A Stochastic Mechanism for Biofilm Formation by Mycoplasma pulmonis

Abstract

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.Biofilm formation can enhance the resistance of pathogens to antimicrobial agents (6) and immune surveillance (36). This has been attributed in part to the encasement within an extracellular matrix of the biofilm that isolates and protects the bacteria from the host immune response. The extracellular matrix of a biofilm is complex and usually contains lipid, protein, DNA, and exopolysaccharide (1, 2, 4, 38). Most studies have focused on the complexity of the molecular systems involved in the formation of the matrix constituents. The formation of biofilms by some bacteria is dependent on two-component and quorum-sensing systems that regulate macromolecules necessary for attachment and matrix synthesis (24). A recent trend is to describe biofilm formation in the context of a multicellular structure that develops as a result of programmed gene expression (7,21).Few studies address the formation of biofilms from the viewpoint of simplicity. The recent finding that mycoplasmas can form biofilms (19) provides us with a model in which microorganisms using minimally regulated genetic systems, or perhaps stochastically modulated genetic systems, can form biofilms. The mycoplasmas are the smallest and simplest of self-replicating cells (11,22). Although some mycoplasmas can produce complex structures such as an attachment organelle, their genomes encode only a few hundred proteins. There are no known two-component regulatory systems or other recognizable global regulators in most species of mycoplasmas, including Mycoplasma pulmonis. The lack of such regulators can be attributed to the survival of the mycoplasmas in only one environmental niche: the animal host.The variable surface antigen (Vsa) proteins of the murine respiratory pathogen M. pulmonis are associated with virulence (34) and media...

The Vsa Shield of Mycoplasma pulmonis Is Antiphagocytic

“…Alternatively, the mycoplasmas producing a short Vsa may bind to MH-S cells nonspecifically. Mycoplasmas producing a short Vsa protein are proficient at biofilm formation and bind well to many surfaces, including the epithelium and other host cells (37,39,40). It is important to note that over the course of the killing assays we did not observe a decrease in the numbers of AM, indicating that the mycoplasmas were not detrimental to these cells.…”

“…Jones and Hirsch grew the mycoplasmas as a biofilm attached to glass coverslips, which were scraped to harvest the cells (27). Growing the mycoplasmas in this manner selects for organisms producing a short Vsa protein (37). Because the Vsa protein would have been short, the large numbers of mycoplasmas bound to the macrophages, as well as the observation that AM were capable of killing any bound mycoplasmas without the addition of trypsin or antimycoplasma antibody, are consistent with the findings of the current study.…”

“…The protein makes up 8 to 10% of the total protein produced by M. pulmonis (37). The variable region consists of a series of tandem repeats whose number varies at a high frequency through slippedstrand mispairing (SSM).…”

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

“…The variable surface antigens (Vsa proteins) of Mycoplasma pulmonis modulate numerous properties of the mycoplasma, including susceptibility to complement, susceptibility to phage, and the abilities to hemadsorb and to form a biofilm (11)(12)(13). Differences in the size of Vsa result from the loss or gain in the number of tandem repeat units via slipped-strand mispairing in the gene's 3Ј repetitive region (10).…”

“…Generally, biofilms are structurally organized into honeycombed regions and areas of outgrowths referred to as mushrooms or towers (16). Biofilms formed in vitro by the mycoplasmas contain all of the molecular and structural features found in biofilms that are formed by other bacteria (9,11). The tower structures contain mycoplasmal cells that are densely packed together relative to the honeycombed region.…”