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Eukaryotic Cell, February 2009, p. 197-206, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00202-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biofilm Formation by Pneumocystis spp.{triangledown} ,{dagger}

Melanie T. Cushion,1,2* Margaret S. Collins,1,2 and Michael J. Linke2

University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 560, Cincinnati, Ohio 45267-0560,1 Cincinnati VAMC, 3200 Vine Street, ML151, Cincinnati, Ohio 452202

Received 19 June 2008/ Accepted 16 September 2008

Pneumocystis spp. can cause a lethal pneumonia in hosts with debilitated immune systems. The manner in which these fungal infections spread throughout the lung, the life cycles of the organisms, and their strategies used for survival within the mammalian host are largely unknown, due in part to the lack of a continuous cultivation method. Biofilm formation is one strategy used by microbes for protection against environmental assaults, for communication and differentiation, and as foci for dissemination. We posited that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system comprised of insert wells suspended in multiwell plates containing supplemented RPMI 1640 medium supported biofilm formation by P. carinii (from rat) and P. murina (from mouse).Dramatic morphological changes accompanied the transition to a biofilm. Cyst and trophic forms became highly refractile and produced branching formations that anastomosed into large macroscopic clusters that spread across the insert. Confocal microscopy revealed stacking of viable organisms enmeshed in concanavalin A-staining extracellular matrix. Biofilms matured over a 3-week time period and could be passaged. These passaged organisms were able to cause infection in immunosuppressed rodents. Biofilm formation was inhibited by farnesol, a quorum-sensing molecule in Candida spp., suggesting that a similar communication system may be operational in the Pneumocystis biofilms. Intense staining with a monoclonal antibody to the major surface glycoproteins and an increase in (1,3)-β-D-glucan content suggest that these components contributed to the refractile properties. Identification of this biofilm process provides a tractable in vitro system that should fundamentally advance the study of Pneumocystis.

* Corresponding author. Mailing address: University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 560, Cincinnati, OH 45267-0560. Phone: (513) 861-3100, ext. 4417. Fax: (513) 475-6415. E-mail: Melanie.Cushion{at}uc.edu

{triangledown} Published ahead of print on 26 September 2008.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, February 2009, p. 197-206, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00202-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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