Oral and vaginal candidiasis are two of the most common forms of human fungal infections and detailed investigations into these complex diseases are greatly facilitated by the use of animal models. Here, we report the creation of a new low-estrogen murine model of concurrent oral and vaginal C. albicans colonization that resembles human candidal carriage, which reduces the numbers of experimental mice by half, allows the study of delicate host–pathogen interactions during the natural state of C. albicans colonization, and facilitates the assessment of potential mucosal vaccine candidates.
One of the unique features of our model is that Candida
‘colonization’ rather than ‘disease’ can be established at two mucosal sites simultaneously in the same mouse. The concept of ‘colonization’ versus ‘infection’ (or ‘disease’) is an important issue. Some regard ‘infection’ as similar to ‘colonization’ in that the simple presence of an organism signifies that someone is ‘infected’, irrespective of any symptomatic evidence. However, most regard ‘infection’ as similar to ‘disease’, where clinical symptomatic or histological evidence (signs, symptoms, invasion, cell damage, host immune cell infiltrates) are present in addition to colonization. In this paper, ‘colonization’ is defined as the presence of fungal cells demonstrated histologically and by culture but without (a) pseudomembranous or erythematous lesions, (b) signs of epithelial damage, (c) deep epithelial penetration by hyphae, or (d) PMN/lymphocyte infiltration. Nearly all current oral or vaginal murine Candida
models establish a ‘disease’ state at one or other site and are based on immunosuppressive therapies or high estrogen doses [1
]. In our model there is no evidence of these four criteria (), strongly indicating a ‘colonization’ rather than a ‘disease’ phenotype.
In respect of our vaginal data, the low levels of estrogen used (10 μg weekly/mouse) are in contrast to most current murine models of vaginal candidiasis that typically use between 100 and 500 μg estrogen weekly/mouse [7
]. Although these high estrogen levels permit the establishment of persistent vaginal infections with relatively high fungal burdens, they are also likely to have undesirable host effects and significantly influence the defense response [19
]. In support of this, when we administered pilocarpine (to stimulate saliva flow for antibody studies) to mice given 100 μg of estrogen, we often observed severe vaginitis and secondary bacterial infections, and occasionally death. In our low estrogen model, no adverse health effects to pilocarpine were observed and the mice remained healthy. This indicates a less profound effect of estrogen in our model and, therefore, probably a more relevant host physiological and immunological response to the fungus when colonized.
In respect of our oral data, low-dose estrogen appeared to enhance oral colonization, although for C. albicans
529L the oral fungal burdens were less affected by estrogen than vaginal burdens, which were estrogen-dependent (). This was demonstrated by the ability of C. albicans
529L to orally colonize estrogenized mice for at least 5 weeks () but not non-estrogenized mice (data not shown). Low estrogen doses may subtly alter the finely balanced host–fungus equilibrium at oral sites to promote fungal adherence and colonization by acting upon the host or the pathogen [19
]. For example, the action of estrogens may affect estrogen receptor (ER)-β expression, the predominant ER subtype present in human oral tissues, and/or saliva secretion and composition [20
]. In addition, C. albicans
produces an estrogen binding protein (EBP) [21
] and estrogens may thus enhance fungal colonization directly [22
]. Recently, it has been suggested that the mechanism for estrogen-sensitive vaginal colonization by C. albicans
includes a functional ligand-EBP interaction within the yeast [23
Fungal burdens were noticeably higher both orally and vaginally with the fresh clinical strains 529L as compared with laboratory strains SC5314 or 3153 ( and ) and persisted for up to 6 weeks (). Oral colonization burdens were generally 1–2 log higher (102
CFU per ml saliva or per oral or vaginal swab) than previously described oral models without immunosuppression [12
], which are compatible to C. albicans
carriage levels found in humans (although there is more variability vaginally) [24
]. Given that C. albicans
virulence might be tissue specific and dependent upon a strains origin [26
], one reason why 529L exhibited enhanced colonization and persistence over the laboratory strains SC5314 or 3153 might be that 529L is a fresh human oral isolate. Thus, 529L might be innately adapted to the in vivo
environment and may naturally express more relevant adherence/virulence moieties that enhance mucosal colonization. This hypothesis was partially supported by additional experiments, whereby although fresh clinical isolates consistently colonized initially, by week 5 only 2/6 colonized one or both mucosal sites at similar levels to 529L (). However, another laboratory strain (NCPF 3516) was as effective as the two additional clinical strains at establishing colonization. Nonetheless, colonization for 5 weeks or longer by fresh human isolates was greater (n
= 3/7, with higher CFU counts) than by laboratory strains (n
= 1/3, with lower CFU counts). Further evidence for in vivo
adaptation is supported by the passaging experiments, whereby passaged SC5314 colonized both mucosal sites in all animals for up to three weeks (). An alternative explanation that primary infection makes the mice more susceptible to secondary challenge seems less likely since reinoculation by non-passaged strains did not lead to enhanced colonization. Together, the data suggest that although fresh isolation of a strain from an in vivo site may enhance mucosal colonization, it is not a fundamental criterion in order to establish colonization.
Since SC5314 is the wild-type strain used in the majority of gene mutation studies to assess virulence phenotypes, our model using passaged SC5314 provides an option of using this strain to investigate the effect of gene disruption on mucosal colonization at both anatomical sites over a 3-week period. However, detailed analysis of persistent colonization (up to 6 weeks) will be limited with SC5314. On the other hand, our model could be potentially very useful in studying host–pathogen colonization interactions of fresh clinical isolates, which may be more relevant than studying laboratory isolates. Moreover, advances in gene disruption techniques now allow the relatively quick generation of null mutants in clinical wild-type strains; therefore, our model could be widely utilized to study C. albicans colonization in clinically relevant strains if desired.
One of the primary objectives of this study was to establish a concurrent mucosal colonization model of sufficient longevity that could be utilized for immunization studies to study the efficacy of potential vaccine candidates. We found that immunization with Sap2, a known virulence factor of C. albicans
associated with mucosal infection [18
] significantly reduced fungal burdens both orally and vaginally (P
< 0.001 and P
< 0.0001, respectively) (). This finding corroborates previous studies [28
] demonstrating that Sap2 is an immunogenic antigen capable of inducing protective responses against C. albicans
colonization and infection, and tentatively supports its targeting as a potential vaccine candidate. The data demonstrate proof-of-principle that our concurrent mucosal model can be used for both immunological and potential vaccine studies and permits the detailed analysis of host–fungal interactions during the natural state of Candida