Biofilm Formation by Pneumocystis spp.

Pneumocystis spp. can cause a lethal pneumonia in hosts withdebilitated immune systems. The manner in which these fungalinfections spread throughout the lung, the life cycles of theorganisms, and their strategies used for survival within themammalian host are largely unknown, due in part to the lackof a continuous cultivation method. Biofilm formation is onestrategy used by microbes for protection against environmentalassaults, for communication and differentiation, and as focifor dissemination. We posited that the attachment and growthof Pneumocystis within the lung alveoli is akin to biofilm formation.An in vitro system comprised of insert wells suspended in multiwellplates containing supplemented RPMI 1640 medium supported biofilmformation by P. carinii (from rat) and P. murina (from mouse).Dramaticmorphological changes accompanied the transition to a biofilm.Cyst and trophic forms became highly refractile and producedbranching formations that anastomosed into large macroscopicclusters that spread across the insert. Confocal microscopyrevealed stacking of viable organisms enmeshed in concanavalinA-staining extracellular matrix. Biofilms matured over a 3-weektime period and could be passaged. These passaged organismswere able to cause infection in immunosuppressed rodents. Biofilmformation was inhibited by farnesol, a quorum-sensing moleculein Candida spp., suggesting that a similar communication systemmay be operational in the Pneumocystis biofilms. Intense stainingwith a monoclonal antibody to the major surface glycoproteinsand an increase in (1,3)-β-D-glucan content suggest thatthese components contributed to the refractile properties. Identificationof this biofilm process provides a tractable in vitro systemthat should fundamentally advance the study of Pneumocystis.

INTRODUCTION

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INTRODUCTION

Organisms in the fungal genus Pneumocystis cause an often-lethalpneumonia in mammals with a compromised immune status. To date,there have been five formally described species, although allmammalian species examined to date appear to host at least onePneumocystis 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 culturesystem. Although the incidence of frank pneumonia caused bythese organisms has decreased in developed countries such asthe United States and European countries, mounting evidencepoints to new niches being exploited by these fungi. The presenceof P. jirovecii in patients with underlying chronic diseasessuch as chronic obstructive pulmonary disease has been suggestedto be a comorbidity factor (42-44). A series of recent casereports have identified P. jirovecii as a significant causeof infection in patients being treated with tumor necrosis factoralpha inhibitors for rheumatoid arthritis and other chronicdiseases (22, 25, 28, 54). Other studies in animals and humanssuggest that Pneumocystis organisms are also present in thelungs of nonimmunocompromised hosts (24, 60). In contrast, theprevalence of Pneumocystis pneumonia in developing countries,such as those in sub-Saharan Africa, remains high and poorlycontrolled (37, 46). The strategies used by these organismsto grow and survive in the context of an intact or debilitatedhost defenses are largely unknown.

Morphological analysis of histologic sections of human and rodentlung tissue, redolent of layers within a microbial biofilm (Fig.1), as well as recent studies showing the ability of P. cariniito thrive under reduced oxygen conditions in vitro (26) ledus to posit that Pneumocystis spp. follow a pathway to biofilmformation within the host lung, similar to that of other fungalpathogens. Since oxygen availability has been shown to influencethe metabolic activity in different zones within Pseudomonasaeruginosa biofilms (61), it seemed reasonable to suggest thatthe masses of Pneumocystis cells that accumulate within an infectedmammalian lung lumen also are exposed to a similar oxygen gradientand subsequently have evolved survival mechanisms for hypoxicconditions.

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FIG. 1. P. jirovecii in apparent biofilm structures within an infected human lung. Grocott methenamine silver staining of postmortem lung from a patient with AIDS is shown. (A) P. jirovecii filling the alveolar lumen (arrow) and in an apparent biofilm formation (double arrows). Bar, 100 µm. (B) Higher magnification of P. jirovecii within the alveolar lumen. Note the close apposition of organisms resembling a biofilm structure. Bar, 10 µm. (C) P. jirovecii in the alveolar lumen, illustrating a biofilm-like structure with a base (horizontal, packed organisms) with a protuberance into the lumen. Bar, 10 µm.

To explore our hypothesis, we devised an in vitro system thatsupported the transformation of two different species of Pneumocystisinto biofilm-like structures. The process of formation was welldefined and reproducible, resulting in adherent films composedof unique morphological structures. That the biofilms held viableand infective organisms was confirmed by ATP content and fluorescentviability staining and the ability to cause pneumonia in immunosuppressedrodent lungs. The establishment of a biofilm system for Pneumocystiswill facilitate studies of life cycle, survival mechanisms,and host cell and parasite interactions and a wide variety ofapplications heretofore unavailable to the research community.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Organisms. Pneumocystis carinii (from rats) and P. murina (from mice) wereused immediately after isolation and purification from infectedrodent lungs or from cryopreserved aliquots. Infected lungswere removed from rats or mice after approximately 8 weeks ofchronic administration of dexamethasone as an immunosuppressant(14). Organisms were released from lung tissue by homogenizationfollowed by washing, hypotonic lysis (to reduce erythrocytesand other host cells), and then centrifugation at 1,000 x g.The organisms were enumerated by tinctorial methods using arapid Giemsa stain, Hema3 (Fisher Diagnostics, Middletown, VA),and expressed as the number of Pneumocystis nuclei/ml (13).Each nucleus represents one organism. Organisms were cryopreservedin RPMI 1640 containing 7.5% dimethyl sulfoxide (7). Each batchwas assayed for ATP content and plated on nutrient agar (36°C),on Sabouraud dextrose agar (25°C), and in RPMI 1640 witha 20% serum supplement (36°C).

Biofilm formation. RPMI 1640 medium (Gibco Invitrogen, Grand Island, NY) with penicillin(200 U/ml)-streptomycin (200 µg/ml)-amphotericin B (0.5µg/ml) (Cellgro, Herndon, VA), vancomycin (5 µg/ml)(Fluka, Sigma-Aldrich), 20% calf serum (Atlanta Biologicals,Lawrenceville, GA), and vitamins and amino acids as previouslydescribed (14) and containing the desired organism concentrationwas placed in one of several types of tissue culture insertwells, including polyethylene terephthalate (PET) track-etchedmembrane cell culture inserts (no. 35-3090; Becton DickinsonInc., Franklin Lakes, NJ), Transwell (no. 3460; Costar, Corning,Corning, NY), and Millicell-CM hydrophilic polytetrafluoroethylene(PTFE) membranes (PICM-01250 [12 mm] and PICM-03050 [30 mm])and Millicell-HA insert wells with mixed cellulose ester membranes(PIHA-01250 [12 mm]) (Millipore Corp., Billerica, MA), suspendedin each of a well in a 6- to 12-well multiwell polystyrene plateor a 24-well polystyrene plate. The same medium was added tothe outside well. Lab Tek II chamber slides (Nunc, Rochester,NY) were used in some cases for microphotography. After 24 hof incubation at 36°C in a water-jacketed incubator with5% CO₂, the medium in the outside well was aspirated and discarded.The medium within the insert well was carefully removed witha pipette. The biofilm was washed to remove nonadherent organismsby the addition of 500 µl of the medium or phosphate-bufferedsaline (PBS). The media in the outside well and insert werereplenished. This point was considered "day 1" for the biofilmstudies. "Day 0" refers to the measurement of the inocula usedto seed the inserts. The medium in each outside well was replaceddaily to renew nutrients and remove waste products.

Biofilm quantification. Biofilm formation was measured by a ATP bioluminescence assayused extensively by our lab to assess the viability of Pneumocystis(5, 7-9, 27). The assay is similar in concept to the XTT reductionassay used for fungal biofilm growth, which assesses mitochondrialfunction, but is a more sensitive and rapid method. The bioluminescenceassay is based on the ATP-driven reaction of the substrate luciferinwith the enzyme luciferase, which results in the evolution oflight as relative light units (RLU) (ATPlite detection kit;Perkin-Elmer). The light evolved is proportional to the amountof ATP in the sample and, correspondingly, to the number ofcells from which it was extracted (5). Both unattached organismsfound in the supernatant (planktonic) and those attached tothe substrates (sessile/biofilm) were measured. Three wellswere used for each time point. For sampling of planktonic organisms,supernatants were removed from each insert by gentle pipettingand centrifuged to pellet the cells, followed by addition of100 µl of ATP lysis solution. The biofilms were then washedwith PBS, and 100 µl of ATP lysis solution was added directlyonto the insert membrane, mixed, and allowed to incubate atroom temperature for 10 min. A 50-µl volume from eachphase was removed and placed into wells of a 96-well white plate(Greiner Scientific, Ocala, FL) containing the luciferin-luciferase(substrate-enzyme) reaction buffer. Plates were incubated atroom temperature with gentle agitation for 60 min to permitfull evolution of light, which is stable for up to 5 h. Luminescencewas measured using a POLARStar Optima plate reader (BMG Labtechnologies)with an output of RLU. Quench controls, to control for unanticipatedinterference of additives with the luciferin-luciferase reaction,were included in each assay.

Phase-contrast and bright-field microscopy. Biofilms were monitored on a daily basis, and formation wasphotodocumented using a Nikon Diaphot inverted phase microscopeequipped with a SPOT image capture system (Diagnostic Instruments,Inc.). Morphological assessments were performed under phase-contrast,bright-field, and fluorescent microscopy using an Olympus BH2microscope. For bright-field examination, supernatants wereremoved and biofilms were washed twice with PBS to remove anyresidual planktonic cells. The biofilms were harvested by scrapingthe inserts with a pipette and depositing the material on amicroscope slide and then were allowed to air dry. The slideswere stained with a rapid Wright-Giemsa stain, Hema3 (FisherDiagnostics, Middletown, VA), for 1 min in each reagent, a modificationof our standard method in which staining is for 10 to 20 s each.Magnification was assigned by calibration with an objectivemicrometer (Nikon Inc., Melville, NY).

CSLM. Pneumocystis biofilm structure and viability were evaluatedat various time points after staining with 20 µM FUN-1and 10 µM concanavalin A (ConA)-Alexafluor 488 (MolecularProbes, Invitrogen Detection Technologies, Carlsbad, CA) followingprocedures established for evaluation of Candida biofilms (4),The dyes were added to the culture media, and after 30 min at37°C, the medium was removed, cells were washed twice withPBS, and the biofilm was embedded in Fluromount-G (SouthernBiotech). The FUN-1 dye is processed during the incubation periodinto strikingly red fluorescent inclusion bodies. This conversionof the dye is accompanied by a reduction in the diffuse greencytoplasmic fluorescence. In combination with the ConA-Alexafluor488 staining, live cells will exhibit discrete sequestered redfluorescence that will appear orange to yellow in combinationwith the green fluorescence of the ConA counterstain. Confocalscanning laser microscopy (CSLM) was performed with an LSM 510laser scanning microscope (Zeiss, Germany). ConA staining wasobserved using the 488-nm laser line and a 505- to 550-nm band-passemission filter. FUN-1 staining of live cells was visualizedusing the 543-nm laser line with a 560-nm long-pass filter.The gain was set to unstained organisms as "black" to controlfor any autofluorescence. To determine the three-dimensionalbiofilm structure, a series of horizontal (x-y) optical sectionswith a thickness of 0.18 µm at 0.50-µm intervalswas taken throughout the biofilms. Eight-bit confocal imagesof green (ConA) and red (FUN-1) fluorescence were obtained usingmultitracking, which reduced the likelihood of bleedthrough.The images were captured and processed using Zeiss LSM ImageBrowser software.

Surface glycoprotein staining. All Pneumocystis species contain a family of glycoproteins thatare a major antigenic component on the surface of all developmentalforms of these organisms, the major surface glycoproteins (MSGs)(57). Biofilms were evaluated for the presence of MSGs by stainingwith a monoclonal antibody (RA-E7) directed to an epitope ofthe protein component of these molecules (35). Biofilms wererecovered from the insert surface by scraping with a pipettetip, air dried, and fixed with acetone. The biofilms were probedfor the expression of MSG by an indirect immunofluoresence assayas previously described (34). Briefly, the slides were blockedwith 1% bovine serum albumin (BSA) in PBS (PBS-BSA) for 60 minand then incubated in a 1/500 dilution of RA-E7 in PBS-BSA for60 min at 37C. The slides were washed in PBS and then reactedwith an Alexafluor 594-labeled F(ab')₂ fragment of goat anti-mouseimmunoglobulin G (heavy and light chains) (Molecular Probes,InVitrogen, Eugene, OR) diluted 1/1,000 in PBS-BSA for 60 minat 37C. After washing, the slides were mounted with fluorescentmounting medium and examined under oil immersion with a NikonE600 epifluorescence microscope. Controls included additionof the Alexafluor 594-labeled F(ab')₂ fragment of goat anti-mouseimmunoglobulin G conjugate directly on the biofilm preparations,monoclonal antibody RA-E7 alone, and PBS without additionalreagents. No red autofluorescence was observed.

Effects of farnesol on the viability and formation of P. carinii biofilms. Farnesol (Acros Organics, Thermo Scientific Fisher, NJ), a quorum-sensingmolecule elucidated by Candida albicans, was added at the timeof inoculation of P. carinii into PET inserts at a concentrationof 100 µM (48) and then assayed for ATP content of thesupernatant and biofilm cells over a 1-week period. Two separateexperiments were performed with two batches of P. carinii. Resultswere expressed as the percent ATP versus that for untreatedP. carinii inoculated in the same insert system. Statisticswere evaluated using GraphPad Prism v. 4 (GraphPad Software,Inc., San Diego, CA).

Measurement of β-1,3-Glucan. Measurement of the (1,3)-β-D-glucan content in P. cariniibiofilms over time was conducted using the Glucatell kineticassay, following the vendor's instructions (Associates of CapeCod, Inc., East Falmouth, MA). The reaction is based on activationof factor G, a serine protease zymogen, which in turn activatesthe proclottin enzyme that cleaves pNA from the chromogenicpeptide substrate, creating a chromophore that absorbs at 405nm. The rate of optical density increase is measured againststandards. At each time point, supernatants from duplicate wellswere removed and inserts were washed twice with PBS. The membraneswere then scraped with a pipette tip and the contents combinedand air dried within a sterile biosafety cabinet. The driedsamples were resuspended in 1 ml of 1 M sodium hydroxide andshaken at room temperature for 1 h. Samples were prepared as10-fold dilutions in pyrogen-free water in test tubes certifiedto be free of glucan (Associates of Cape Cod). Glucan standardswere prepared in the same manner according to the vendor's instructions.Samples were read at 2-min intervals for 1.2 h (40 cycles).The (1,3)-β-D-glucan was measured as slope/minute, rawdata – blank, and calculated using a linear regressioncurve of standards from 25 to 200 pg/ml. The correlation coefficientfor the studies presented was 0.992, which is within the acceptablerange suggested by the vendor (>0.980). Glucan content wasexpressed as pg/ml. Statistical analysis was not performed sincethe duplicate wells were pooled to provide sufficient materialfor assay.

Passage of biofilms. The ability to passage biofilms was evaluated using small (12-mm,Corning Transwell) and large (30-mm, Falcon, BD) inserts (0.4-µmpore size) that could both be contained in a six-well polystyreneplate. P. carinii was inoculated onto the small inserts andwashed after 24 h, and the medium was replaced. Each small insertwas then placed inside the larger insert well, to which 1 mlof the RPMI+ medium was added. The smaller insert hung withinthe larger insert, permitting communication between the supernatantsbut not the insert membranes. Both inserts were placed withina well of a six-well polystyrene plate. The outer well contained2 ml of the RPMI+, which was replenished daily. The insertswere sampled for ATP content every 3 to 4 days and examinedmacroscopically for evidence of organism transfer and biofilmformation, by detection of the formation of a white film. Thelarger insert that was then populated with the first-passageorganisms was removed and placed in another of the six-wellpolystyrene wells with fresh medium. An uninoculated small insertwas then hung within the confines of the large insert that containedthe organisms. A representation of this process is in Fig. S1in the supplemental material.

Inoculation of passaged biofilms into P. carinii-naïve rats. The ability of the passed organisms to induce infection in immunosupressedrats was evaluated. Biofilms grown on Millicell-CM 12-mm insertswere passed on day 15 of culture by transfer of the small insertto a large insert. After 1 week, the small inserts were removedand the entire contents of each of the large insert wells (passagedorganisms) were harvested, pooled, and cryopreserved. One halfof the pooled organisms were inoculated into each of three immunosuppressedSprague-Dawley rats (3.35 x 10⁵ [log 5.52] nuclei each) as previouslydescribed (12). Uninoculated immunosuppressed rats served ascontrols. Immunosuppression continued for an additional 75 days.The rats were then sacrificed and the lungs processed for microscopicenumeration of P. carinii (7).

RESULTS

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RESULTS

Insert composition and ATP content. Several insert types from different manufacturers were assayedfor their ability to support biofilms over a 2-week period (Table1.). The data are expressed as the total ATP as RLU within awell, which included measurements of both the planktonic andbiofilm cells and as a ratio of the ATP content of the adherentcells/total ATP. Inspection of the RLU for each insert typerevealed various levels of decreases for the different insertsfrom day 1 to day 14, although by day 10, all systems experiencednotable drops in ATP. Inserts composed of hydrophilized PTFE(Biopore-CM from Millipore) and Millicell-HA cellulose rapidlyformed tightly adherent biofilms that were able to maintainATP levels higher than those of P. carinii in other insert typesover 10 days. The kinetics of biofilm formation among each inserttype were generally similar in that the percent ATP contentassociated with the adherent populations (adherent/total ATP)generally increased or remained stable over the 2- to 3-weektime period while the planktonic ATP levels decreased, withthe exception of the PET inserts, where little biofilm formationwas observed except at day 10. Of the two matrices that bestsupported biofilm formation, the PTFE inserts are suitable foraddition of coatings such as collagen or vitronectin, are easilyprepared for electron microscopy, and are compatible with cellularand/or fluorescent stains. The cellulose inserts are opaqueand cannot be used for other applications, and they were notused in further experiments.

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TABLE 1. Effect of support matrices on the formation of P. carinii biofilms^a

The Pneumocystis biofilms could be observed macroscopically. Titration of P. carinii inoculum numbers at 1 x 10⁶ to 1 x 10⁸using the Millicell-CM PTFE inserts showed little differencesin ability to form biofilms over 2 weeks, with the ATP levelsincreasing in the biofilm compartment over time with each inoculum(data not shown). In each case, the biofilms began as smalladherent clusters, which grew larger over time regardless ofinoculum size. The P. carinii biofilms could be observed macroscopicallyas thick white films with visible clumps, as illustrated wheninoculated into Lab Tek II chambers (Fig. 2A) or the Millicell-CMPTFE inserts (Fig. 2B).

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FIG. 2. Macroscopic biofilm formation by P. carinii. (A) Duplicate wells showing biofilm formation after 1, 5, and 10 days on a Lab Tek II chamber slide. (B) PTFE inserts (12 mm) without inocula (left) and after 14 days of biofilm formation (right). Photographs were taken with a 35-mm SLR digital camera on the bench top. Bar, 1 cm.

Phase-contrast monitoring of the biofilms. The biofilms of P. carinii and P. murina both began as microscopicclusters, which grew over time and coalesced with other clusters,often forming connecting branches that produced three-dimensionalstructures. Figure 3 illustrates this process for P. murina,but it should be noted that P. carinii biofilms followed thesame progression. As shown in Fig. 3B and C, small microclustersof P. murina were observed during the first day and week ofculture on insert membranes by inverted phase microscopy. Theseclusters continued to increase in size up until the end of theexperiment, on day 10. Evident in Fig. 3D are the very large,expansive clusters that formed on the insert as a biofilm butalso extended up into the supernatant.

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FIG. 3. Temporal development of P. carinii biofilms on inserts. Organisms were inoculated onto cell culture insert membranes at a density of 2 x 10⁷/insert, placed in multiwell plates, allowed to adhere for 24 h, washed with PBS, and placed back in the well with fresh medium. (A) Uninoculated membrane of insert. (B) Small microclusters (arrows) forming 24 h after washing; the inset shows a magnified microcluster. (C) Seven-day biofilm. Note larger branching clusters attached to membrane (arrows); the inset shows a magnified cluster. (D) Ten-day biofilm, showing an example of a large cluster (note lower magnification). Bars, 100 µm (A to C) and 200 µm (D).

The large clusters produced by either species were highly refractileand contained hundreds to thousands of organisms enmeshed withina matrix. Examples of clusters formed by P. murina are shownin Fig. 4A and B, and examples of those formed by P. cariniiare shown in Fig. 4C. Many of the clusters formed elongatedextensions, as seen in Fig. 4A and C. Smaller extensions couldbe seen on the ends of some branches, as seen in Fig. 4B. Themore robust biofilms were formed by P. murina organisms inoculatedas fresh isolates (as in Fig. 4A and B). The macroscopic filmswere quite impressive, spreading across the membrane in thickbranching clusters often spanning several low-power microscopicfields. These clusters were autofluorescent when excited at488 nm (Fig. 5).

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FIG. 4. Biofilm formation by P. murina and P. carinii with highly refractile large clusters. (A and B) Large clusters of P. murina from 10-day-old biofilms viewed unstained under phase-contrast microscopy. Note that the clusters spanned well beyond the microscopic field (x10). (C) Cluster of P. carinii at day 14 showing elongated extensions. White arrows indicate elongated morphologies in both P. murina and P. carinii biofilm structures. The white arrowhead in panel B indicates a small bud-like extension. Bars, 10 µm.

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FIG. 5. Autofluorescence of a P. carinii biofilm cluster. (A) Differential interference contrast image of a cluster with anastomosed extension from a 7-day-old biofilm; (B) same cluster showing autofluorescence at 488-nm excitation. Bar, 10 µm.

The process of biofilm formation. After 24 to 48 h in the insert system, the organisms retainedthe density and staining characteristics that we and othershave observed in standard tissue culture samples (see Fig. 7A).However, in the biofilm system, after 3 to 4 days a noticeablechange began to occur (Fig. 6 A). The clusters, especially thecysts, took on a highly refractile property around the peripheryof the cysts, with some focal points of refractility withinthe cysts that resembled spores (Fig. 6A). Over time (10 to14 days), the refractile property encompassed the entire cluster(Fig. 6 B). After 2 to 3 weeks, elongated and refractile branchescould be observed (Fig. 6C). The refractility coincided withpoor uptake of the stain, as can be observed in the images shownin Fig. 7B to H.

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FIG. 7. The morphology of Pneumocystis changes dramatically during biofilm formation. (A) P. carinii from the supernatant of a 3-day-old standard short-term culture stained with Hema3, illustrating the differences in morphology from the biofilm structures. (B to H) Images were taken from 16-day-old biofilms inoculated with P. murina (obtained as a fresh isolate). The images were obtained from films on inserts that were scraped with a pipette tip, aspirated, air dried, and stained with Hema3, a rapid Wright-Giemsa stain, as described in Materials and Methods. Images were viewed under oil immersion. Bars, 10 µm. (B) P. murina cluster showing a cyst-like structure with a stalk (arrow). (C) A cyst in a chain with intracystic forms. (D) Refractile cluster beginning to form extensions (arrow). (E) Two large clusters forming a linkage or bridge structure (arrow). (F) A series of cyst-like structures forming a chain (arrows). (G) Cyst containing a structure which appears to be trophic forms undergoing binary fission or conjugation (arrow). (H) Cyst-like forms containing intracellular spore-like morphologies (arrows). (I to L) Images were taken from P. carinii biofilms at day 9 of the first passage. (I) Cluster mass seemingly comprised of three clusters that have joined in the center (arrow). (J) Cyst-like structures in a cluster with obvious intracellular contents resembling spores (arrows). (K and L) Chain-like forms. Bars, 10 µm.

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FIG. 6. Process of biofilm formation monitored by phase microscopy. Phase microscopy of wet mounts (unstained) of P. carinii biofilms is shown. (A) Cluster of P. carinii after 4 days in the Millicell-CM insert system. Arrows indicate focal points of refractility. (B) P. carinii after 10 days in the insert system. Arrows indicate peripheral refractility. (C) P. carinii after 14 days in the insert system. The refractile property encompassed the entire cluster, with elongated extensions beginning to occur (arrows). Bar, 10 µm.

The morphology of Pneumocystis changes dramatically during biofilm formation. The images in Fig. 7 (except for Fig. 7A) were obtained fromfilms on inserts that were scraped with a pipette tip, aspirated,air dried, overstained with Hema3 (a rapid Wright-Giemsa stain),and viewed under oil immersion (13). Biofilm images from bothP. murina and P. carinii and primary and passaged biofilms areshown to illustrate the similar morphologies between the twospecies and between the primary and passaged structures. Theimages in Fig. 7B through H were taken from a 16-day P. murinabiofilm. Those in Fig. 7I through L were samples from a passagedP. carinii biofilm after 9 days on the inserts. Immediatelyapparent from the images is the difference in staining patternscompared to organisms that were obtained from aspiration ofstandard supernatant cultures (Fig. 7A) versus the biofilms(Fig. 7B to L). Whereas most of the supernatant-derived organismsstained readily, showing blue cytoplasm with reddish-purplenuclei, the structures from biofilms excluded much of the stain,were highly refractile, and formed chains and elongated structures(Fig. 7F, K, and L). A striking feature was the linkages formedbetween organism clusters, which appeared to facilitate theanastamosis of the separate biofilm clusters into larger, moreexpansive mats (Fig. 7E and I). This morphology was common amongthe more mature biofilms (>7 days). Many of the sphere-shapedforms contained apparent spore-like structures that were highlyrefractile (Fig. 7C, G, H, and J). The structure shown in Fig.7G contained what appeared to be organisms undergoing binaryfission (or conjugation) within an enclosed sphere. Many ofthe organism chains had elongated tips (Fig. 7D). In other cases,clusters contained cyst-like structures that were recognizablebut that were larger and more aberrant than typically observedin standard suspension cultures (Fig. 7B, C, G, and H). We proposeadoption of the term "allomorphs" to describe these oddly shapedstructures, as suggested for alternative forms of Histoplasmacapsulatum (18).

CSLM. FUN-1 is routinely used to assess the integrity of fungal biofilms(1, 2, 4). All cells, live or dead, take up the dye, resultingin a diffuse green fluorescence. Metabolically active cellsrapidly process the dye, which causes a shift from green tobright red fluorescence that is sequestered in intracellularinclusion bodies. ConA-Alexafluor is used to highlight the mannose-containingextracellular matrix. Biofilms stained in this manner can beused to assess viability and for measurement of the biofilmthickness.

After 7 days of culture in the Lab Tek II chambers, the biofilmsconsisted of mostly metabolically active cells showing the redinclusion bodies (Fig. 8) or a yellow green fluorescence indicatingdual staining with the ConA-Alexafluor stain. The green- oryellow-staining areas indicate mannose or glucose componentsof extracellular material or surface carbohydrates. The imagesalso show tunnels throughout the structure (top and side), indicatingthat there is spatial heterogeneity in the three-dimensionalstructure. The thickness of the biofilm at day 7 was 14.59 µm,somewhat less than that of a typical fungal biofilm (e.g., 25to 40 µm for a 72-h biofilm of Trichosporon asahii) (17),but this was not unexpected since Pneumocystis organisms donot appear to form the typical hyphal masses produced by otherfungi in biofilms.

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FIG. 8. Merged orthogonal images of P. carinii biofilms with FUN-1 and ConA-Alexafluor. After 7 days of culture in Lab Tek II chambers, FUN-1 and ConA-Alexafluor 488 were added to the P. carinii culture medium and cells were incubated, washed, and mounted in Fluromount-G. The images were merged to produce this representation. The center panel shows viable organisms (red, arrows) overlaid with ConA-staining mannose residues (arrowheads) to produce the yellow color. The side panels show the structure and thickness of the biofilm, approximately 14.59 µm. Bar, 10 µm.

Effects of farnesol on the viability of P. carinii biofilms. Exposure of C. albicans to 300 µM farnesol prior to establishmentof a biofilm rendered the fungus incapable of making a biofilm(48). At a much lower concentration, 100 µM, farnesolwas able to dramatically decrease ATP levels in P. carinii inboth the supernatant and adherent compartments of the biofilmsystem to less than 2% of that in untreated controls after 7days of exposure (Fig. 9).

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FIG. 9. ATP content of P. carinii biofilms exposed to farnesol. Farnesol (100 µM) was added to the inserts with the inocula. Biofilms were fed with medium containing the farnesol on a daily basis. Bars reflect the percent ATP content compared to untreated control biofilms over a 7-day period. Error bars indicate standard deviations.

Measurement of (1,3)-β-D-glucan. The increasing refractility of the Pneumocystis biofilms wasa prominent feature of biofilm formation. Since P. carinii isknown to possess a glucan synthase gene, that for Gsc-1, andcontains (1,3)-β-D-glucan in its cell wall (19, 32), wehypothesized that this compound could be a key component inthe morphological changes that the organisms underwent duringthe process. The (1,3)-β-D-glucan content in duplicateP. carinii biofilms was measured over time using the Glucatellkinetic assay. Recently, the assay has been used for the diagnosisof P. jirovecii infection with apparent strong sensitivity andspecificity (29, 39, 55). Figure 10 shows that the amount ofglucan (pg/ml) steadily increased over time in the P. cariniibiofilms, resulting in a 61% increase. The (1,3)-β-D-glucanin a single P. murina biofilm assay increased by 75% but wasmore erratic in the kinetics than for P. carinii (data not shown).

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FIG. 10. (1,3)-β-D-glucan content in P. carinii biofilms. Duplicate Millicell-CM inserts were harvested over a 3-week period and the (1,3)-β-D-glucan content measured using the Glucatell assay kit as described in Materials and Methods. Day 0 refers to the (1,3)-β-D-glucan content of the inoculum.

Biofilms produce an abundance of MSGs. Biofilms reacted with a monoclonal antibody specific for Pneumocystisspp. and targeted to a common epitope within the P. cariniiMSG family of proteins to produce a robust fluorescence (Fig.11), suggesting that these surface glycoproteins may play aprominent role in the morphogenetic conversion to adherent biofilms.Indeed, these mannose- and glucose-containing proteins couldbe a major component of the extracellular matrix or could contributeto the refractile properties of the biofilms.

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FIG. 11. MSGs are abundant on the surface of P. carinii biofilms. Staining reactivity of monoclonal antibody RA-E7, directed to a protein epitope of the MSG family of antigens, with a cluster from a 7-day P. carinii biofilm is shown. Bar, 10 µm.

Inoculation of passaged biofilms into P. carinii-naïve rats. The ability of the passed organisms to induce infection in immunosupressedrats was evaluated by inoculation of passed organisms into immunosuppressedrats (Table 2), the standard animal model protocol that ourlaboratory uses for cryopreserved organisms (12). The cyst-to-trophratio of slightly more than 1:1 was lower than those reportedfor natural infections, which usually range from 1:5 to 1:20,but the levels of organism burdens were within the range formoderate infections (59), showing that the passaged fungi wereviable and remained infective (59). The lower organism burdensmay have been due to the lower numbers in the inocula used inthe present study than in the standard model (3.35 x 10⁵ versus1 x 10⁷).

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TABLE 2. Organism burdens of immunosuppressed rats inoculated with passaged P. carinii biofilms

DISCUSSION

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DISCUSSION

Biofilms are organized microbial communities enmeshed withina matrix of exopolymeric substances and attached to biotic orabiotic matrices. Fungal and bacterial pathogens exist predominantlywithin biofilms, rather than as free-floating, planktonic organisms.There are a myriad of advantages of biofilm formation for microbes,including the exchange of plasmids within the closely apposedcommunity, reduced susceptibility to antimicrobial agents, resistanceto host immune clearance, and dispersion of populations fromaggregates in infection foci. A significant proportion of microbialpathogens causing human disease have been shown to use a biofilmstrategy during the infective process (47). While bacteria havebeen the focus of most biofilm studies, increased appreciationof the widespread nature of these microbial communities havelead to several studies reporting this phenomenon in pathogenicfungi, including Cryptococcus neoformans (38), Aspergillus fumigatus(45), Trichosporon asahii (17), Candida spp. (21, 31, 36, 47,49-52), and Blastoschizomyces capitatus (15).

Within the mammalian lung, Pneumocystis trophic forms adhereto type I pneumocytes and spread along these cells, which compriseover 90% of the alveolar surface. Pneumocystis cells grow inthree-dimensional clusters that fill the alveolar lumen space(Fig. 1.). There, different developmental forms are in closeapposition with one another and form layer upon layer of cellswithin a dense matrix that could be construed as a biofilm.We postulated that like other fungi, Pneumocystis spp. formbiofilms, and this process could be established in an in vitrosystem.

In the present study, biofilms were formed by two species ofPneumocystis on inert matrices of insert wells suspended insupplemented RPMI 1640 medium. There were specific requirementsfor the composition of the matrix, with a hydrophilized PTFEmembrane providing the most robust growth. Two species of Pneumocystis,P. murina and P. carinii, were evaluated since differences inability to form biofilms have been reported to exist among Candidaspecies (47). One difference between the two species was theobserved ability of P. murina to form more extensive biofilms,especially from organisms that were freshly isolated from theimmunosuppressed mouse lung, than their P. carinii counterparts,but the process of biofilm formation and the morphological structuresformed were strikingly similar.

The structure of these biofilms resembled those of other fungiwhen analyzed by fluorescence labeling and CLSM, with three-dimensionalstructures that indicated channels for nutritional exchangewith spatial heterogeneity. The biofilms reached an averagethickness of about 15 µm, which was less than that forother fungi. However, it is clear that Pneumocystis spp. donot form the typical hyphal elements that contribute to thefungal biomass, but they did produce morphologies unique tothe biofilm setting. A novel finding in this study was the observationthat both species of Pneumocystis were able to undergo morphologicaltransformations that resulted in similar but unusual and oddlyshaped forms, which we termed "allomorphs" after the name givento alternative phenotypic forms of Histoplasma. capsulatum (18).Formation of these allomorphs could be in response to changesin pH, oxygen availability, or other environmental stimuli uniqueto this environment.

The formation of Pneumocystis biofilms followed a reproducibleprocess that could be observed at the light microscopic level.Clusters of organisms gained refractile properties, formed elongatedextensions as they spread across the insert membrane, and ultimatelyproduced large three-dimensional structures that were visibleat the macroscopic level. A moderate (61%) increase in (1,3)-β-D-glucancontent over time suggested that this polymer may play a rolein the biofilm process and could contribute to the increasedrefractility. The signature family of surface glycoproteinsof Pneumocystis spp. were produced in the biofilms. These mannose/glucose-richglycoproteins have often been observed to enmesh the organismswithin the lung alveoli and could function as a component ofthe extracellular matrix, an important component of fungal biofilms.

Like for biofilm formation by various Candida species, metabolicactivity was highest within the first 24 h after inoculation,followed by a plateau thereafter to 48 h (3, 49). Our studieswere carried out much further, to 21 days in some cases, andshowed significant decreases in ATP content over these extendedtimes. A decrease in metabolic activity in fungal biofilms isa common characteristic. However, visual inspection of the biofilmsat these later time points showed horizontal and vertical expansionof the biofilm mass and increasing complexity of the biofilmmorphology, suggesting that the decrease in metabolic activitydid not halt the biofilm formation.

Another striking finding of these studies was the ability topass the biofilms and that the passaged P. carinii retainedthe ability to produce infections in the immunosuppressed ratmodel. The method of passage was not an active one, where thebiofilms were scraped and used to inoculate naïve inserts.Rather, inserts with biofilms were placed in naïve wellsthat subsequently became populated with organisms, both in thesupernatant and on the membranes. Detachment of cells from bacterialbiofilms has been shown to play an important role in disseminationof infection (56), and such a mechanism may play a role in thedissemination of the organisms in this system, as the supernatantsof the two inserts were in communication. Alternatively, thepossibility of specialized cells that may disseminate the infectioncannot be ruled out. In C. albicans biofilms treated with antifungalregimens, a subpopulation of perister cells arise that are antifungaltolerant and able to produce new biofilms (33), providing aprecedent for such phenotypic variation.

Quorum sensing is a chemical means of cell-to-cell communicationwithin prokaryotic and eukaryotic biofilms. The process hasbeen implicated in preventing overpopulation, controlling competitionfor nutrients, and mediating dissemination, an especially importantprocess in Candida biofilms (47). Two quorum-sensing molecules,farnesol and tyrosol, have been biochemically and functionallydefined in Candida biofilms (6, 23). At high concentrations,farnesol blocks the morphological transition from yeast to filaments,while tyrosol accelerates germ tube formation, suggesting thatthe fungus can react both positively and negatively in responseto environmental stimuli. The close apposition of the Pneumocystisorganisms in the biolayers creates an ideal environment forexchange of chemical signals that could mediate mating or changesin cell cycle. Sensitivity to relatively low concentrationsof farnesol supports the contention that a similar communicationsystem may be operational in Pneumocystis biofilms. Alternatively,quorum-sensing molecules play a role in staving off other microbialpredators, as in the case of the inhibitory effects of farnesolon the swarming motility in Pseudomonas aeruginosa strains isolatedfrom cystic fibrosis patients (40). Farnesol could also be actingin this manner by reducing the viability of the P. carinii.

In contrast to a previous study that reported to have establisheda long-term culture method for P. carinii using culture insertsthat required daily supplementation with S-adenosylmethionine(41), we found that this supplement was not required and, ifadded, caused a dramatic fall in viability to undetectable levelsby 24 h (data not shown). In addition, the morphological changesdocumented herein were not reported in the other study, andit is doubtful that these two systems are comparable.

We propose that biofilm formation, documented here in an invitro setting, occurs in the mammalian lung and confers severaladvantages for the survival of Pneumocystis. The multilayeredstructure and morphological changes offer protection from hostimmune responses, such as oxygen free radicals, by the natureof the extracellular matrix and refractile layer, through thecreation of a barrier between the organism in this matrix andthe alveolar lumen, where host immune cells reside. Phagocytosiswould be physically hindered by the large and extensive structuresthat could not be engulfed by macrophages. Glucan in the cellwall of P. carinii has been shown to mediate inflammatory cytokineproduction through NF- ${kappa}$ B-dependent mechanisms (19), and the increasedelucidation of (1,3)-β-D-glucan by Pneumocystis biofilmscould further exacerbate this process, thereby increasing thepathology of the immune responses associated with the pneumonia(58). Like for other fungal biofilms, it is anticipated thatthe biofilm structures will decrease the efficacy of anti-Pneumocystisdrugs. Preliminary data in our laboratory suggest that thisis the case.

The ability of Pneumocystis to survive in an environment withreduced availability of oxygen (26) is in keeping with the microenvironmentsproduced by the layers of the biofilms and would be an additionalsurvival mechanism that coincides with the production of thesestructures. Finally, little is known about the way in whichPneumocystis organisms disseminate from one alveolus to thenext to produce pneumonia or new areas of colonization. It isreasonable to speculate that the large extensive mats of thebiofilm facilitate the process of emigration to other alveoliusing a process similar to that of other fungi which use hyphalor pseudohyphal extension or by detachment of portions of thebiofilm to seed uninfected alveoli. Alternatively, specializedcells could migrate from the established biofilms to colonizeother areas of the lung.

The significance of the novel in vitro system that supportsPneumocystis biofilms described here is severalfold. It providesinvestigators with organisms that remain viable in vitro forweeks, permitting approaches such as the establishment of atransformation system as a genetic tool, long-term assessmentof anti-Pneumocystis drugs with the ability to passage the treatedorganisms to evaluate whether the agents are static or cidal,and an opportunity to perform life cycle studies to better understandthe metabolic capacities of this family of fungal organisms.Moreover, the concept of biofilm formation by Pneumocystis providesnew insights into the mode of replication and disseminationin the mammalian lung and offers a potential survival strategyfor these organisms in the hostile milieu of this environmentby affording protection from the immune response and other defensesby the host or a shelter from therapeutic agents.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Service, Departmentof Veterans Affairs, and the National Institutes of Health (grantsR01AI076104 and R01AI050450).

FOOTNOTES

* 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

Published ahead of print on 26 September 2008.

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

REFERENCES

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