pathogen interactions in the genomic era.
Animal models of candidiasis Animal models represent powerful tools to elucidate the molecular and cellular pathogenesis of candidiasis (previously reviewed in [7,8]). The principal advantage in studying animals instead of human beings is that the animal and its environment can be controlled [7], allowing a precise cause-and-effect longitudinal analysis of host– pathogen interactions. In addition, these models obviate the often problematical procurement of tissue samples from human patients. The usefulness of animal models of candidiasis includes not only the study of pathogenesis but also the in vivo assessment of novel antifungals, immunomodulators and potential Candida vaccines [9,10]. Ideally, an animal model of candidiasis to be used in studying pathogenesis should not only reproduce as faithfully as possible the process of colonization and invasion at a specific anatomic portal of entry, but also closely match the specific immune defects or hormonal conditions associated with infection at that particular site. The experimental infection should be sufficiently protracted to sequentially reveal the participation of Candida virulence attributes and the recruitment of host defenses. Although other species including rats, rabbits and monkeys have been used, murine models of candidiasis are advantageous because of the economy of scale, the ready www.sciencedirect.com
Animal models in the analysis of Candida host–pathogen interactions de Repentigny 325
availability of reagents and methods for immunological analysis, as well as the ability to generate knockout and transgenic mice that reproduce specific immune perturbations of the host. Without detracting from their relevance as animal hosts for studying the pathogenesis of candidiasis, a difference which needs to be taken into consideration is that contrary to humans, mice are not normally colonized by C. albicans and must therefore be experimentally infected. By contrast, humans are colonized by C. albicans shortly after birth and candidiasis is most frequently of endogenous origin. Therefore, the ability to establish colonization with C. albicans in mice before any immunocompromising procedure would more closely mimic the onset and progression of candidiasis in humans. Very few animal models of candidiasis achieve this goal, with the notable exception of the infant mouse model [11,12], in which compromising agents are administered to mice persistently colonized with C. albicans in infancy. A growing array of clinically relevant animal models of mucosal or systemic candidiasis have been established and validated over the past decade [13–34], representing the most frequent forms of candidiasis encountered clinically (Table 1). Persistent oral mucosal carriage of C. albicans can be achieved in normal mice treated with glucocorticoids or irradiation, in mice infected with a mouse retrovirus complex and developing murine AIDS, or in transgenic mice expressing the genome of HIV-1, while persistent vaginal candidiasis can be maintained in animals treated with estrogen. The recently characterized model of oro-esophageal candidiasis in transgenic mice
expressing HIV-1 closely mimics the clinical and pathologic features of candidal infection in human AIDS, and thus provides a novel opportunity to study the pathogenesis of mucosal candidiasis in HIV-infection under controlled conditions in a small laboratory animal. Systemic candidiasis in the neutropenic host can be reproduced in rabbits with chemotherapy-induced persistent granulocytopenia, or in mice treated with cyclophosphamide or anti-granulocyte monoclonal antibody. Notably, a murine model of systemic candidiasis has been recently established in allogeneic bone marrow transplant recipients, further broadening the scope of clinically relevant susceptible hosts. While some models of systemic candidiasis rely on intravenous inoculation of C. albicans, oral or oral-intragastric inoculation more closely mimic the predominant gastrointestinal portal of entry leading to systemic dissemination. In a second category of experimental models for the analysis of Candida host–pathogen interactions, animals with intact immunity or targeted immune defects are employed [9,26,27,30,35–64] (Table 2). Intact mice were first used to explore the pathogenesis of candidiasis and remain useful to define immune responses to C. albicans in the normal host. Congenitally immunodeficient mice with individual or combined defects of T-cells, B-cells, NK cells and phagocytes have provided a foundation for understanding the critical roles of Th1 CD4þ T-cells, CD8þ T-cells, gd T-cells, macrophages and polymorphonuclear leukocytes in host defense against mucosal and systemic candidiasis. Most recently, the rapid development and availability of transgenic and knockout mice
Table 1 Clinically relevant animal models for the analysis of Candida host–pathogen interactions. Clinical form of candidiasis
Predisposing factor
Experimental model Animal
Immune suppression or alteration
Route of infection
Mucosal Oro-pharyngeal
Glucocorticoids Irradiation HIV-infection
Normal adult mice Normal adult mice Normal adult mice Transgenic mice
Cortisone Irradiation Murine AIDS AIDS-like disease
Oral Oral Oral Oral
[13,14] [15,16] [17] [18]
Gastrointestinal
HIV-infection
Infant mice Transgenic mice Normal adult mice
Murine AIDS AIDS-like disease Irradiation, bone marrow transplantation
Oral-intragastric Oral Oral-intragastric
[19] [18] [20]
Bone marrow transplantation
References
Vaginal
Estrus Estrus Estrus
Macaques Normal adult mice Normal adult rats
Estrogen Estrogen Ovariectomy þ estrogen
Vaginal Vaginal Vaginal
[21] [22,23] [24–27]
Systemic
Neutropenia
Normal adult rabbits Normal adult mice Infant mice SCID mice
Intravenous Intravenous Oral-intragastric Oral
[28,29] [30] [11,12] [31,32]
Bone marrow transplantation
Normal adult mice
Cytosine arabinoside Cyclophosphamide Cortisone þ cyclophosphamide Anti-granulocyte monoclonal antibody Irradiation, bone marrow transplantation
Intravenous
[20,33,34]
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326 Host–microbe interactions: fungi
Table 2 Experimental models of candidiasis in animals with intact immunity or targeted immune defects for the analysis of Candida host–pathogen interactions. Clinical form of candidiasis
Immune defect
Mucosal Oro-pharyngeal
None Innate and/or adaptive immunity
Experimental model
References
Animal
Immune suppression or alteration
Route of infection
Normal adult mice nu/nu mice bg/bg nu/nu mice
None T-cells PMNs þ NK cells þ T-cells T-cells, NK-cells
Oral Oral Oral
[35–38] [39–41] [42,43]
Oral
[43–46]
Transgenic mice Gastrointestinal
Innate and/or adaptive immunity
nu/nu mice bg/bg nu/nu mice
T-cells PMNs þ NK-cells þ T-cells
Oral Oral
[41] [42]
Vaginal
Adaptive immunity
nu/nu mice Knockout mice
T-cells TCR, or CD4þ cells
Vaginal Vaginal
[47] [47,48]
Systemic
None
Normal adult mice Normal adult mice Congenitally immuno-deficient mice SCID mice bg/bg nu/nu mice
None None
Intravenous Intra-peritoneal
[9,26,27,30,49–53] [54,55]
T- and B- cells PMNs þ NK-cells þ T-cells Cytokines TCR B-cells C3 Fas molecule TLRs
Intravenous Oral
[9] [42]
Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous
[56–60] [61] [62] [63] [30] [64]
Innate and/or adaptive immunity
Knockout mice
C3, complement component C3; PMNs, polymorphonuclear leukocytes; TCR, T-cell receptor; TLRs, Toll-like receptors.
with targeted immune defects has prompted a more closely focused assessment of the role of specific components of the host immune response to C. albicans. Although they can provide significant insights into the pathogenesis of candidiasis, these approaches using congenitally immunodeficient mice or the depletion of specific cell populations or cytokines in intact or knockout mice have significant potential limitations. First, by selectively probing discrete components of the host immune response to C. albicans, the role of only single or a few factors potentially involved in protection against Candida can be assessed in these animal models, and their involvement may be masked or underestimated by intact redundant defense mechanisms. Second, these animal models are of limited use to analyze the broad range of immune perturbations which favor candidiasis in clinically relevant immunocompromised hosts. Nevertheless, it may be possible in the future to combine these complementary approaches by establishing clinically relevant models of candidiasis with targeted knockouts of specific components of host immune mechanisms. In order integrate data on Candida pathogenesis from different laboratories and minimize experimental variation, these animal models should be selected and performed according to procedures appropriate to the pathogenic mechanism under study. Current Opinion in Microbiology 2004, 7:324–329
In vivo assessment of putative Candida virulence factors Animal models are also being widely employed to determine the role of putative Candida virulence factors in the pathogenesis of candidiasis, by demonstrating both virulence gene expression of wild-type strains in vivo as well as loss of virulence of isogenic null mutants of the gene of interest prepared by the Ura-blaster technique. A majority of assessments of isogenic null mutants have been conducted by comparing differences in survival and organ burdens of C. albicans to those of wild-type strains, after intravenous inoculation of normal adult mice. Less frequently but of greater potential relevance to the pathogenesis of candidiasis, the virulence of null mutants and wild-type strains have been compared in clinically relevant models of candidiasis. A rigorous re-evaluation has now brought into question the use of URA3 as a selectable marker for disruption and virulence assessment of C. albicans genes in animal models of candidiasis [49,65,66]. The URA3 gene of C. albicans encodes orotidine 50 -monophosphate (OMP) decarboxylase, which catalyses the conversion of OMP to uridine 50 -monophosphate, the last step in the de novo pyrimidine biosynthetic pathway. Ura-auxotrophic mutants of C. albicans are avirulent after intravenous www.sciencedirect.com
Animal models in the analysis of Candida host–pathogen interactions de Repentigny 327
inoculation of normal mice, suggesting that within the uridine-poor environment of mouse tissues, small differences in expression of URA3 may affect virulence. Indeed, a survey of C. albicans strains with Ura-blastergenerated genetic disruptions revealed variably reduced OMP decarboxylase activity compared to that of the wildtype, suggesting a positional effect on the level of URA3 gene expression [65]. In addition, the level of expression of URA3 affects the adhesion properties of C. albicans [67]. These findings therefore suggested that the decreased virulence observed in strains constructed with the Urablaster cassette cannot accurately be attributed, in all cases, to the targeted gene disruption [65]. In elegant studies re-evaluating the role of the HWP1 gene of C. albicans in systemic candidiasis, it was shown that this gene indeed contributes to the virulence of C. albicans, but that the genomic location of the URA3 selectable marker may influence URA3 gene expression and therefore confound interpretation of the role of the virulence gene of interest using the Ura-blaster technique [49,66]. This same group went on to show that confounding effects caused by differing genetic contexts of the URA3 gene can be avoided by placing URA3 at the same genomic location in the strains to be compared [49,66]. These limitations of Ura-blaster technology and the URA3 marker have recently led to the development of a GAL1 selection system in which the deletion of one or both copies of GAL in the C. albicans genome does not affect virulence in an intravenous-inoculation model of systemic candidiasis in normal mice [68]. GAL1 is thus an attractive alternative to URA3 that may be increasingly employed as a selection marker to study gene function in C. albicans [68]. Alternatively, homozygous gene deletions in C. albicans can now be generated by a PCR-based method that obviates the need to first clone a gene before its disruption ([69]; reviewed in [70]). Sequencing of the C. albicans genome has recently led to the construction of whole-genome DNA microarrays for in vitro transcription profiling of C. albicans after exposure to azole antifungals [71] or while undergoing the yeast-tohyphal transition [72]. The ability to also apply genomewide microarray analysis of C. albicans gene expression to experimentally infected animals could potentially provide a broad understanding of the adaptation of the fungus and expression of the required virulence determinants at each step of colonization and infection. Proof-ofprinciple for such an approach has recently been achieved by Fradin et al. [73], who were able to identify unique sets of different fungal genes specifically expressed at different stages of exposure of C. albicans to human blood. Differentially expressed genes included those that are involved in the general stress response, anti-oxidative response, glyoxylate cycle, as well as putative virulence attributes. In addition, numerous C. albicans genes that were highly expressed in human blood were also detected in the blood of normal adult mice infected intravenously www.sciencedirect.com
[73]. These included genes encoding enzymes of the protein synthesis machinery, carbohydrate metabolism, hyphal-specific genes and stress response genes.
Conclusions The power of analysis of Candida host–pathogen interactions using animal models of candidiasis has made a quantum leap forward in the genomic era, with the concurrent development of clinically relevant animal models, construction of C. albicans mutants with targeted mutations of putative virulence factors, the application of microarrays and other emerging technologies [70] to comprehensively assess C. albicans gene expression in vivo, and construction of transgenic and knockout mice to simulate specific host immunodeficiencies. The opportunity to combine these powerful tools to analyze tissue-specific Candida–host interactions, either as a commensal or as a pathogen, will yield an unprecedented wealth of new knowledge on the molecular and cellular pathogenesis of candidiasis.
Acknowledgements The author is supported by a grant from the Canadian Institutes of Health Research HIV/AIDS Research Program (HOP-41544), and wishes to thank Sylvie Julien for expert manuscript preparation.
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