Biofilms Protect Mycoplasma pulmonis Cells from Lytic Effects of Complement and Gramicidin

The length of the tandem repeat region of the Vsa protein ofMycoplasma pulmonis has previously been shown to modulate thesusceptibility of mycoplasmas to killing by complement: cellsthat produce a short form of the Vsa protein are highly sensitive,and cells producing the long Vsa protein are resistant. In contrastto their differing susceptibilities to complement, the mycoplasmaswere highly sensitive to gramicidin irrespective of the lengthof the Vsa protein produced. We show here that when encasedwithin a biofilm, cells of M. pulmonis producing a short formof the Vsa protein were more resistant to complement and gramicidinthan mycoplasmas that were dispersed. The resistance appearedto be localized to those mycoplasmas within tower structuresof the biofilms. Biofilm formation may be a mechanism that protectsmycoplasmas from host immunity.

INTRODUCTION

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INTRODUCTION

Biofilms are communities of microorganisms encased in an extracellularmatrix of polysaccharide, lipid, DNA, and protein (7). A majorfunction of a biofilm is to protect the microbial cells fromantimicrobial agents (3) and immune surveillance (15). Generally,biofilms are structurally organized into honeycombed regionsand areas of outgrowths referred to as mushrooms or towers (16).Biofilms formed in vitro by the mycoplasmas contain all of themolecular and structural features found in biofilms that areformed by other bacteria (9, 11). The tower structures containmycoplasmal cells that are densely packed together relativeto the honeycombed region.

The variable surface antigens (Vsa proteins) of Mycoplasma pulmonismodulate numerous properties of the mycoplasma, including susceptibilityto complement, susceptibility to phage, and the abilities tohemadsorb and to form a biofilm (11-13). Differences in thesize of Vsa result from the loss or gain in the number of tandemrepeat units via slipped-strand mispairing in the gene's 3'repetitive region (10). Mycoplasmas that produce a Vsa proteincontaining about 40 tandem repeats do not form biofilms (11)and are resistant to killing by complement but susceptible tothe antimicrobial protein gramicidin (12, 13). Mycoplasmas thatproduce a Vsa protein containing a few tandem repeat units,such as M. pulmonis strain CT182-R3, are efficiently killedby both complement and gramicidin. It has been proposed thatthe long Vsa proteins sterically hinder the access of largermolecules, such as complement, to the mycoplasma cell membranewhile permitting the access of smaller molecules such as gramicidin(12).

The mycoplasmas used in these previous studies were dispersedinto the reaction medium and were readily accessible to complementand gramicidin. M. pulmonis strain CT182-R3 grows as a biofilm(11). To determine whether the biofilm was protective, we incubatedintact mycoplasma biofilms or mycoplasma cells that were dispersedfrom biofilms with complement or gramicidin. We found that thebiofilm protected the mycoplasmal cells. Furthermore, the resistanceappeared to be localized to the tower structures in the biofilms.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Mycoplasma strains. M. pulmonis strain CT182-R3 produces a VsaA protein containingthree tandem repeats and has been shown to be sensitive to killingby complement (12, 13). The starter stocks of strain CT182-R3used for these experiments are the stocks used in previous publications.The mycoplasmas were grown for 48 h on 22-mm by 22-mm glasscoverslips (11) in 12-well polystyrene tissue culture plates,or in 6-well plates where indicated (Corning Incorporated, Corning,NY), in mycoplasma broth supplemented with 20% heat-inactivated(HIA) whole horse serum (HyClone).

Complement killing assays. Mycoplasma biofilms that had adhered to tissue culture plateswere washed three times with phosphate-buffered saline (PBS)and either left intact or dispersed by scraping and pipetting.The dispersed cells or the biofilms were incubated with guineapig serum (GPS; Colorado Serum Company, Denver, CO) or HIA-GPSat a final concentration of 10% at 37°C for 30 min in thepresence of Mg²⁺ and Ca²⁺ at final concentrations of 5 mM and1 mM, respectively. The biofilms were then dispersed by scrapingand pipetting. The mycoplasmas were gently sonicated to disruptcell aggregates and serially diluted to determine the numberof CFU recovered from the reactions. The percentage of CFU recoveredfor each replicate well was represented as the fraction (x 100)of CFU recovered after complement treatment relative to theCFU surviving treatment with HIA-GPS. Data were analyzed bythe Student t test.

In some experiments, the GPS-treated biofilms were washed withPBS, either left intact or dispersed into PBS, and incubateda second time with a 10% concentration of GPS or HIA-GPS for30 min at 37°C. After the second round of treatment, themycoplasmas from the reactions were dispersed and sonicated,and the fraction of surviving CFU was determined. Data wereanalyzed by one-way analysis of variance, with pairwise multiplecomparisons performed by the Student-Newman-Keuls method.

Gramicidin killing assays. Mycoplasmal biofilms adhering to 12-well plates were washedthree times with PBS. Five hundred microliters of gramicidinfrom Bacillus aneurinilyticus (Sigma), at a concentration of100 ng/ml in PBS, or PBS alone, was added to the wells. In oneexperimental group, the biofilm was scraped and dispersed bypipetting immediately after the addition of gramicidin. In anotherexperimental group, the biofilm was left intact during the incubationperiod. After incubation for 30 min at 37°C, 1.5 ml of PBSwas added to each well, and the biofilms were scraped and dispersedby pipetting. This fourfold dilution reduced the concentrationof gramicidin to about the MIC for M. pulmonis (12). The reactionmixtures were immediately diluted 100-fold in mycoplasma broth,sonicated, and serially diluted to determine the number of CFUrecovered. The percentage of CFU recovered was expressed asthe fraction (x 100) of CFU from the wells treated with gramicidinrelative to the CFU recovered from wells incubated with PBSonly. The reactions were performed in triplicate for each groupin an experiment, and the experiments were performed twice.Data were analyzed by the Student t test.

Fluorescence microscopy. The biofilms that were grown on 22-mm by 22-mm glass coverslipswere washed three times in sterile PBS. For the reactions withcomplement, the biofilms were incubated with a 10% concentrationof GPS or HIA-GPS for 30 min at 37°C as described above.For reactions with gramicidin, the biofilms were incubated withgramicidin at a concentration of 500 ng/ml or with PBS for 30min at 37°C.

After the biofilms were incubated with complement or gramicidin,the glass coverslips were washed three times in PBS and incubatedin PBS containing 20 µg of Hoechst 33342 and 500 ng ofpropidium iodide (PI) per ml for 30 min. The coverslips werewashed twice in PBS and mounted on glass slides with a solutioncontaining 50% glycerol and 50% Prolong Gold mounting medium(Molecular Probes). Digital images were acquired at a magnificationof x1,600 with a Leica HC fluorescence microscope using theChroma 86012v2 4',6'-diamidino-2-phenylindole or Texas red filterset. Since intact cells are impermeable to PI but permeableto Hoechst 33342, mycoplasma cells that bound PI were interpretedas dead cells.

RESULTS

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RESULTS

Mycoplasmas in a biofilm are relatively resistant to complement. When mycoplasmas were incubated with GPS a single time, about60% of the mycoplasmas were recovered from biofilms that wereleft intact (Fig. 1A) while 25% of the mycoplasmas were recoveredfrom biofilms that were dispersed prior to incubation with GPS(P < 0.001). In separate experiments, the percentage of CFUrecovered from GPS-treated biofilms that were left intact andincubated a second time with GPS did not differ from the percentageof CFU recovered from biofilms that were incubated with GPSonly once (Fig. 1B). About 50% of the CFU were recovered. Incontrast, when the GPS-treated biofilm was dispersed and incubateda second time with GPS, only 6% of the mycoplasmas were recovered(P < 0.001). Similar results were obtained using 6-well platesinstead of 12-well plates. About 50% of the mycoplasmas wererecovered from biofilms that were incubated a single time withGPS, while only 10% of the mycoplasmas that were dispersed fromthe first reaction were recovered when they were incubated asecond time with GPS (data not shown).

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FIG. 1. Killing of mycoplasmal biofilms by complement or gramicidin. Results are mean percentages of CFU recovered from biofilms after incubation with GPS (A and B) or gramicidin (C). Error bars, standard errors of the means. Asterisks indicate statistical significance. (A) Intact biofilms (n = 10) or dispersed biofilms (n = 10) were incubated with GPS. (B) Intact biofilms were incubated with GPS (n = 9) as for panel A. Surviving biofilms were washed, left intact, and incubated again with GPS (n = 9). Dispersed survivors from the intact biofilms were washed, dispersed, and incubated again with GPS (n = 6). (C) Intact biofilms were incubated with gramicidin (n = 6). Dispersed biofilms were dispersed immediately after the addition of gramicidin (n = 6).

Mycoplasmas in a biofilm are relatively resistant to gramicidin. The partial resistance of mycoplasmal biofilms to killing bygramicidin was similar to the resistance to killing by complement(Fig. 1C). About 64% of the mycoplasmas were recovered whenintact biofilms of strain CT182-R3 were incubated with gramicidin.In contrast, only 20% of the mycoplasmas were recovered if thebiofilm was scraped and dispersed immediately after the additionof gramicidin to the wells (P < 0.05).

Mycoplasmas in towers of biofilms are resistant to complement and gramicidin. The relative resistance of the biofilm to lysis by complementor gramicidin appeared to reside in the tower structures (Fig.2). Fluorescence microscopy of biofilms that were incubatedwith GPS or gramicidin and stained with PI and Hoechst 33342revealed a large number of cells in the honeycombed region ofthe biofilm stained with PI (Fig. 2A and E). Very few cellsin the honeycombed region stained with PI in control biofilmsthat were incubated with HIA-GPS or PBS (Fig. 2C and G). Littleuptake of PI was detected in the cells within the tower structuresof the biofilms that were incubated with either GPS (Fig. 2B),HIA-GPS (Fig. 2D), gramicidin (Fig. 2F), or PBS (Fig. 2H). However,some of the cells within the towers stained with PI, indicatingthat the cells within the towers were accessible to PI.

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FIG. 2. Fluorescent images of biofilms of M. pulmonis. Red, DNA of cells stained with PI; blue, DNA of cells stained with Hoechst 33342. Images on the left show the honeycomb regions of the biofilms. Images on the right show the tower structures of the biofilms. Biofilms were incubated with either GPS (A and B), HIA-GPS (C and D), gramicidin (E and F), or PBS (G and H). The scale bar is in red.

DISCUSSION

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DISCUSSION

The length of the Vsa tandem repeat region modulates susceptibilityto complement (12, 13), and all strains of M. pulmonis testedto date are sensitive to gramicidin when the cells are dispersed(5, 6, 12). The results presented here indicate that biofilmformation protects mycoplasmas from antimicrobial agents andthe innate immune system. Within the honeycomb region of thebiofilm, cells are sensitive to lysis by complement or gramicidin,but cells in the towers are relatively resistant. Although gramicidinis produced by Bacillus aneurinilyticus, the murine host ofM. pulmonis produces other antimicrobial peptides. Mycoplasmascan form aggregates on epithelial or subepithelial surfacesin vivo (14). It follows that if such aggregates constitutea biofilm, the mycoplasmas will be partially protected fromcomplement and antimicrobial peptides. Since other mycoplasmascan grow as biofilms (9), our findings may be applicable toa wide range of mycoplasma species and perhaps other bacterialpathogens.

The mechanisms that contribute to the resistance of the biofilmsare unknown. The amount of information available on the interactionsof complement and biofilms is limited. Biofilms protect Pseudomonasaeruginosa from killing by serum (1), and the level of complementactivation is lower than that by planktonic cells (8). Complementis clearly being activated by the mycoplasmal biofilm, sincethe cells in the honeycomb region are lysed. Additionally, theresistance of the mycoplasmas encased within a biofilm to killingby GPS did not result from individual cells acquiring resistance,because 90% of the mycoplasmas from GPS-treated biofilms werekilled when they were dispersed and incubated with GPS a secondtime. Therefore, the resistance imparted by the biofilm residesin the structure of the biofilm.

The resistance to complement and gramicidin was limited to thetower structures, a region where the cell density is highestand abundant amounts of polysaccharide are detected (11). Plausibly,either the cell density per se or the polysaccharides in thetowers of the mycoplasma biofilms could contribute to the resistance.Extracellular polysaccharide can protect bacteria from the lyticeffects of antimicrobial peptides (2, 15). Nevertheless, theintegrity of the mycoplasmal cells in the honeycombed regionswas compromised by the complement terminal lytic complex, whilethe towers hindered the access of at least one of the complementcomponents to the cells within the towers. The towers also hinderedthe access of gramicidin, a small pore-forming peptide, to themycoplasmas. Consistent with these results, antibodies to VsaAdo not penetrate the towers of the biofilms, and it seems likelythat antibody-mediated killing of the mycoplasmas would be hindered(11). These results suggest that the formation of towers invivo could modulate resistance to complement and antimicrobialpeptides secreted by host immune cells and could also protectthe mycoplasmas from adaptive immune responses. In support ofthis, M. pulmonis cells that produce a short form of the Vsaprotein (VsaH) and grow as a biofilm (11) have been isolatedfrom the lungs of experimentally infected mice (4).

ACKNOWLEDGMENTS

We thank Portia Caldwell and Abha Soni for expert technicalassistance.

This work was supported by Public Health Service grant A164848from the National Institutes of Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Genetics, University of Alabama at Birmingham, KAUL 720, Birmingham, AL 35294-0004. Phone: (205) 934-2794. Fax: (205) 975-4418. E-mail: wsimmons{at}uab.edu

Published ahead of print on 21 May 2007.

Editor: A. Camilli

REFERENCES

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