Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture, Cell Wall Composition and Germination Kinetics Eszter Deak1, Selwyn D. Wilson1, Elizabeth White2, Janice H. Carr3, S. Arunmozhi Balajee1* 1 Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 2 Infectious Diseases Pathology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Clinical and Environmental Microbiology Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America Abstract Infection with Aspergillus terreus is more likely to result in invasive, disseminated disease when compared to other Aspergillus species; importantly this species appears to be less susceptible to the antifungal drug amphotericin B. Unique to this species is the ability to produce specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this organism, we analyzed the phenotype, structure and metabolic potential of these conidia. Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC architecture by electron microscopy revealed distinct morphological differences between the two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were metabolically more active compared to PC. Additionally, AC contained less cell membrane ergosterol, which correlated with decreased susceptibility to AMB as determined using a flow cytometry based analysis. Furthermore, AC exhibited surface patches of b1-3 glucan, suggestive of attachment scarring. Collectively, the findings of this study suggest a possible role for AC in A. terreus pathogenesis. Citation: Deak E, Wilson SD, White E, Carr JH, Balajee SA (2009) Aspergillus terreus Accessory Conidia Are Unique in Surface Architecture, Cell Wall Composition and Germination Kinetics. PLoS ONE 4(10): e7673. doi:10.1371/journal.pone.0007673 Editor: Marta Feldmesser, Albert Einstein College of Medicine, United States of America Received February 2, 2009; Accepted October 8, 2009; Published October 30, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: The funding source and the role of the funders will be present. Eszter Deak was supported in part by an appointment to the Emerging Infectious Disease (EID) fellowship program administered by the Associations of Public Health Laboratories (APHL) and funded by the CDC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fir3@cdc.gov Introduction Aspergillus species remain the predominant etiological agents of invasive fungal infections among patients with hematologic malignancies and recipients of solid-organ and hematopoietic stem-cell transplants [1]. Although Aspergillus fumigatus accounts for a majority of cases of invasive aspergillosis (IA), A. terreus is emerging as a significant pathogen in certain medical centers [2]. Studies also demonstrate that A. terreus can cause more fulminating and disseminating disease than other Aspergillus species, and is refractory to treatment with the antifungal drug amphotericin B (AMB) [3,4]. Species within the genus Aspergillus, including A. terreus, produce asexual conidia that arise from conidiophores (phialidic conidia, PC). These PC are produced by Aspergillus species in the natural environment and on laboratory media but are rarely produced during infection. In addition to PC, A. terreus produces another type of asexual conidia denoted as accessory conidia (AC), which arise directly on hyphae. Uniquely, A. terreus has the ability to produce AC both in vitro and in vivo during invasive infection. Detection of these distinctive structures in body fluids and/or biopsy specimens has been considered ‘‘diagnostic’’ for A. terreus infection [4]. Production of AC by A. terreus isolates in vivo was recorded as early as the 1950s when investigators noted the presence of these structures in the stomach contents of a bovine fetus after mycotic abortion [5]. Raper and Fennell in their monograph on the genus Aspergillus speculated on the possible role of these structures in dissemination of disease [5,6]. Clinically relevant fungi such as Scedosporium species, Fusarium species, Paecilomyces species, and Acremonium species produce adventitial unicellular forms in vivo during infection [7] and it has been postulated that the high proportion of disseminated infection due to these fungi can be attributed in part to these structures. It is not known at this time if a similar function exists for A. terreus AC. It has also been hypothesized that AC might be the source of AMB resistance in this organism. The only previous study comparing the susceptibility of A. terreus AC with that of PC found that both cellular forms exhibited similar susceptibility to AMB [8]. Thus, little is known about the function and role of AC in A. terreus biology and its correlation with pathogenesis and AMB drug resistance. The present study was therefore designed with the following objectives: (1) to estimate AC production of a large number of A. terreus isolates recovered from both clinical and environmental samples, (2) to examine morphological differences between AC and PC, (3) to evaluate germination kinetics and metabolism of AC and PC, (4) to study differences in AMB susceptibilities of AC and PC using a flow cytometry based method, and finally (5) to analyze ergosterol content and b-glucan PLoS ONE | www.plosone.org 1 October 2009 | Volume 4 | Issue 10 | e7673