Factors influencing the cell adhesion and invasion capacity of Mycoplasma gallisepticum
© Fürnkranz et al.; licensee BioMed Central Ltd. 2013
Received: 3 May 2013
Accepted: 31 August 2013
Published: 5 September 2013
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.
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.
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.
For a long period of time, Mycoplasma spp., very small, wall-less prokaryotes, were considered obligate extracellular bacteria until in 1989 Lo et al. described intracellular organisms in an AIDS-patient, which were later identified as M. fermentans. To date, other Mycoplasma spp., such as M. penetrans, M. genitalium, M. pneumoniae, M. suis, M. synoviae, M. bovis, M. hyorhinis, and M. hominis have likewise been shown to be capable of invading non-phagocytic eukaryotic cells. Mycoplasma gallisepticum, the causative agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys has been shown to be able to invade HeLa cells and chicken embryo fibroblasts (CEF) in vitro and chicken red blood cells (RBC) in vitro and in vivo. Furthermore, M. gallisepticum has been proven to cross the mucosal barrier and to spread systemically in vivo. However, even closely related M. gallisepticum strains can differ markedly in their pathogenicity for chickens [13, 14]. The strains Rlow and Rhigh derive from different passages of strain R grown in artificial medium [14, 15]. The low-passage, hemadsorption-positive and virulent strain Rlow (10th passage) was shown to be cell-invasive in vitro and in vivo, whereas the high-passage, hemadsorption-negative strain Rhigh (164th passage) displays a highly reduced virulence and exhibits only marginal cell invasiveness [10–12, 16].
Many publications over the past years addressed the bacterial proteins involved in mycoplasma cell adhesion and invasion ([17–19] for reviews). However, host factors involved in cell invasion have been investigated to a much lesser extent. The extracellular matrix (ECM) occupies the space between animal cells and is composed of secreted proteoglycans and non-proteoglycan polysaccharides or proteins like collagens, fibronectin, and laminin [20, 21]. These macromolecules influence adhesion, migration, proliferation, and differentiation of eukaryotic cells , and they serve also as substrate for the attachment and internalization of pathogenic microorganisms [23, 24]. ECM- and plasminogen-binding properties have also been reported for a limited number of Mycoplasma species like M. penetrans, M. hyopneumoniae[26, 27], M. bovis and M. gallisepticum[29–31]. So far, only for M. fermentans and M. pneumoniae an influence of ECM molecules and plasminogen on adhesion and invasion capabilities has been documented [32–34]. For M. gallisepticum strains Rlow and Rhigh, published data is limited to differential binding properties of the organism to fibronectin , heparin  and plasminogen .
Another host factor that plays a role in bacterial invasion processes is cholesterol, the major component of lipid rafts . Cholesterol seems to play a major role in the invasion process of M. fermentans, as invasion rates were 70% lower in cholesterol-depleted HeLa cells, whereas adhesion rates were not influenced .
In the current study, our aim was to investigate hemadsorption-positive and -negative strains of M. gallisepticum for their capability to adhere to and invade HeLa cells and chicken red blood cells in the presence of selected ECM molecules and plasminogen. The role of cholesterol availability on the host cell membrane for the adhesion and invasion of M. gallisepticum was also examined.
Materials and methods
Cultivation of host cells and bacteria
Escherichia coli DH10B, Streptococcus pneumoniae type strain DSM20566 (DSMZ, Braunschweig, Germany) and Streptococcus canis strains G1 and G2 (obtained from G.S. Chhatwal, Helmholtz Center for Infection Research, Braunschweig, Germany) were used as controls in fibronectin and plasminogen binding assays. M. gallisepticum strains Rlow and Rhigh were originally provided by S. Levisohn, Kimron Veterinary Institute, Bet Dagan, Israel. Mycoplasma cultures were grown in modified Hayflick medium  containing 20% (vol/vol) heat-inactivated horse serum (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) and 100 IU penicillin per ml (HFLX). Solid medium agar plates were produced by adding 1% (wt/vol) bacteriological agar (Agar No. 1; Oxoid Deutschland GmbH, Wesel, Germany) to HFLX. Numbers of viable bacteria [colony forming units (CFU)] were determined as described elsewhere .
Chicken red blood cells (RBC) from female Lohmann Brown chicken, kindly provided by C. Hess (Clinic for Avian, Reptile, and Fish Medicine, University of Veterinary Medicine Vienna, Austria), were washed twice with PBS and working suspensions were adjusted to 2 × 108 RBC per ml in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) containing 10% (vol/vol) fetal calf serum (FCS, Gibco BRL) and 5% (vol/vol) tryptose phosphate broth (Sigma). Since HeLa cells were previously used as model organisms in many mycoplasma invasion assays (e.g. [10, 33, 34]), we included this cell line. For the Gentamicin Invasion Assay (GIA), cells from the human epithelial-like cell line HeLa-229 (ATCC CCL-2.1; Manassas, VA) were washed three times with PBS, trypsinized for 10 min (0.05% trypsin-EDTA; Gibco BRL), subsequently seeded in 24-well cell culture plates (Greiner Bio-One GmbH, Kremsmünster, Austria; 5 × 104 cells per well) and cultured for 2 days at 37°C in a 5% CO2 atmosphere.
Adherence and invasion assays
The high degree of sequence homology between human, bovine and chicken fibronectin renders them experimentally interchangeable , which prompted us to use human fibronectin for adherence and cell invasion assays. All ECM molecules, cellular fibronectin (F2518), plasma fibronectin (F2006), collagen type IV (C5533), collagen type V (C3567) and porcine heparin (H3149), as well as plasminogen (P7999), urokinase plasminogen activator (No.124; American Diagnostica Inc., Stamford, CT), ϵ-aminocaproic acid (EACA) (A2504), and methyl-β-cyclodextrin (MßCD; C4555) were of human origin and were purchased from Sigma-Aldrich, unless otherwise indicated. The molecules were used at the following concentrations: 25 μg/ml for fibronectins, plasminogen, and collagens, 200 μg/ml for heparin, 100 μg/ml for EACA, 20 U/ml for urokinase plasminogen activator and 6 mg/ml for MβCD.
Adherence and invasion rates of M. gallisepticum were analyzed together in a modified GIA as described elsewhere [10, 11]. Briefly, for the GIA using RBC, an overnight culture of M. gallisepticum was centrifuged at 10,000 × g for 10 min, resuspended in DMEM and diluted to reach a final concentration of 4 to 8 × 104 CFU per ml. One ml of this suspension was mixed with 20 μl of a stock solution of the respective ECM molecules. A 250-μl aliquot of this mixture was incubated with 250 μl DMEM containing gentamicin (final concentration 400 μg/ml) for 3 h at 37°C to verify the lethal concentration of gentamicin. The residual 750 μl of the mixture was co-incubated for 2 h at 37°C with 750 μl RBC in DMEM containing 1.5 × 108 RBC. A 500-μl aliquot of this mixture was then diluted 1:10 in PBS and 100-μl samples were plated on HFLX agar plates to count the number of input bacteria. The remaining ECM-mycoplasma-RBC mixture was divided into 2 samples, and centrifuged at 300 × g for 3 min. The resulting pellets (two per ECM molecule) were washed once with PBS to remove non-adherent bacteria, and resuspended in 500 μl DMEM each. One resuspended pellet was further diluted 1:10 in PBS and plated on HFLX agar plates to determine the total number of adherent and intracellular bacteria. The other resuspended pellet was treated with 500 μl DMEM containing gentamicin (final concentration 400 μg/ml) and incubated for 3 h at 37°C. After the gentamicin treatment, 100 μl-samples were plated to count the intracellular bacteria.
The GIA using HeLa cells was carried out in 24-well cell culture plates. If HeLa cell membranes had to be depleted from cholesterol, the cells were preincubated in the presence of 5 mM MβCD for 30 min at 37°C and 5% CO2 as described elsewhere . After thoroughly washing to remove any MβCD, HeLa cells were co-incubated with 500 μl-samples of ECM-treated bacteria (4 to 8 × 104 CFU per ml DMEM) for 2 h at 37°C in a 5% CO2 atmosphere (2 wells per ECM molecule). After 2 h of incubation, the HeLa cells were washed with PBS to remove non-adherent mycoplasma, trypsinized for 10 min (0.05% trypsin-EDTA; Gibco BRL) and the cells of one duplicate well were treated with 1 ml DMEM containing gentamicin (400 μg/ml) to kill all extracellular mycoplasma. After 3 h of incubation, the number of intracellular mycoplasma was determined by plating 100 μl of serial dilutions in PBS onto HFLX agar plates. The other duplicate well was used to determine the number of adherent mycoplasma and treated the same way except that no gentamicin was added. The number of input bacteria was determined from serial dilutions of mycoplasma samples before ECM treatment. All experiments were arranged in duplicate assays and repeated two (Rhigh), or three (Rlow) times, respectively.
Lysates of whole cell protein were produced from 1-ml samples of M. gallisepticum grown in HFLX or E. coli grown in Luria-Bertani (LB) medium according standard methods. For S. pneumoniae, biomass was collected directly from Columbia III agar plates with 5% sheep blood (Becton Dickinson, Heidelberg, Germany). Denatured whole cell proteins were separated by SDS-gel electrophoresis and transferred to nitrocellulose membranes (Whatman, Dassel, Germany). The blots were blocked with 2% blocking reagent (Blotting Grade Blocker, Bio-Rad) and incubated with either anti-fibronectin or anti-plasminogen antibodies (ABIN125555 or ABIN285631; antibodies-online GmbH, Aachen, Germany), diluted 1:1,000 in TBS. Horseradish peroxidase-labeled goat anti-rabbit IgG (P0217; DAKO Hamburg, Germany), diluted 1:2,000, was used as the secondary antibody, and the blots were developed using 4-chloro-1-naphthol (4CN) according to the manufacturer’s instructions (Bio-Rad).
One-ml samples of overnight cultures of Rhigh, Rlow, E. coli, S. pneumoniae and S. canis were washed in equal amounts of PBS and five μl were dropped onto nitrocellulose and allowed to dry. Blocking and further processing was performed as described for Western blots.
Invasion and adhesion frequencies are expressed as the mean ± standard deviation of n independent values. Statistical analysis of the data was performed running the SPSS 18.0 software package (SPSS Inc. Chicago, USA). Group means were compared by Single-factor ANOVA and post hoc Tukey test, and P-values of < 0.05 were considered statistically significant.
However, standard deviations were high and thus differences were not statistically significant. The previously reported low cell invasion rates of Rlow for RBC  could be confirmed and were not notably influenced by any ECM molecule (Figure 1B). A slight decrease in the mean invasion rate was observed in the presence of plasminogen. Rhigh showed almost no cell invasion capabilities in this set-up; only with plasminogen a significantly increased cell invasion (p < 0.05) was observed (0.014 ± 0.003%).
Cell invasion rates of Rlow into HeLa cells were increased by the addition of plasminogen, plasmin and collagen type IV (Figure 2B). The combination of EACA with either plasminogen or plasmin decreased invasion rates. Surprisingly, we also recorded enhanced cell invasion rates of Rlow with EACA alone, compared to the mean invasion rates of Rlow alone (Figure 2B).
To get an insight into the role of cholesterol/lipid rafts concerning the cell invasion and/or adhesion process, HeLa cell membranes were depleted from cholesterol using MβCD prior to the addition of the bacteria. Whereas cholesterol depletion had a positive effect on the adhesion of Rlow (Figure 2A), cell invasion was slightly decreased (Figure 2B). However, the observed differences in cell invasion were not statistically significant.
ECM molecules, like fibronectin or vitronectin, have been described to play a strategic role for pathogenic bacteria. They serve as a bridge component between the pathogen that binds to the ECM molecule and to host cells, which naturally have receptors for these ECM molecules [39, 40], thereby bringing the bacterium in close contact with the host cell. A more sophisticated exploit of interactions between ECM molecules has been described for Streptococcus pyogenes which is able to adhere to collagen via fibronectin bound to the bacterial surface . Plasminogen might act similarly as a bridging component in rare events . However, for cell invasive bacteria plasminogen might be of better use in its activated form, the serine protease plasmin. Besides exhibiting a crucial role in fibrinolysis and ECM remodeling, the proteolytic activity of plasmin could empower the plasmin-equipped bacterium more directly to penetrate host cells or transmigrate through monolayers, thereby assisting to cross the epithelial barrier [42–44]. M. fermentans has been shown to profit from binding to plasminogen or ECM molecules. When co-incubated with HeLa cells, M. fermentans exhibited an increased adherence to HeLa cells when pretreated with collagen type III or IV or plasminogen [32, 34]. In this study, we report the likewise positive effect of plasminogen, as well as a slight enhancement due to fibronectin on the adherence capacity of M. gallisepticum. Fibronectin and plasminogen can of course be found in the serum used in the culture medium, which was proven by the ability to detect these molecules on the surface of Rlow and Rhigh without further adding them (Figures 3 and 4). Nevertheless, to study their distinct effects on the invasion and adhesion capabilities of Rlow and Rhigh, fibronectin and plasminogen were added in abundance. The hemadsorption-positive strain Rlow showed an increased adherence to HeLa cells when preincubated with plasminogen. Strain Rhigh, lacking the major cytadherence proteins GapA and CrmA, exhibits drastically decreased cytadherence capacities that have been well documented . However, it seems that the rather marginal adhesion rates of Rhigh to HeLa cells can be enhanced by plasminogen, plasmin and fibronectin (Figure 2A). The recruitment of plasmin or fibronectin as a bridging molecule could represent a secondary anchoring system of M. gallisepticum besides the primary and accessory cytadhesion proteins like GapA and CrmA. As, on the other hand, adhesion rates of Rhigh with plasmin or fibronectin were still lower than adhesion rates of Rlow alone, it seems that the loss of GapA and CrmA on bacterial site cannot be overcome by the ECM-mediated adhesion.
The positive effect of fibronectin on the adhesion capacity of Rhigh to HeLa cells was surprising, as in 2006 Rhigh was reported to lack fibronectin-binding proteins . When we tested whole cells and whole cell-lysates of M. gallisepticum strains Rlow and Rhigh for fibronectin-binding properties, it turned out that fibronectin could be detected on the surface of mycoplasma whole cells as well as in cell lysates, irrespective of the strain tested. In contrast to May et al., who investigated the TX-114 insoluble proteins of M. gallisepticum Rhigh, we did not exclude the cytoplasmic proteins from our analyses. In M. pneumoniae, also cytoplasmic proteins like EF-Tu and PDHB were shown to appear at the surface of the mycoplasma cell and to bind fibronectin . Also in other Mycoplasma species like M. genitalium, EF-Tu was found to be present at the cell surface . Interestingly, although the EF-Tu proteins of M. genitalium and M. pneumoniae share a 96% identity, the M. genitalium protein does not bind fibronectin. Recently, Balasubramanian et al. showed that 3 amino acid residues (S343, P345, and T357) of the M. pneumoniae EF-Tu were essential for its binding activity to fibronectin, and that these critical amino acids are changed to alanine residues in the EF-Tu of M. genitalium. The EF-Tu of M. gallisepticum, on the other hand, shares a lower overall identity with the EF-Tu of M. pneumoniae, but is higher conserved for these three Fn-binding residues (Q343,K345, T357), leaving it open to speculate about an involvement of M. gallisepticum Rhigh EF-Tu in the adhesion process by fibronectin binding.
Detection of fibronectin on the surface of M. gallisepticum, routinely cultured in HFLX, indicates that the fibronectin concentration in the horse serum is high enough to allow the pathogen to adsorb substantial amounts. Absorbance of protein components from the growth medium by M. gallisepticum has already been reported for transferrin or immunoglobulins  and for the latter IgG-binding proteins have been recognized . Whether binding of fibronectin plays a role for M. gallisepticum in vivo, remains to be elucidated.
The influence of another family of ECM molecules, collagen type IV and V, onto M. gallisepticum adhesion and invasion was also investigated in the present study. Whereas collagen type V revealed no marked effects (data not shown), type IV collagen had a slightly positive effect on the adherence and invasion capacity of Rlow to HeLa cells. For M. fermentans a supporting effect of collagen type III and V on the adherence capacity to HeLa cells had been reported . In contrast, collagen type IV had no effect on M. fermentans adherence. This difference in the mode of action concerning the same family of compounds (collagens) could perhaps be explained by the different nature of the three collagens investigated, as collagen type III and V exhibit fibrillar structures, and are mostly found in extensible connective tissue, whereas collagen type IV is rather linked to the basal lamina. Concomitantly, this difference might also reflect the different natural habitats of M. fermentans and M. gallisepticum.
Plasminogen is not an ECM molecule per se but after its full activation to plasmin, it exerts a serine protease function on the fibrinolytic system, as well as on many ECM molecules. Binding of plasmin or plasminogen to fibrin and other ECM molecules predominantly occurs via its high- and low affinity lysine-binding sites (LBS) , and therefore can be easily blocked by EACA, a lysine analogue. If plasminogen is used as a bridging molecule by M. gallisepticum, then binding of EACA to the LBS of plasminogen should compensate for any positive effect of plasminogen on adherence and/or invasion. The results from the invasion assays support this hypothesis, as Rlow, pretreated with plasmin or plasminogen (Figure 2B), showed higher invasion rates to HeLa cells than the untreated control and addition of EACA indeed had a negative impact on the plasminogen-elevated invasion rates. For M. fermentans, plasminogen was shown to enhance cell invasion as well as cell adhesion [32, 51], whereas no effect was seen with M. pneumoniae. When we analyzed the cell adherence of M. gallisepticum, an increase after treatment with plasminogen or plasmin was observed (Figure 2A). To our surprise, however, EACA had no negative effect on the plasminogen-elevated cell adhesion, but rather boosted the cell adhesion. Until recently, EACA has been described as a potent inhibitor of plasminogen-binding to bacteria, [51, 52], no one has yet investigated the effect of EACA alone on bacterial cell invasion or adhesion capacity. The increase in cell adhesion of Rlow and Rhigh in the presence of EACA alone (Figure 2A) may indicate that another M. gallisepticum surface protein is participating in the multifactorial adhesion process. It is well established that M. gallisepticum contains several cell surface proteins with hemagglutinating (pMGA/VlhA ), or adhesive properties like Mgc2 , α-enolase  or the uncharacterized proteins P30, P48, P50, P80 .
Cholesterol depletion from HeLa cell membranes resulted in a decreased invasion capacity of M. gallisepticum, suggesting that cholesterol-rich regions in the cell membrane like lipid rafts are needed for the invasion process. Many bacteria have been described to enter host cells via receptors being present in lipid rafts and/or caveolae [reviewed by [56, 57]. An alternative strategy for mycoplasma entry could be the engulfment via the lipid rafts. So far, entry into eukaryotic cells via engulfment without the use of receptors has only been described for very small objects like viruses and bacterial toxins . Mycoplasmas belong to the smallest bacteria, they do not contain a cell wall, and their bodies seem easily deformable. M. gallisepticum has been observed to squeeze itself through 220-nm  and apparently even 100-nm pore filters (own observation). As lipid rafts in vivo have diameters up to 700 nm , M. gallisepticum are flexible and small enough to maybe enter the eukaryotic cell by squeezing through lipid rafts. As envisioned by others before [17, 59] the fusogenic properties of the mycoplasma lipidic cell wall might additionally enable the pathogen to interact with the lipid-rich raft domains. While cholesterol depletion lowered the cell invasion rates of M. gallisepticum, adherence capabilities to HeLa cells were improved. This could be explained by the fact, that cholesterol removal from membranes results in dispersion of the raft-associated lipids and proteins , thus destroying the possible entry site, but at the same time generating more sites for the mycoplasma to adhere to. With M. fermentans, plasminogen-preincubated bacteria adhered to cholesterol-depleted HeLa cells as well as to cholesterol-containing cells, but internalization of M. fermentans was almost completely inhibited after cholesterol depletion .
Taking these results together, it seems that M. gallisepticum is utilizing a second anchoring system besides its own primary and accessory cytadhesion proteins like GapA and CrmA that relies on the recruitment of ECM- or ECM-like molecules like collagen type IV, fibronectin or plasminogen/plasmin. Cholesterol depletion of HeLa cells had contrary effects on the adherence and cell invasion of M. gallisepticum, calling us to investigate the role of lipid rafts on the host cell surface in these mechanisms.
We gratefully thank Drs. M. Fulde and G.S. Chhatwal (Helmholtz Center for Infection Research, Braunschweig, Germany) for providing us with S. canis strains. This work was supported by grant P20712 from the Austrian Science Fund FWF.
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