For C. albicans, the relationships between factors that contribute to the three states of existence in the host, (1) low-level asymptomatic persistence, i.e. colonization, in the gastrointestinal tract among inhibitory microbial flora, (2) frequent occurrence of asymptomatic oral and vaginal carriage, and (3) virulence in immunocompetent and immunodeficient hosts, are poorly understood.
In this study, the presence of HWP1 mRNA was found to be correlated with the presence of C. albicans in both asymptomatic carriers and in cases of candidiasis at oral and vaginal sites. In the oral cavity, 37/40 individuals with candidiasis and 28/29 carriers were positive for HWP1 mRNA by the radioactive RT-PCR, whereas 59/59 vaginal samples from carriers and candidiasis cases were positive. RT-PCR results for ACT1 and HWP1 were uniformly negative for the culture-negative oral and vaginal control samples.
To quantitate the levels of HWP1 mRNA during oral candidiasis, real-time PCR was performed on samples (five per group) from individuals with EC or PC. EC is characterized by a reddening of tissue, but not by the raised white aphthae, consisting of mixtures of fungi and epithelial cells, that are found in PC. An HWP1 message was detected in all samples, with levels that were two- to 20-fold lower than those of the ACT1 message. Equivalent levels of HWP1 mRNA were found in individuals with PC or EC [PC, median transcript level of 106 (interquartile ranges 106–188); EC, median 128 (interquartile ranges 128–138), relative to an arbitrary transcript level of 1000 for ACT1]. Quantification of HWP1 mRNA from carriers by real-time PCR was attempted, but was inconsistent and non-reproducible, probably because the amount of message was below the necessary threshold for reliable detection by this method (results not shown).
The RT-PCR results showed that carriers and candidiasis cases were equivalent in terms of the expression of HWP1
. Furthermore, the five oral specimens in which HWP1
mRNA was not detected were derived from both groups, indicating that cases were not more likely to have HWP1
mRNA than candidiasis carriers. In this respect, HWP1
gene expression results are similar to frequencies seen for SAP2
, which are the SAP
genes most commonly expressed during candidiasis and carriage (Naglik et al., 1999
). However, in the case of HWP1
, the results suggested an increased level of HWP1
mRNA in candidiasis cases. HWP1
mRNA in candidiasis probably exceeded that in asymptomatic conditions, based on the uniformly positive results of RT-PCR in samples from symptomatic, but not asymptomatic, infections. Thus the presence of HWP1
gene expression appears to parallel the tissue burden, as reflected in the higher levels of c.f.u. in the symptomatic group.
Superimposed upon questions regarding mechanisms of persistence in health are the relative contributions of yeasts, hyphae and pseudohyphal growth forms of C. albicans
to disease, to carrier states on mucosal surfaces, and to colonization in the lower gastrointestinal tract. The enhanced adherence and invasive properties of hyphal forms relative to yeast forms (Sundstrom, 2006
), and the attenuated virulence of mutants unable to form hyphae (Lo et al., 1997
), support the belief that equates hyphal forms with invasiveness and yeast forms with commensalism; however, little experimental support exists for this dogma today. Hyphae, pseudohyphae and yeast are found in the same tissue specimens in candidiasis (Sundstrom, 2006
), and hyperfilamentous mutants are also attenuated in virulence (Laprade et al., 2002
; Bahn et al., 2003
), leading to the view that reversible bud–hypha transitions are required for candidiasis. Demonstration of growth morphology during gastrointestinal tract colonization and the carrier state in health is not possible, because of the small numbers of fungi amongst large numbers of normal bacteria in sites that are not easily amenable to specimen collection.
mRNA is abundant in hyphae compared to yeast, an implication of the presence of HWP1
mRNA in carriers is that hyphal forms are present in the host in the absence of symptoms. This agrees with our previous report, which concluded that hyphae are probably present in most samples, based on widespread expression of SAP4
(Naglik et al., 2003
). However, HWP1
mRNA may also arise from pseudohyphal growth forms (Snide & Sundstrom, 2006
), or possibly from yeast forms, although experimental data to support the presence of HWP1
mRNA in yeast forms has not been reported.
The high frequency of HWP1 gene expression in oral and vaginal specimens from carriers strongly implicates Hwp1 and hyphae in the ability of C. albicans to establish and maintain its presence on mucosal surfaces of human hosts. The results do not support a predominance of yeast forms with absent hyphal forms in the asymptomatic carrier state or in seropositive individuals who lack C. albicans in oral or vaginal specimens. It is also unlikely that PC and EC differ with respect to the morphological composition of C. albicans in the tissue, since the measurement of HWP1 gene expression by real-time PCR in EC versus PC showed equivalent levels in both groups.
Specific host determinants that contribute to persistence and candidiasis are also poorly understood. It is not known whether loss of host immunity alone is enough to permit candidiasis, or if upregulation of specific virulence attributes is required. Known immune responses in healthy adults include humoral antibodies to polymannose surface proteins, and cell-mediated immunity to candidal antigens, as demonstrated by positive delayed-type hypersensitivity skin-test reactions to undefined factors in crude extracts that develop early in life (Shannon et al., 1966
; Munoz & Limbert, 1977
; Lehmann & Reiss, 1980
To determine if healthy adults also mount immune responses to Hwp1, antibody titres in oral-culture-negative subjects were compared to those of oral carriers and candidiasis cases by ELISA of rHwp1 (Staab et al., 2004
). In contrast to HWP1
gene expression, immune responses to Hwp1 did not correlate with tissue burden in oral samples. Using the non-parametric Kruskall–Wallis analysis of variance, it was found that there were equivalent titres of serum IgG and salivary IgA anti-Hwp1 antibodies in the oral-culture-negative group as well as in the other groups, and there were equivalent titres among the groups also (). Hwp1, like polymannose, and unlike other antigens that stimulate host responses associated with candidiasis (Strockbine et al., 1984
), is a common target of host responses to C. albicans
that are recognized to result from long-term colonization. The presence of Hwp1 immune responses in healthy adults does not rule out a role for Hwp1 in the pathogenesis of candidiasis.
Salivary IgA and serum IgG median antibody titres in the three patient populations Values shown are median antibody titres, with interquartile ranges (IQRs) in parentheses.
The common occurrence of mucosal candidiasis accompanying HIV infection raises the possibility that HIV infection might have affected the antibody responses to Hwp1 seen in this study. Although some of the individuals that contributed samples to the RT-PCR results were HIV infected (Naglik et al., 2003
), no differences were found between HIV-positive and -negative groups with respect to the presence of Hwp1 mRNA, as detected by RT-PCR. It was not possible to address the effect of HIV infection on salivary anti-Hwp1 IgA levels, because patients providing saliva samples for IgA determinations were not asked about their HIV status. Moreover, those attending the Oral Medicine clinic at Guy's Hospital were deemed to be at low risk for HIV. Given the equivalent anti-Hwp1 antibody levels found in oral-culture-negative controls compared to those of other groups, it is unlikely that the presence of HIV affected the anti-Hwp1 antibody results in this study.
Like the virulence attributes of bacterial pathogens that establish long-term associations with human hosts (Falkow, 2006
), Hwp1 appears to be important both in establishing and maintaining the commensal state, as shown by the universal presence of adaptive immune responses, and in candidiasis, as shown by the probable increase in gene expression in this study, by animal studies (Sundstrom et al., 2002
), and by in vitro
adherence assays (Staab et al., 1999
). Developmental regulation of Hwp1 implicates hyphal and pseudohyphal forms in both commensalism and candidiasis. C. albicans
serves as a model organism to elucidate fungal determinants that play a dual role in the establishment of long-term, permanent associations with human hosts, and in tissue invasion leading to disease.