Factors influencing the cell adhesion and invasion capacity of Mycoplasma gallisepticum

Abstract

Background: The cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, Rhigh and Rlow, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.

Results: Collagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain Rhigh, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that Rhigh as well as Rlow are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.

Conclusion: ECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.

Figure 1

Influence of ECM molecules on adhesion and cell invasion of M. gallisepticum incubated with RBC. Mycoplasma gallisepticum R_low (solid bars) and R_high (open bars) were incubated with chicken RBC alone (w/o) or in combination with the ECM molecules fibronectin (Fn), plasminogen (Plg), plasminogen and urokinase plasminogen activator resulting in plasmin (PU), ϵ-aminocaproic acid (EACA), ϵ-aminocaproic acid and plasminogen (EP), and ϵ-aminocaproic acid, plasminogen, and urokinase plasminogen activator (EPU). The values for the adhesion (A) or cell invasion rates (B) represent the means of at least three independent experiments performed in triplicate (R_low) or duplicate (R_high) ± standard deviations. A significant difference to RBC alone (p < 0.05) is indicated by an asterisk (*).

Figure 2

Influence of ECM molecules on adhesion and cell invasion of M. gallisepticum incubated with HeLa cells. Mycoplasma gallisepticum R_low (solid bars) and R_high (open bars) were incubated with HeLa cells alone (w/o) or in combination with the ECM molecules fibronectin (Fn), plasminogen (Plg), plasminogen and urokinase plasminogen activator resulting in plasmin (PU), ϵ-aminocaproic acid (EACA), ϵ-aminocaproic acid and plasminogen (EP), ϵ-aminocaproic acid, plasminogen, and urokinase plasminogen activator (EPU), collagen type IV (Coll IV) or heparin (Hep). Furthermore, HeLa cells were pre-treated with methyl-β-cyclodextrin (MβCD). The values for adhesion (A) or cell invasion rates (B) represent the means of at least three independent experiments performed in triplicate (R_low) or duplicate (R_high) ± standard deviations. Significant differences (p < 0.05) are indicated by asterisks.

Figure 3

Detection of fibronectin in cultures of R_low and R_high. The occurrence of fibronectin in whole washed cells and cell lysates of M. gallisepticum strains R_low and R_high was analyzed in Western blot (A) and dot blot (B) assays by use of a fibronectin-specific antibody. Purified fibronectin (Fn) as well as the reference culture S. pneumoniae DSM20566 (+) were used as positive controls, while E. coli DH10B (−) served as a negative control. A high molecular weight band comigrating with purified fibronectin could be detected in cell lysates of R_low and R_high(A). Sizes of the molecular weight markers (MWM) are given in kDa next to the arrows. Dot blot analyses (B) indicated the binding of fibronectin to the bacterial cell surface of both M. gallisepticum strains, R_low and R_high.

Figure 4

Detection of plasminogen in cultures of R_low and R_high. R_low and R_high were also equally well stained on dot blot assays using a plasminogen-specific antibody (A). As positive controls Streptococcus canis strain G1 (+) [38] and plasminogen (Plg) were used, while S. canis strain G2 (−) [38] or HFLX medium served as negative controls. On Western blots, prominent protein bands of 158, 89, 64 and 53 kDa were stained equally well in cell lysates of R_low and R_high(B). MWM, molecular weight marker.