The extent of differences inherent amongst the aspergilli is often underappreciated, but is evidenced by phenotypic features such as color, hydrophobicity, rate of germination, cell size and surface structure. For instance, although both A. fumigatus
and A. nidulans
conidia contain two hydrophobins, RodAp and RodBp (A. fumigatus
), RodAp and DewAp (A. nidulans
), and the rodA
gene of A. fumigatus
has been shown to complement homologous RodAp mutations in A. nidulans
, the rodB
gene is unable to complement A. nidulans
DewAp mutations in spite of having the same molecular mass and similar signal sequence.21
Analysis of physicochemical properties in the RodAp knockouts of the two species also suggests that there may be other differences in the composition of their outer cell walls, i.e., lipid and glycoprotein content, that are unmasked by disruption of the rodlets upon germination.22
Similarly, the asexual conidia of A. fumigatus
and A. terreus
are distinct in color and cell size and recent studies in our laboratory indicate differential rates of germination and hydrophobicity between these conidia.1
In this study, we further demonstrate that A. terreus
PC have an average of two nuclei compared to A. fumigatus
conidia, which often contains one nucleus. Additionally, PC of both species exhibit distinct cell surface characteristics including β-glucan expression. Specifically, there was no β-glucan staining on the surface of dormant A. fumigatus
PC, in contrast to the irregular staining apparent on dormant A. terreus
PC. Furthermore, early germlings of A. fumigatus
displayed concentrated β-glucan staining at the tips while this was not observed on the tips of A. terreus
germlings. Finally, in late polar growth, dectin-1 labeling was uniform throughout the surface but considerably diminished in A. fumigatus
, while staining of A. terreus
hyphae was punctuate and intense. No significant differences were observed between the clinical and environmental isolate tested. These species specific characteristics hold the potential to elicit differential immune responses to these organisms and will need to be tested in future experiments.
Our previous study indicated that A. terreus
AC have enhanced β-glucan display compared to dormant A. terreus
suggesting that these conidia differ markedly as well. In the present study, β-glucan display was continuous on the surface of AC throughout the stages of germination. In contrast, β-glucan display showed an irregular staining pattern on dormant A. terreus
PC. β-glucan display was enhanced during swelling and early germ tube formation, and subsequently diminished. In A. fumigatus
, conidia swell upon induction of germination, in the process losing a hydrophobin or rodlet layer, as well as pigments such as melanin. These surface molecules conceal β-glucans that serve as binding sites for Dectin-1, as well as other pathogen-associated molecular patterns (PAMPs) that can be recognized by immune cells, i.e., Toll-like receptor 4 (TLR4) and mannose receptors, thus modulating stage specific immune responses.17,20,21,23
Here, we show that the two different conidial types of A. terreus
have differential β-glucan display and accordingly are recognized differently by immune cells. The influence of differential patterns of β-glucan presentation on pathogenicity for AC versus PC, and the consequences of altering these patterns, requires deeper investigation.
Phenotypically, A. terreus
AC are lighter in color compared to PC. Previous SEM and TEM studies from our laboratory demonstrated that the cell surface of AC appear to be devoid of a yet to be characterized fungal pigment in contrast to the PC surface.1 A. fumigatus
conidia lacking the pigment melanin are white in color and a recent study demonstrated that albino A. fumigatus
conidia induced significantly more proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC), as compared to melanized wild-type conidia.23
Similarly, in the present study, we found that production of cytokines/chemokines elicited by AC was greater than that elicited by PC, both ex vivo and in vivo in a mouse model. This heightened proinflammatory response could be attributed to “albino” AC (lacking pigment) that display strong cell surface β-glucan. Since we only inactivated the conidia by heat killing and did not perform additional blocking experiments with Laminarin (for β-glucan), we cannot at this time rule out the presence of other stimulatory PAMPs like mannan derivatives on the AC surface. However we did use A. fumigatus
as a control, since staining of A. fumigatus
with Dectin-1 has been well characterized in previous studies. An additional limitation of this study was that we did not elucidate fungal burden or survival and thus mice were sacrificed at 18 h post-challenge. Future studies, powered to understand survival after challenge with PC and AC need to be performed.
Our studies show that A. terreus
AC are multinucleate and may contain as many as seven nuclei. To ensure that the AC were not breaking “dormancy” upon detachment from the hyphae, thus inducing cell cycle stages to progress within the conidial structures and the multiple nuclei observed, Hoechst staining was performed on AC still attached to the hyphae. These AC were also found to contain multiple nuclei, again varying in number. In A. oryzae
, multi-nucleation of conidia conferred greater viability and resistance to UV radiation and freeze-thaw treatment, thus resulting in better adaptation to adverse environmental conditions and could ensure conidial preservation.24
Although such viability experiments were not performed in the present study, there is some evidence that AC are at least more resistant to antifungal drugs than PC. If indeed multinucleation imparted viability benefits to AC, this would be a significant virulence factor for A. terreus
as it would allow the organism to survive the harsh environment of the host during infection.
Alternatively, multinucleation could suggest a rudimentary form of conidia that have not yet evolved cell cycle control mechanisms, the evolutionary benefit of which might be rapid germ tube emergence. In fact, our previous studies demonstrated that AC germinated much more rapidly than either A. fumigatus
or A. terreus
PC, and this study found that the AC formed multiple germ tubes (). Similar to our study, in A. oryzae
, multinucleate conidia had a higher germination efficiency than uninucleate conidia.24
Additionally, late stage germination of both A. terreus
PC and AC culminates in production of hyphae on which grow more AC, which in turn polarize in multiple directions to quickly form several more hyphal extensions. In spite of differences in germination potential between the two conidial forms, once both conidia achieved germination, the ensuing developmental stages appear to proceed comparably resulting in similar mycelial masses as assessed visually under a microscope (data not shown). The ability for each AC to produce multiple hyphae and subsequently more AC, thus ultimately more hyphae in a very short period of time perhaps sets the stage for rapid invasion and dissemination during infection in spite of inducing strong immune responses.
In summary, our study demonstrates phenotypic differences between A. fumigatus and A. terreus, specifically β-glucan display. Additionally, we demonstrate differences in cell surface β-glucan between the two A. terreus asexual conidia, elucidate the role of AC in inducing inflammatory responses in a mouse model of aspergillosis, and demonstrate multinucleation and hyperpolorization in these structures, all of which may contribute towards the pathogenicity of this organism.