Biofilm Formation by Pneumocystis spp

Abstract

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)-␤-Dglucan 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.Organisms in the fungal genus Pneumocystis cause an oftenlethal pneumonia in mammals with a compromised immune status. To date, there have been five formally described species, although all mammalian species examined to date appear to host at least one Pneumocystis species. Those that have been described are P. jirovecii from humans (20), P. carinii (53) and P. wakefieldiae (10, 11) from rats, P. murina (30) from mice, and P. oryctolagi (16) from rabbits.Limited progress has been made in understanding the life cycle, transmission, and natural history of any Pneumocystis species, due in large part to the absence of a continuous in vitro culture system. Although the incidence of frank pneumonia caused by these organisms has decreased in developed countries such as the United States and European countries, mounting evidence points to new niches being exploited by these fungi. The presence of P. jirovecii in patients with underlying chronic diseases such as chronic obstructive pulmonary disease has been suggested to be a comorbidity factor (42-44). A series of recent case reports have identified P. jirovecii as a significant cause of infection in patients being treated with tumor necrosis factor alpha inhibitors for rheum...

Pneumocystis carinii Interactions with Lung Epithelial Cells and Matrix Proteins Induce Expression and Activity of the PcSte20 Kinase with Subsequent Phosphorylation of the Downstream Cell Wall Biosynthesis Kinase PcCbk1

“…Recent studies also support the notion that Pneumocystis forms biofilms (2). In many infecting organisms in vivo, biofilms may contain microorganisms and organism-derived proteins, as well as host proteins, including matrix proteins.…”

Fungal Morphogenesis

“…4) . Although filamentous growth is not essential for the formation of biofilms (Hawser and Douglas 1994;Reynolds and Fink 2001;Martinez and Casadevall 2007;Cushion et al 2009), hyphae strengthen the biofilm structure and provide strong and expanded scaffolds for the deposition of extracellular matrix materials and other cells (Ramage et al 2002;Richard et al 2005). Hyphae of C. albicans are known to penetrate host tissues and even denture materials, and thus it is not surprising that there are many hyphal-specific adhesins that help anchor hyphae firmly to the substrate.…”

Candida parapsilosis complex water isolates from a haemodialysis unit: biofilm production and in vitro evaluation of the use of clinical antifungals

“…The high frequency of biofilm-producing isolates found in our study was perhaps due to a higher capacity of isolates from environmental sources to form biofilms in comparison with isolates obtained from clinical sites. In nature, fungi and pathogenic bacteria exist predominantly in biofilms, rather than as planktonic or free-floating organisms (Costerton et al 2003, Cushion et al 2009). Currently, the percentage of isolation of different species that constitute the complex C. parapsilosis: C. orthopsilosis, C. metapsilosis and C. parapsilosis have not yet been described.…”