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Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

A. Yavlovich, M. Tarshis, S. Rottem
DOI: http://dx.doi.org/10.1111/j.1574-6968.2004.tb09488.x 241-246 First published online: 1 April 2004


Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.

  • Mycoplasma pneumoniae
  • Bacterial invasion
  • Atypical pneumonia
  • Respiratory diseases
  • A549 cells
  • HeLa cells

1 Introduction

Mycoplasmas, the smallest and simplest self-replicating bacteria, are pathogens causing a wide variety of diseases. Most mycoplasmas depend on adhesion to host tissues for colonization and infection and adherence is the major virulence factor [1, 2]. The most intensively studied mycoplasma system is that of Mycoplasma pneumoniae. While this organism is known to be a significant cause of acute respiratory illness, the significance of chronic infections is undetermined. In humans, M. pneumoniae has been detected several months after clinical recovery from acute pneumonia [3] and even after therapy with effective antibiotics, M. pneumoniae can still be cultured from respiratory secretions [4, 5]. Recently it has been shown in a murine model that M. pneumoniae can establish a chronic pulmonary infection for up to 18 months after inoculation [6], and the organism was detected in adults with chronic asthma [7]. M. pneumoniae has classically been considered as an extracellular pathogen interacting exclusively and directly with the host cell surface [38]. The bacteria colonizes the bronchial passages adhering to the base of the cilia [8]. This adherence is considered to be an essential step in tissue colonization and subsequent disease pathogenesis [9]. The bacteria has a polar, tapered cell extension at one of the poles containing an electron-dense core in the cytoplasma [10]. This structure, termed the tip organelle, functions as an attachment organelle. A surface 169 kDa protein designated P1 [11] and a 30 kDa protein designated P30 [12] are densely clustered at the tip organelle and are directly associated with the adherence process. In addition, several accessory proteins that promote adherence were identified [13]. On the host cell surface it seems that M. pneumoniae interact with several types of receptors such as sialoglycoconjugates, a sialic acid-free glycoprotein and sulphated glycolipids containing terminal Gal(3SO4)β1-residues [14, 15].

Some bacterial pathogens are capable of entering eukaryotic host cells where the host cytoplasm serves as a permissive environment for intracellular bacterial persistence and growth [1618]. Although it is widely accepted that most mycoplasmas are strict surface parasites adhering to the surface of epithelial cells, indications that some Mycoplasma species are capable to invade host cells were presented awhile ago [19]. Recently, it has been shown that Mycoplasma penetrans is an invasive species [1] and that under certain conditions, Mycoplasma gallisepticum and Mycoplasma fermentans turned to be invasive, being able to survive within non-pagocytic cells [20, 21]. In the present study we show that when a human lung carcinoma cell line (A549) was infected with M. pneumoniae, low but significant levels of bacteria were detected within the host cells. Such internalization may be a mechanism by which this bacteria is able to escape immune surveillance and resist antibiotic treatment establishing a carrier state that may result in a chronic lung disease.

2 Materials and methods

2.1 Bacteria, host cells and growth conditions

Mycoplasma pneumoniae (M129) were cultured in plastic flasks (Nunc, Roskilde, Denmark) containing 60 ml of a modified Chanock medium supplemented with 20% horse serum [22] for 96 h at 37 °C. For metabolic labeling, the bacteria were grown in a medium containing 0.5 μCi ml−1 of [9,10(n)-3H]palmitic acid (53.0 Ci mmol−1). Growth was monitored by recording pH changes in the growth medium. The bacteria were collected by centrifugation at 12,000g for 20 min, washed twice and resuspended in a cold solution of 10 mM Tris–HCl in 250 mM NaCl (pH 7.5; TN buffer) to a protein concentration of 1 mg ml−1. The human lung carcinoma cell line (A549, ATCCslash # CCL-185) was maintained in F12 medium and HeLa cells (human adenocarcinoma cells originating from the cervix, ATCCslash # CCL-42.1) in DMEM medium. The media were supplemented with 10% FCS (Biological Industries, Israel) and the cultures were incubated at 37 °C in 5% CO2.

2.2 Adherence of M. pneumoniae

[3H]Palmitate-labelled M. pneumoniae cells were added to confluent monolayers of HeLa or A549 cells at a multiplicity of infection (MOI) of 100 and incubated at 4 or 37 °C for various periods of time. The cell cultures were then washed twice with phosphate-buffered saline (PBS) to remove non-adherent bacteria, trypsinized (2.5 μg ml−1 trypsin) for 10 min at 37 °C and resuspended in PBS. Aliquots were transferred to vials containing scintillation liquid and counted.

2.3 Plasminogen binding assay

A qualitative determination of plasminogen (Pg) binding to M. pneumoniae and M. fermentans was performed by immunoblot. Mycoplasma cells (0.5 mg cell protein) were incubated with 25 μg of Pg in TN buffer. After 1 h of incubation at 37 °C, the cells were pelleted, washed three times and resuspended in the TN buffer. The bound Pg was released from the mycoplasmas by incubating them in 10 mM of η-amino caproic acid (ηACA) for 20 min at 37 °C. The intact cells were removed by centrifugation at 8000g and the supernatant fluid containing the Pg released was immobilized on a nitrocellulose membrane using a BioRad bio-dot SF microfiltration apparatus that focuses sample to a thin line instead of a circle. The nitrocellulose membranes were processed by: (i) blocking for 1 h at room temperature with skim milk; (ii) incubation for 16 h at 4 °C with goat anti-human Pg anti-serum and (iii) incubation at room temperature for 1 h with horseradish-peroxidase conjugated mouse anti-goat IgG. Blots were developed by using the O-dianisidine substrate (Sigma) according to the manufacturer's recommendations.

2.4 Internalization of M. pneumoniae

Internalization of M. pneumoniae into host cells was determined by the gentamicin resistance assay [23] and by confocal laser scanning microscopy (CLSM) of immunofluorescence stained preparations [24]. For the gentamicin resistance assay, A549 or HeLa cell cultures were seeded in 24-well plates at a density of 105 cell per well. After 24 h of incubation (37 °C, 5% CO2), the cultures were inoculated with 10 μl of M. pneumoniae suspension in TN buffer (MOI of 100, or 1000). The infected cell cultures were incubated for up to 48 h, washed twice with PBS and incubated for an additional 2 h in DMEM medium (1 ml well−1) containing 400 μg ml−1 gentamicin with or without 0.01% Triton X-100 (TX-100). The medium was then removed and the cell cultures were trypsinized for 3–4 min, resuspended, diluted in DMEM medium and plated. As it was crucial to obtain a single-cell suspension, the trypsinization efficiency was routinely checked by microscopic examination, and when HeLa or A549 cell aggregates were visualized they were broken by passing the cell suspension through a syringe needle.

For immunofluorescence staining, a polyclonal rabbit anti-M. pneumoniae anti-serum, kindly provided by J.G. Tully (NIAID, Bethesda, MD), was used. A549 or HeLa cells were infected with M. pneumoniae at a MOI of 100 or 1000 for up to 48 h as previously described [23]. Washed monolayers of the infected cells on coverslips were fixed at room temperature for 10 min with freshly prepared 4% formaldehyde in phosphate-buffered saline (pH 7.4, PBS) containing 2 mM MgCl2. The cells were then washed twice with PBS and incubated for 5 min in 50 mM NH4Cl to quench free aldehyde groups. After two additional washings with PBS, the cells were permeabilized by incubation for 3 min with 0.2% TX-100 in PBS–BSA buffer. The coverslips were then overlaid for 20 min at room temperature with 2% normal horse serum, and after the excess serum was removed the cells were incubated for 60 min at room temperature with rabbit polyclonal anti-M. pneumoniae anti-serum diluted 1:120 in PBS–BSA buffer. Non-bound antibody was removed by dipping the coverslips four times in PBS and the cells were then incubated for 60 min at room temperature with goat anti-rabbit Alexa 488-conjugated IgG serum (Molecular Probes), diluted 1:150 in PBS–BSA buffer. The coverslips were rinsed with PBS and mounted in a solution containing 90% glycerol, 3% DABCO (1,4-diazabicyclo-[2,2]-octane) as an anti-fading agent and 0.1% sodium azide. Foci of fluorescence were observed by confocal microscopy as described earlier [24].

2.5 Analytical methods

Protein was determined by the method of Bradford [25] using bovine serum albumin as standard. Fixation of host cells or of M. pneumoniae was obtained by treatment with 4% paraformaldehyde (PFA) for 3–5 min at room temperature. Immunofluorescent samples were analyzed using a Zeiss LSM 410 confocal system with axiovert 135M inverted microscope and 40×/1.3 PlanNeofluar oil immersed lens. Fluorescence of Alexa 488 (488 nm line of argon laser with 515 nm emission filter) was recorded simultaneously with differential interference contrast (DIC) bright field images. Z-series of optical sections were acquired at spacing steps of 0.6-μm intervals from the surface through the vertical axis of the cells and then stored for further image analysis. The viability of A549 and HeLa cells invaded by M. pneumoniae was observed with the Viability/Cytotoxicity Kit (Molecular Probes). The assay is based on the simultaneous determination of live and dead cells with two fluorescent probes that measure two recognized parameters of cell viability–intracellular esterase activity and plasma membrane integrity.

3 Results and discussion

It is well established that M. pneumoniae adheres to host cell surfaces [1, 3, 4]. In the present study we show that M. pneumoniae strain M129 initially adheres to native HeLa cells as small clusters which subsequently grew into large bacterial aggregates as visualized by CLSM of immunofluorescence stained preparations (Fig. 1(a)). When M. pneumoniae was incubated with PFA fixed-HeLa cells, adherence of the bacteria was lower by 35–45% than that recorded with native HeLa cells (Table 1), and the bacteria were distributed all around the cell surface of the denatured host cells (Fig. 1(b)), suggesting that the receptors for M. pneumoniae on native HeLa cells form clumps upon interaction with the bacteria but remain distributed in PFA fixed host cells. Ideally, adherence of M. pneumoniae would be measured with a human neonatal lung epithelial cell line as adherence to lung epithelial cells is the major step in the pathobiology of this microorganism [3]. Such a cell was not available and we have employed in further studies A549, a cell line derived from an adult lung carcinoma. The A549 line is well differentiated, with many of the characteristics of type II alveolar pneumocytes, including surfactant production [26]. Binding of M. pneumoniae to HeLa and A549 cells was quantified by using [3H]palmitate-labelled mycoplasmas. The binding results obtained with [3H]palmitate-labelled M. pneumoniae were similar to those obtained with [3H]tymidine-labelled mycoplasmas (data not shown). Furthermore, binding of [3H]palmitate-labelled M. pneumoniae to host cells was inhibited by increasing amounts of unlabelled mycoplasmas in the binding assay reaching almost 80% at a 10-fold excess of unlabelled M. pneumoniae. These results indicate that binding of [3H]palmitate-labelled M. pneumoniae represent binding of the microorganism rather then an exchange of radiolabelled lipids between M. pneumoniae and the host cells. Similar adherence levels of M. pneumoniae to HeLa and A549 cells were observed (Table 1). Adherence at 4 °C was about 40–50% of the adherence level recorded at 37 °C, and almost identical levels were obtained with exponential and stationary phase mycoplasmas (data not shown).

Figure 1

Confocal micrographs demonstrating binding of M. pneumoniae to native (a) and PFA-treated (b) HeLa cells.

View this table:
Table 1

Adherence and internalization of M. pneumoniae

Host cellsAdherenceInternalization
(CPM10−6 cells) after:(CFU 10−6 cells) after:
2 h2 h24 h
HeLa9940 ± 900<200<200
HeLa – PFA-treated5500 ± 520<200<200
A5499090 ± 9504200 ± 50016000 ± 1200
A549 – PFA-treated3800 ± 300<200<200
  • M. pneumoniae cells were grown to mid-exponential phase of growth in a medium containing [3H]palmitate. HeLa or A549 cells were infected at a multiplicity of infection of 100 and incubated at 4 or 37 °C for various periods of time. Binding was calculated from the residual radioactivity associated with washed tissue culture cells incubated with the bacteria at 4 °C. Internalization was determined by the gentamicin resistance assay as described in Section 2. Data represent means ± SD from four independent experiments performed in triplicates.

To further study M. pneumoniae internalization, it is essential to differentiate between bacteria adhering to a host cell and those which have been internalized. For this purpose we used the immunofluorescence qualitative technique. This rather simple and reliable technique is based on the differential fluorescent staining of internalized bacteria and those that remain on the cell surface, and is capable to differentiate between bacteria adhering to a host cell and those which have been internalized [24]. After interacting M. pneumoniae with host cells, the infected host cells were permeablized by TX-100 to allow antibodies to penetrate the host cells and stain both extracellular and intracellular mycoplasmas. Using this non-destructive, high-resolution method, infected host cells were optically sectioned, following fixation and immuno-fluorescent labeling, to localize mycoplasmas within the host cell. Single-cell imaging of HeLa cells infected with native M. pneumoniae were permeabilized, incubated with anti-M. pneumoniae antibodies and immunofluorescently stained with a second FITC conjugated-antibody. The results revealed foci of fluorescence only on the cell surface, corresponding to the extracellularly bound mycoplasmas (data not shown), suggesting that M. pneumoniae did not invade HeLa cells under the conditions tested in this study. Similar results were obtained previously with M. fermentans and HeLa cells [21]. Nevertheless, when HeLa cells were infected with M. fermentans preincubated with plasminogen (Pg) in the presence of the Pg activator urokinase, the organism was detected also within the host cells [21]. The possibility that a Pg-dependent internalization process in HeLa cells can be detected also with M. pneumoniae was therefore tested. Fig. 2 shows that the levels of Pg bound to M. pneumoniae, determined by the immunoblot assay, was almost the same as the levels bound to M. fermentans, suggesting that M. pneumoniae is capable of binding Pg. However, when images of HeLa cells infected with the Pg-bound and urokinase-treated M. pneumoniae, foci of fluorescence were detected only on the cell surface (Fig. 3(a)).

Figure 2

Immunoblot analysis of plasminogen binding to M. pneumoniae and M. fermentans. Mycoplasmas (0.5 mg protein) were incubated with (+) or without (−) 25 μg Pg for 1 h at 37 °C. Pg-bound cells were washed and the bound Pg was released by treating with 10 mM ηACA as described in Section 2. The released Pg (1:1, 1:10 and 1:100 dilutions) was immobilized and reacted with anti-human Pg.

Figure 3

Confocal micrographs demonstrating binding and internalization of M. pneumoniae by HeLa and A549 cells. Following infection of HeLa or A549 cells with M. pneumoniae for 0 and 24 h, the cells were washed, fixed and immunostained with anti-M. pneumoniae antibodies as described in Section 2. (a) HeLa cells infected with Pg + uPA-treated M. pneumoniae; (b) A549 cells infected with native M. pneumonie. Each picture is a single image made through the mid ZY plane of a single HeLa or A549 cell.

Whereas M. pneumoniae did not invade HeLa cells, images of A549 cells infected with native M. pneumoniae revealed fluorescence in the cytoplasm and perinuclear regions of the host cells (Fig. 3(b)), suggesting that M. pneumoniae was able not only to adhere, but also to invade A549 cells. The respiratory origin of the A549 cells versus the cervical origin of HeLa cells may explain this difference. Differences in the capability of bacteria to adhere and invade different epithelial cell lines were described before [16, 27].

Internalization of M. pneumoniae by A549 cells was a time- and temperature-dependent process, observed as early as 20 min post-infection and reached a maximal level after 24 h of incubation (data not shown). Internalization was observed at 37 °C but not when the infected A549 cells were incubated at 20 or 4 °C. Thus, for determining the binding of M. pneumoniae, the incubation was carried out at 4 °C. The level of bacteria intertnalized by A549 cells was the same with native M. pneumoniae or with M. pneumoniae pretreated with Pg with or without urokinase. However, internalization was not detected in control A549 cells infected with PFA fixed (4%, 5 min) M. pneumoniae (data not shown).

As the intracellular fluorescence observed within A549 cells infected with M. pneumoniae may represent released mycoplasmal antigen rather than intact and viable microorganisms, the ability of M. pneumoniae to invade A549 cells was corroborated by the gentamicin resistance assay. In this assay, extracellular bacteria are killed, while the intracellular bacteria are shielded from the antibiotic effect due to limited penetration of the gentamicin into the eukaryotic host cell. This method has been previously used to study the invasion of M. penetrans into HeLa cells [23]. M. penetrans was relatively stable to gentamicin, but was made susceptible to the antibiotic by adding low concentration of TX-100 to the medium [23]. With M. pneumoniae, a very good killing effect of adhered bacteria was obtained with 400 μg ml−1 gentamicin even without TX-100 and thus this concentration was used in further studies. Since M. pneumoniae is susceptible to detergent lysis as the host cells, we have plated dilutions of the infected host cells directly onto solid Chanock media without lysing them beforehand. Accordingly, each mycoplasma colony obtained represents an infected host cell rather than a single intracellular bacterium [23]. We defined internalized M. pneumoniae as bacteria that survived two washings of the infected host cells and 2 h of exposure to 400 μg ml−1 gentamicin. Table 1 shows the results obtained after 2 and 24 h incubation with HeLa or A549 cells at 37 °C. Whereas binding of M. pneumoniae to HeLa and A549 cells, determined with [3H]palmitate-labelled mycoplasmas at 4 °C, was to about the same extent, internalization was apparent only with A549 cells. No viable mycoplasmas were detected in PFA-treated cells although the treated cells bind significant amounts of the bacteria (Table 1). Our results are in agreement with recent findings, obtained by confocal scanning microscopy and electron microscopy, showing intracellular location of fluorochrome-labelled M. pneumoniae in hepatocytes infected by this organism in vitro [28]. The internalized M. pneumoniae survived within the host cells for prolonged periods of time. Thus, when A549 cells were infected with M. pneumoniae for 4 h, washed, treated with gentamicin and further incubated for up to 72 h, constant levels of viable mycoplasmas were detected within A549 cells for 48 h, slowly declining afterwards (data not shown). Throughout this period, the host A549 cells were viable (data not shown). Low levels of viable mycoplasmas were detected more than a week post-infection, results that may explain why these bacteria establish a carrier state or could account for the difficulty in clearing infections by antibiotic treatment [5].

Mycoplasma pneumoniae respiratory infection in humans is regarded as a self-limited infection, and the resolution of acute infection within a few weeks without treatment partly supports this concept. However, the organism has been detected by culture in the respiratory tract for up to several months after clinical recovery [4] even in patients treated with effective antibiotics [5] and in a murine model some individuals developed chronic pulmonary inflammation, and elicit chronic pulmonary function abnormalities [6]. These findings supported the notion that a subset of individuals may handle this infection in a less favorable manner developing chronic pulmonary abnormalities. Our observations that M. pneumoniae is internalized by a lung epithelial cell line and that the bacteria survive for extended periods of time within the host cells may explain how M. pneumoniae escape immune surveillance and produce chronic lung disease. This would be consistent with the lack of M. pneumoniae antibody in asthmatic patients from whom M. pneumoniae was detected in their respiratory tract [29].


We thank A. Katzenell for excellent technical assistance.


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