In this paper, we present the first characterization of a small heat shock protein in C. albicans and demonstrate its requirement for intracellular stress protectant homeostasis, environmental stress adaptation, Cek1 phosphorylation, host-pathogen interactions and virulence of this major human fungal pathogen.
As yet, few investigations have focused on the role of small Hsps (sHsps) in microbial pathogenicity. However, it is known that expression levels of sHsps generally increase in response to environmental stresses 
. Therefore, sHsps may play an important role during microbial infection. Indeed, the novel sHsp encoding C. albicans
gene orf19.822 (HSP21
) has been shown to be strongly upregulated under such environmental stress conditions, including thermal, oxidative and acetic acid stress as well as in several models of infection (Table S1
). sHsps are defined by a central α-crystallin domain, flanked by a variable C-terminal extension and a non-conserved N-terminal arm and are phylogenetically, structurally and functionally distinct from classical HSPs. In silico
analysis of the Hsp21 amino acid sequence revealed the presence of such an sHsp-typical core α-crystallin domain, flanked by C- and N-terminal regions. Strengthening this finding, promoter analysis led to the detection of two heat shock elements (HSEs), one non-standard HSE (nHSE) as well as one stress responsive element (STRE). It has recently been shown that transcription of Hsp-encoding genes, such as HSP70
, is regulated by binding of the heat shock transcription factor Hsf1 to HSEs in C. albicans
, specifically in response to thermal stress. The nHSE, on the other hand, was shown to be non-functional 
. It is therefore likely that expression of HSP21
may also be regulated by Hsf1 or other heat shock transcription factors via the HSEs in its promoter. The role of the nHSE remains unclear, although it might be important for HSP21
expression under stress conditions other than heat shock. During exposure of C. albicans
wild type cells to weak acid stress HSP21
is amongst the most strongly induced genes and it has been proposed that HSP21
expression is regulated by Mnl1 
. However, in contrast to Mnl1, Hsp21 was not required for resistance to acetic acid stress (data not shown).
HSP21 has no orthologue in the non-pathogenic yeast S. cerevisiae. Indeed, sequence similarities to Hsp21 on the protein level were detected exclusively for four uncharacterized proteins in fungal species belonging to the CUG clade, which translate this codon to serine instead of leucine. Interestingly, the first three best hits were found in C. dubliniensis, C. tropicalis and C. parapsilosis (), which are pathogenic fungi, indicating that Hsp21 orthologues may play a role in the virulence of these non-albicans species. The remaining protein, with the lowest homology (Hsp18) belonged to the non-pathogenic yeast Pichia stipitis. The relatively low identity of 39% might point to a divergent function in this yeast.
Interestingly, despite robust transcriptional induction of HSP21
upon heat shock 
, a hsp21
Δ/Δ mutant displayed only moderate sensitivity to short-term heat shock. This phenomenon is reminiscent of the S. cerevisiae HSP70
mutant, which has a similar phenotype, i.e. a growth defect at higher temperatures but wild type tolerance to short-termed heat shocks 
. C. albicans
Hsp21 might therefore have comparable functions to Hsp70 or cooperate with C. albicans
Hsp70, for example by transferring partially unfolded client proteins to the Hsp70/Hsp100 disassembling machinery. Such a cooperation has been shown to exist between the S. cerevisiae
sHsp Hsp26 and the major Hsps Ssa1 (Hsp70) and Hsp104 
. As there is no S. cerevisiae
Hsp26 orthologue in C. albicans
, it is tempting to speculate that CaHsp21 may have taken over a similar function.
Although relatively tolerant to surviving short term heat shock, hsp21
Δ/Δ was unable to grow at elevated temperatures. Moreover, HSP21
contributes to growth under oxidative and nutrient stress, but not osmotic or cell wall stress. Therefore, Hsp21 is specifically required for growth under particular environmental conditions. Heat shock proteins (including sHsps) function by binding to and stabilizing their clients, preventing their unfolding and aggregation 
. Although further studies are required to reveal the full repertoire of Hsp21 clients, we have identified the mechanistic outcome of HSP21
deletion: disrupted homeostasis of the three major cellular stress protectant molecules: glycerol, glycogen and trehalose. Osmotic stress induced strong glycerol accumulation, with simultaneous downregulation of glycogen and trehalose levels. Conversely, thermal stress did not effect glycogen levels but stimulated glycerol production and high levels of trehalose accumulation. Only oxidative stress elicited a detectable increase in glycogen levels. Cellular homeostasis of all three molecules was mis-regulated in the hsp21
Δ/Δ mutant. Osmotic stress resulted in lower glycerol induction than in the wild type, however this defect did not manifest as a higher level phenotype – hsp21
Δ/Δ grew well under osmotic stress. On the other hand, reduced glycogen levels in hsp21
Δ/Δ cells under oxidative stress correlates well with heightened sensitivity to this stress.
The dominant cellular function of Hsp21 appears to be thermal stress adaptation (). The hsp21
Δ/Δ mutant produced significantly less trehalose than the wild type under long-term elevated temperature. Trehalose is an important stress-protective molecule with chaperone-like functions and is specifically produced during heat and oxidative stress 
. Therefore, Hsp21 is involved in thermal-induced trehalose synthesis, possibly via stabilizing metabolic enzymes such as Tps1–3. Interestingly, glycerol was over-produced by hsp21
Δ/Δ cells in response to thermal stress. This directly demonstrates that, although incapable of growth, hsp21
Δ/Δ cells were metabolically active under thermal stress and indicates that Hps21 rather fine-tunes the cellular balance of stress protectant molecules in response to environmental conditions.
Model of Hsp21-dependent adaptation to elevated temperature.
In agreement with these conclusions, mutants defective in genes encoding key metabolic enzymes for the synthesis of trehalose (Tps1, Tps2) phenocopied HSP21
-deletion. The trehalose 6-phosphate synthase Tps1 and the trehalose 6-phosphate phosphatase Tps2 form a complex together with the stabilizing proteins Tps3 and Tsl1 
. A tps1
Δ/Δ mutant has previously been shown to be defective in trehalose production, hyphal formation, resistance to oxidative stress and virulence in vivo
. Interestingly, tps1
Δ/Δ did not grow at 42°C on glucose but grew normally on glycerol 
. Disruption of TPS2
in C. albicans
leads to defective trehalose accumulation, thermosensitivity, sensitivity to oxidative stress, and attenuated virulence in mice 
. However, the capacity for hyphal formation was unaffected in this mutant 
. Therefore, the thermal sensitivity of the hsp21
Δ/Δ mutant is most likely due to impaired trehalose synthesis.
Interestingly, the defect of the hsp21
Δ/Δ mutant to grow at 42°C could be completely bypassed by simultaneously applying osmotic stress. In a recent publication it has been shown for the filamentous fungus Aspergillus fumigatus
, that deletion of the UPR-regulating transcription factor HacA results in a similar phenotype, i.e. inability of a ΔhacA
mutant to grow at elevated temperatures (45°C) which is reversed by supplementation of the medium with sorbitol or KCl 
. The authors conclude that osmotic stabilization of the medium compensates for reduced cell wall integrity of the ΔhacA
mutant. However, C. albicans hsp21
Δ/Δ did not exhibit defects in cell wall integrity ( and data not shown) and mutant cells did not lyse upon thermal stress, but rather remained viable and metabolically active. Osmotic stabilization of cellular integrity, in this case appears improbable. An alternative explanation is that osmotic stress of hsp21
Δ/Δ resulted or the induction of other heat shock protein(s) (such as HSP12
), or stress responsive pathways (such as Cek1, see below) thereby compensating for the lack of Hsp21.
has been shown to be upregulated in the absence of the adenylyl cyclase Cyr1 
. Therefore, HSP21
lies downstream of the cyclic AMP pathway.
To determine which pathway(s) Hsp21 functions in, we performed a systematic Western blot analysis of the three main stress responsive MAP kinase pathways (Mkc1-, Cek1- and Hog1-mediated pathways) in C. albicans
wild type and hsp21
Δ/Δ strains under a range of stress conditions. Of the three MAP kinases, Cek1 phosphorylation in response to thermal stress was found to be Hsp21-dependent. Significantly, dual challenge of cells with osmotic and thermal stress bypassed Hsp21-dependent Cek1 phosphorylation. Therefore, the Cek1 phosphorylation state of C. albicans
directly correlates with the ability to grow under elevated temperatures. In line with this, C. albicans CEK1
has previously been shown to be induced by high temperatures 
. These data suggest that Hsp21 functions upstream of Cek1 in a temperature-responsive pathway. It remains to be investigated whether Cek1 is responsible for activation of trehalose synthesis in response to elevated temperatures.
Furthermore, Cek1 has been shown to be required for hyphal formation on solid Spider, SLAD and serum agar, and for full virulence in a mouse model of systemic candidiasis 
. These phenotypes correlate well with those observed for hsp21
Δ/Δ. In contrast to deletion of HSP21
, however, cek1
Δ/Δ was found to be unattenuated in resisting killing by neutrophils and macrophages 
, indicating that Hsp21 has further cellular functions, possibly by stabilizing additional client proteins.
Deletion of Hsp21 also affected hyphal growth and hypha-associated processes. The hsp21
Δ/Δ mutant formed shorter hyphae, smaller hyphal colonies than the wild type and exhibited reduced capacity to invade semi-solid agar. These morphological defects are likely to have contributed at least partially to the reduced damage capacity of hsp21
Δ/Δ during infection of endothelial and oral epithelial cell monolayers. Although the epithelial/endothelial adhesion and initial invasion properties of the mutant were unaffected, the reduced damage capacity of the hsp21
Δ/Δ mutant may be due to a compromized capacity to undergo subsequent inter-cellular invasion 
Importantly, the hsp21Δ/Δ mutant strain was avirulent in a mouse model of hematogenously disseminated candidiasis and displayed attenuated virulence in an alternative embryonated egg infection model. Based on our detailed functional analysis of HSP21, a number of mechanisms are likely to account for the reduced virulence of hsp21Δ/Δ.
Mutants with morphological defects generally exhibit reduced virulence in both murine and in ovo
infection models 
. Therefore, it is possible that reduced hyphal formation in vivo
may at least partially account for the mutant’s virulence attenuation.
Several studies have demonstrated a correlation between reduced capacity to damage host cells and attenuated virulence 
. Infection of embryonated eggs was performed via the chorio-allantoic membrane (CAM). The CAM is a thin, highly vascularized membrane composed of two epithelial cell layers, held together by connective tissue 
. Therefore, the reduced capacity of hsp21
Δ/Δ to damage epithelial and endothelial cells is likely to have contributed to attenuated virulence in ovo
. Similarly, following murine intravenous infection, C. albicans
must traverse the endothelial lining of blood vessels in order to infect deeper organs. It is therefore possible that the reduced endothelial damage potential of hsp21
Δ/Δ may account for decreased virulence during disseminated candidiasis.
Finally, the role of Hsp21 in adaptation to environmental stresses likely plays a crucial role in C. albicans
Δ/Δ was unable to grow at elevated temperature, exhibited greatly increased sensitivity to oxidative stress and was killed more efficiently by human neutrophils. As neutrophils play a key role in killing C. albicans
, it seems likely that the increased sensitivity of hsp21
Δ/Δ to these phagocytes contributed to the strongly reduced virulence of the mutant. Moreover, host cells and tissues are known to induce stress-defensive mechanisms, such as the generation of reactive oxygen (ROS) and/or nitrogen species (RNS). Although a febrile response is unlikely to reach the high temperatures used for in vitro
thermal stress experiments, it is likely that in vivo
, a combination of stresses act simultaneously on invading microbes. Indeed, histopathologic examinations, together with CFU counts of end-point surviving kidney homogenates demonstrated that very few fungal cells remained in the kidneys of mice infected with hsp21
Δ/Δ. We therefore conclude that, unlike wild type and hsp21
Δ/Δ survived poorly in the hostile milieu of the mammalian host.
In summary, this study represents the first characterization of a small heat shock protein (Hsp21) in the human fungal pathogen C. albicans and establishes its role in adaptation to distinct environmental stresses via cellular trehalose homeostasis and Cek1 activation, immune evasion and virulence.