the ACs continued to increase rapidly within two hours after which absorbance reached a plateau (Figure 3). In contrast, at two hours the PCs showed only a slight increase in XTT-formazan production with the XTT conversion rate being slower than compared to ACs even after seven hours of incubation (the last time point tested in this study). This finding was in keeping with another study where the XTT conversion of A. terreus PC was lower when compared to A. flavus and A. fumigatus and was attributed to poor metabolic activity at this early time point (Walsh et al, 2007). Collectively, our study suggests (at least with the strains tested) that ACs have robust metabolic activity when compared to PCs and that this metabolic activity begins earlier in ACs. Our study demonstrates that AC, in comparison with PC, can become metabolically active, germinate rapidly and are more adherent. In vitro experiments demonstrated that these AC can be readily dislodged from hyphae (data not shown). Thus, it is tempting to speculate that during infection in vivo these structures, once pinched off from hyphae at the site of infection, can disseminate to other body sites where they can establish infections swiftly by virtue of their adherence and germination properties. In keeping with this premise, previous studies have shown that A. terreus isolates have a propensity to cause more disseminated and fulminating infections than other Aspergillus species [19]. Accessory conidia have less cell membrane ergosterol content and higher MICs to AMB Our analysis of ergosterol levels by previously described methods [12] demonstrated that AC had lower cell membrane ergosterol content when compared to PC (32.3% PC versus 10.1% AC for CLF 52; 37.6% PC versus 4.8% AC for CLF 29; p = 0.01; representative of three separate experiments). Since it has been shown that cell membrane ergosterol content may correlate with antifungal drug susceptibility to AMB [4,20], we tested the AMB drug susceptibilities of both conidial forms by a FUN 1 based FCM assay [14]. Results of this assay showed that both A. terreus AC had AMB MICs that were higher than MICs of PC (AC mean MIC = 2 mg/ml; PC mean MIC = 8mg/ml). These results differ from that of a recent study that found no difference in AMB MICs of both conidial forms [8] and may be in part due to the differences in methodologies: we employed a fluorochrome based FCM assay while Lass-Flo¨ rl and co-workers generated MICs with the CLSI M38A microdilution method. Another reason that our Figure 3. Metabolic activity of phialidic conidia and accessory conidia using the XTT assay. XTT absorbance (OD490) of phialidic (dashed line) and accessory conidia (solid line) was measured at 0, 0.5, 1, 2, 3, 4, 5, 6, and 7 hours after subtraction of background OD (OD of wells with XTT alone). Error bars represent standard error of experiments performed in triplicate. doi:10.1371/journal.pone.0007673.g003 A. terreus Accessory Conidia study results may differ from the previous study could be that we assayed MICs at 4 hours versus the CLSI study that requires 48 hours of incubation before the results can be read. Since ACs had a propensity to be metabolically active within two hours and PCs did not, we also generated AMB MICs employing the CLSI broth microdilution method [13] to exclude the effect of differences in metabolic activity of the cellular forms on MIC interpretation at an early timepoint employing a FCM method. Although the AMB MIC generated by the CLSI method was at least two fold lower than MIC generated by the FCM method, both ACs had at least four fold higher MICs when compared to PC (mean MIC = 4 mg/ml for AC versus 1 mg/ml for PC). Thus in our study, ACs had a lower susceptibility to AMB when compared to PCs by both the FCM and the traditional CLSI based method. The finding that AC have lower cell membrane ergosterol that correlates with higher MICs to AMB may be in part why A. terreus isolates fail to respond to AMB therapy in vivo [21] and needs further investigation with more isolates. Differential binding of the b1-3 glucan antibody to the two conidial forms The cell wall architecture of the asexual conidia of A. terreus has never been studied, and that of A. fumigatus is only now being understood. Recent studies with A. fumigatus resting conidia have demonstrated that low concentrations of b1-3 glucan are accessible on the surface and only conidial germination unmasks the b1-3 glucan surface moieties [22]. We found that resting A. terreus phialidic conidia (similar to A. fumigatus – Figure 4A) demonstrate low b1-3 glucan labeling (Figure 4B) indicating that, as with other fungi, a cell wall layer may be enveloping the b1-3 glucan moiety, thus efficiently masking A. terreus conidia from immune recognition [22,23,24,25]. In contrast, while the surface of the AC was also largely shielded from b1-3 glucan exposure (as observed by low b1-3 glucan labeling), a patch of the AC was intensely labeled with b1-3 glucan antibody indicating significant levels of b1-3 glucan at this site (Figure 4C indicated with an arrow). We speculate that this region would have been previously attached to the hyphae and therefore represents the attachment scar. The question arises as to why AC exhibit scarring while PC do not. The answer may lie in mode of attachment of these structures on hyphae. Phialidic conidia are never borne directly on hyphae, but rather are pushed out or bud from within the phialide, the flask shaped structures comprising the reproductive zones of the conidiophore. In contrast, AC arise directly from hyphae, and this type of attachment may result in prominent b1-3 glucan scars when AC are pinched off the hyphae. Regardless, we demonstrate for the first time the existence of unique b1-3 glucan rich patches on AC that may represent glucan rich attachment scars. In the pathogenic yeast Candida, it has been shown that bud and birth scars are rich in surface b1-3 glucan, and that this glucan exposure appears to be sufficient to activate Dectin-1 (a b-glucan specific receptor) and trigger potent antifungal inflammatory responses in macrophages [25]. In A. fumigatus, genetic deletion of a polyketide synthase involved in pigment biosynthesis (PksP) results in surface exposure on resting conidia, rendering the PksP mutants vulnerable to efficient macrophage phagocytosis [24,26]. Further, given that b-glucans can trigger inflammatory responses in macrophages, it would be noteworthy to analyze the macrophage cytokine response after exposure to AC and PC in addition to assaying for differences in phagocytosis, and experi- ments are under way in our laboratory to test both these hypotheses. In summary, this study presents preliminary evidence indicating a possible role for accessory conidia in A. terreus pathogenesis PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7673