Robert Koch-Institut, Nordufer 20, D-13353 Berlin, Germany1
Division of Oral Medicine, Pathology, Microbiology and Immunology, Kings College London (Guys Campus), London, UK2
Author for correspondence: Bernhard Hube. Tel: +49 30 4547 2116. Fax: +49 30 4547 2328. e-mail: HubeB{at}rki.de
Keywords: SAP, virulence factors, fungal infections
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Overview |
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Opportunistic fungal infections: from commensal to pathogen |
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Virulence attributes of C. albicans |
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The virulence factors expressed or required by C. albicans to cause infections may well vary, depending on the type of infection, the stage and site of infection, and the nature of the host response. Thus, C. albicans must be highly adapted to an existence on and within the host, which indicates that this fungus possesses virulence attributes distinct from those of the closely related, but non-pathogenic yeast Saccharomyces cerevisiae. Although a number of potential virulence factors have been suggested for C. albicans, cell morphology, adhesion factors, phenotypic switching and extracellular lipolytic or proteolytic activity have long been recognized as the most credible (Odds, 1994 ). Extracellular proteolytic activity had already been discovered in the mid-sixties (Staib, 1965
) but it was not until the early nineties, when molecular methods were introduced into the Candida field, that scientists began to understand the genetic complexity of this fungus. For instance, a gene (SAP1) encoding an extracellular proteinase was cloned in 1991 and was thought to be responsible for the observed secretory aspartic proteinase (Sap) activity of C. albicans (Hube et al., 1991
). However, a detailed study of the C. albicans genome in recent years indicated that SAP1 was just the tip of an iceberg and revealed that the fungus possesses an arsenal of ten SAP genes that encode extracellular proteinases. The fact that C. albicans can encounter a large number of different tissues during the infection process in vivo may provide clues as to why it has evolved to possess such a large repertoire of SAP genes.
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The SAP gene family |
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Possible target proteins of C. albicans proteinases in vivo |
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C. albicans Saps may also act on host proteolytic cascades with numerous effects, which have no obvious advantage for the fungus. For example, Sap2 can activate host protein precursors of the blood clotting cascade (Kaminishi et al., 1994 ), inactivate the epidermal cysteine proteinase inhibitor cystatin A (Tsushima et al., 1994
), and cleave human endothelin-1 precursor (a vasoconstrictive peptide) to alter vascular homeostasis (Tsushima & Mine, 1995
). Such activities may be responsible for the enhanced overall circulating proteolytic activity observed in traumatized mice challenged with C. albicans (Neely et al., 1994
). Furthermore, Candida proteinases have been shown to activate the proinflammatory cytokine interleukin-1ß from its precursor, suggesting a role for Saps in the activation and maintenance of the inflammatory response at epithelial surfaces in vivo (Beausejour et al., 1998
).
These studies suggested that Sap2, in contrast to the highly substrate-specific enzymes produced by certain bacteria, has very broad substrate specificity and may have multiple targets in vivo. However, this raises the question of why, if a single proteinase has such a range of activities and functions, does C. albicans need a family of ten SAP genes? Magee et al. (1993)
postulated that the Sap isoenzymes might have a variety of functions in vivo, which may be called upon at different sites, and during different stages and types of C. albicans infection.
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SAP gene expression in vitro and in vivo |
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Under most proteinase-inducing conditions in the laboratory, the major proteinase gene expressed in C. albicans yeast forms is SAP2, which was found to be regulated by a positive feedback mechanism: peptides resulting from proteolysis of high-molecular-mass proteins were proposed to lead to the induction of SAP2 gene expression (Hube et al., 1994 ). In contrast, SAP1 and SAP3 were discovered to be differentially expressed during phenotypic switching in certain strains (Morrow et al., 1992
; White et al., 1993
). However, later studies indicated the regulation of SAP3 during switching was not absolute (Hube et al., 1994
; White & Agabian, 1995
; Smolenski et al., 1997
). Expression of SAP8 is temperature-regulated (Monod et al., 1998
) and SAP9 and SAP10 are constitutively expressed under most environmental conditions in both yeast and hyphal forms (A. Felk, W. Schäfer & B. Hube, unpublished results). Since most aspartic proteinases are only active under acidic conditions, it was perhaps a surprising discovery that the SAP46 genes were almost exclusively expressed during hyphal formation at neutral pH, even in defined protein-free media (Hube et al., 1994
; White & Agabian, 1995
). These studies demonstrated that the SAP gene family was differentially expressed in vitro and further suggested that, in contrast to the induction of SAP2, expression of other SAP genes was not dependent on the presence of exogenous protein or peptides.
The in vitro demonstration of distinct SAP expression patterns in yeast, hyphal and phenotypically switched cells indicated that proteinase expression was a highly regulated and tightly controlled process. However, this did not address the issue of whether the Sap family contributed to the pathogenicity of C. albicans in vivo. For this reason it was crucial to first ascertain whether the proteinases were also differentially expressed during C. albicans infections; this was demonstrated using in vitro and animal infection experimental models.
In vitro experimental models of oral (Schaller et al., 1998 ) and cutaneous (Schaller et al., 2000
) C. albicans infections suggested that SAP13 were the main proteinases expressed during superficial infections. This supposition was supported by the detection of SAP1 and SAP2 transcripts in a rat vaginitis model (De Bernardis et al., 1995
). In contrast to mucosal infection models, experimental models of systemic C. albicans infections correlated SAP46 expression with systemic disease (A. Felk and others, unpublished results; Staib et al., 2000
). However, using in vivo expression technology, Staib et al. (1999)
also demonstrated SAP2 expression in late stages of systemic infections. Taken together, these in vitro experimental and animal model data correlated SAP gene expression with C. albicans virulence and demonstrated differential SAP gene expression during different types of C. albicans infection.
Whether these models were representative of proteinase expression during human mucosal and systemic infections was not known. However, high titres of anti-Sap antibodies have been observed in sera of candidosis patients, indicating the presence of Sap antigen during infection (Rüchel, 1992 ). In addition, Sap antigens have been detected in biopsies of oral epithelial lesions collected from HIV-infected patients (Schaller et al., 1999a
) and in almost all organs of immunocompromised patients who had died of systemic C. albicans infections (Rüchel et al., 1991
). Nevertheless, only two studies have investigated the expression of the SAP gene family during human C. albicans infections. Schaller et al. (1998)
showed that the SAP genes were indeed expressed during oral candidosis. Moreover, Naglik et al. (1999)
and J. R. Naglik and others (unpublished results) were able to demonstrate differential expression of the SAP gene family, with SAP1, SAP3 and SAP7 transcripts predominantly being expressed in patients with oral C. albicans infection as opposed to oral C. albicans carriers.
Therefore, differential expression of the proteinase family has been demonstrated in culture media, during in vitro and animal experimental infection models and during human C. albicans infections. Such differential expression suggested a more specific role or function for the different SAP genes. However, although these studies correlated SAP gene expression with the ability of C. albicans to cause infection, they did not directly demonstrate that Sap proteinases actually contributed to the pathology or virulence of C. albicans infections.
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Contribution of Saps to the pathogenesis of C. albicans infections |
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Although Sap2 was the dominant C. albicans extracellular proteinase in vitro, the in vivo expression pattern of SAP genes suggested that Sap2 was not the only proteinase and certainly not the most dominant proteinase acting in vivo. Therefore, to determine to what extent the different Saps contributed to the different types of Candida infection, mutants lacking (Table 1) or overexpressing (Dubois et al., 1998
; Kvaal et al., 1999
) distinct SAP genes were created and used in experimental infections. These studies showed that not only were distinct Sap isoenzymes required during different types of infection, but also that not all Candida proteinases were important for virulence per se (Dubois et al., 1998
). Using SAP-deficient mutants, Schaller et al. (1999b
) demonstrated that SAP13, but not SAP46, contributed significantly to experimental C. albicans infections of artificial oral mucosa. A major role for SAP13, but not SAP46, was also demonstrated in experimental vaginal infections using SAP-disrupted mutants (De Bernardis et al., 1999
). Using a gene misexpression strategy in the switching strain WO-1 in which white-phase cells misexpressed the opaque-specific gene SAP1, Kvaal et al. (1999)
demonstrated in a cutaneous mouse model that SAP1 caused a dramatic increase in cutaneous infection, probably as a result of increased adherence to, and cavitation of, the skin.
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These data clearly demonstrate that Saps contribute to the pathogenesis of Candida infections. However, it should be noted that mutants lacking individual SAP genes rarely exhibit a full avirulent phenotype in a particular infection model. This may indicate a synergistic effect of a number of proteinases during certain infections, or the involvement of other virulence factors. Nonetheless, these studies illustrate that not all of the proteinases are required at the same time or stage of the infection process and not all contribute to the same types of infection. Determining the precise roles and functions of the C. albicans proteinases in vivo should provide a big step forward in identifying which proteinases, or set of proteinases, may contribute to, or even be responsible for, specific types of C. albicans infection.
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Why does C. albicans possess a SAP gene family? |
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Firstly, the different Sap isoenzymes may have adapted and evolved to function in different tissues and environments. Such a view may also explain how the gene family evolved: C. albicans may have duplicated a successful factor (an ancestral proteinase), which in turn adapted to different host environments and thus evolved into homologous but functionally distinct Sap isoenzymes. This hypothesis may be supported by the fact that all the SAP genes encode similar amino acid sequences (Fig. 1). However, since only SAP1 and SAP4 are clustered in tandem and all other SAP genes are located on five different chromosomes, this gene duplication event probably occurred sometime in the distant past. Alternatively, it is possible that the high genetic flexibility of C. albicans may have accelerated this widespread distribution of the SAP genes.
Although the overall similarity of the SAP genes is high, differences in their promoter sequences indicate that expression of the various SAP genes is controlled by different SAP-specific transcriptional regulators. This would suggest that the SAP genes might have evolved to possess distinct properties and functions, which indeed appears to be the case. For example, different Saps have been shown to have different pH optima for activity (Borg-von Zepelin et al., 1998 ). Sap2 acts mainly at acidic pH values around pH 4·0, Sap46 are significantly active at physiological pH and Sap3 still has activity at pH 2·0. This provides C. albicans with a range of proteolytic activity from pH 2·0 to 7·0, a property that may prove essential for the specific adaptation of individual Saps to different host environments.
It seems surprising that all secreted proteinases of C. albicans are aspartic proteinases and that neither extracellular serine-, metallo- nor cysteine proteinases have been identified in pathogenic strains of C. albicans. In contrast, other human pathogenic fungi, such as the filamentous fungus Aspergillus fumigatus, secrete several classes of different proteinases, including aspartic, serine and metalloproteinases, although none of these enzymes has been definitively associated with virulence (Monod et al., 1999 ). This may disadvantage C. albicans since aspartic proteinases (with a few exceptions, such as human renin) are usually only active in acidic pH ranges. However, this may be compensated for not only by the adaptation of particular Sap isoenzymes to function at higher pH values, but also by the ability of C. albicans to actively acidify its surrounding environment to provide microniches optimal for Sap activity during infection. These properties may be essential for the fungus when infecting the vaginal mucosa or the oral cavity.
Secondly, the C. albicans SAP gene family may have evolved to allow coordinated regulation of the proteinases with other virulence factors. This may not only explain why certain SAP genes are induced in the absence of exogenous protein or peptides, but also the differences between the SAP promoter sequences. Key transcriptional factors would permit the expression of specific Saps when C. albicans needs to call upon other virulence factors or when it encounters different host environments. Therefore, the same transcriptional factor may regulate several genes to allow the simultaneous expression of a combination of virulence factors to respond to local environmental challenges. For example, SAP46 were found to be regulated during the yeast-to-hyphal transition, which in turn is known to be regulated by key transcriptional factors such as Efg1 (Ernst, 2000 ). It is currently not precisely known why mutants lacking EFG1 are avirulent in several infection models. However, it seems that Efg1 is a global regulator not only of hyphal formation, but also of a number of other virulence attributes, including proteinase expression (Schröppel et al., 2000
). A lack of this factor would subsequently cause severe defects in the virulence potential of C. albicans. In addition, SAP1 and SAP3 were shown to be regulated during phenotypic switching in particular strains, which involves the rapid change of a large number of different phenotypes and is presumably regulated via a master switch mechanism (Soll, 1997
). This specific coordinated regulation of the SAP gene family with various virulence factors indicates that the proteinases may have evolved to specifically enhance the pathogenic ability of C. albicans.
A third possible reason for a family of proteinase genes is that the concomitant expression of a number of similar but functionally distinct SAP genes, rather than the expression of a single SAP gene, may result in a synergistic effect to promote colonization or infection. Thus, several Saps may act in unison to carry out a series of tasks to possibly provide C. albicans with a biological advantage.
Finally, providing an arsenal of SAP genes has the advantage of having a second enzyme in line when one fails (i.e. the enzyme may be functionally redundant), is removed or is otherwise lost. There is evidence for such compensatory mechanisms in C. albicans. For instance, disruption of SAP1 and SAP3 genes, which may be required for oral infection, lead to increased expression of SAP5 and SAP8, suggesting that C. albicans may be attempting to compensate for the loss of key genes by upregulating alternative genes (Schaller et al., 1999b ). Such balanced regulation is likely to be a general phenomenon in C. albicans.
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Conclusion and future directions |
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However, there are still many unanswered questions that need to be addressed. (1) How is proteinase expression regulated? The signal transduction pathways that control the differential expression of SAP genes are currently unknown and no receptor has yet been implicated in the regulation of C. albicans proteinase secretion. (2) What are the actual targets of Saps during human infections? Although the possible targets of the proteinases have been deduced from in vitro studies, these need to be confirmed in the in vivo environment. Furthermore, as the substrate specificity of the Saps is so broad and most Sap antigen is detected within the C. albicans cell wall (Rüchel et al., 1991 ; Schaller et al., 1999a
), how does the fungus manage to protect itself from proteolytic digestion? (3) The precise roles and functions of the SAP genes, especially SAP7, SAP9 and SAP10, during human infections are currently unknown, as is the reason why Sap9 and Sap10 have C-terminal GPI-anchoring sequences. Do these two proteinases have functions similar to the GPI-anchored yapsins of S. cerevisiae (Komano et al., 1999
) or do they contribute to C. albicans infection? (4) The immunological consequences of Sap secretion and other interactions of the proteinases with host factors in vivo are largely unknown and need to be addressed.
In summary, this review has endeavoured to elucidate the possible reasons why C. albicans is equipped with a family of proteinase genes and suggests a number of possible explanations for their evolution in light of the information available. However, there are several shortfalls that need to be tackled before the biological significance of this gene family is fully understood. With access to the complete genome sequence of C. albicans and with the development of new and exciting tools such as DNA microarray analysis, our understanding of the SAP gene family and its interaction with the human host should rapidly increase.
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ACKNOWLEDGEMENTS |
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