Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells
© Cano et al; licensee BioMed Central Ltd. 2009
Received: 18 December 2008
Accepted: 3 August 2009
Published: 3 August 2009
Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction.
The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence.
Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.
Klebsiella pneumoniae is the most common Gram-negative bacterium causing community-acquired pneumonia and up to 5% of community-acquired urinary tract infections [1–3]. Community-acquired pneumonia is a very severe illness with a rapid onset, and despite the availability of an adequate antibiotic regimen, the outcome is often fatal. The observed mortality rates are about 50% . Capsule polysaccharide (CPS), siderophores, lipopolysaccharide (LPS) and adhesins are virulence factors identified for this pathogen. However, most of the studies have focused on the role of CPS in Klebsiella virulence. Early studies suggested that an extracellular toxic complex mainly composed of CPS triggers extensive lung tissue damage [5, 6] and data indicate that there might be a correlation between the production of this extracellular complex and Klebsiella virulence [5, 6]. Similar to CPSs from other pathogens, Klebsiella CPS is responsible for resistance to complement mediated killing  and impedes adhesion to and invasion of epithelial cells  by sterically preventing receptor-target recognition of bacterial adhesins [9, 10]. Recently we have demonstrated that CPS mediates resistance to antimicrobial peptides (APs), trapping APs and thus acting as a bacterial decoy [11, 12].
Few studies have analysed cellular features of the interaction between lung epithelium and K. pneumoniae, the role of virulence factors such as CPS, and the relevance of this interaction in vivo. We have recently shown that an isogenic CPS mutant activates host cellular inflammatory responses and that CPS might prevent this activation through blockage of bacterial uptake . Moreover, Klebsiella infection increases the expression levels of Toll-like receptors 2 and 4 (TLR2 and TLR4) . This increased expression of TLRs results in an enhancement of the cellular response upon stimulation with Pam3CSK4 or lipopolysaccharide, TLR2 and TLR4 agonists, respectively . In this study, we show for the first time that K. pneumoniae exerts a cytotoxic effect on airway epithelial cells that is associated with the presence of CPS.
K. pneumoniae strains 52145 and 1850 are clinical isolates belonging to serotypes O1:K2 and O1:K35, respectively . K. pneumoniae strain 43816 (ATCC 43816) belongs to serotype O1:K2. K. pneumoniae 52K10 is a derivative of strain 52145 which lacks CPS . K. pneumoniae strains were cultured in Luria-Bertani (LB) medium at 37°C.
CPS purification and quantification
Cell-bound CPS was purified by the phenol-water method . Briefly, bacteria were grown in 1 l LB-broth in 2 l flasks in an orbital shaker (180 rpm) for 24 h at 37°C. Cells were removed by centrifugation and washed once with PBS. The pellet was extracted with phenol, and polysaccharides present in the aqueous phase were precipitated by adding 5 volumes of methanol plus 1% (v/v) of a saturated solution of sodium acetate in methanol. After incubation for 24 h at -20°C, the pellet was recovered by centrifugation, dissolved in distilled water, dialysed against water and freeze-dried. For further purification, this preparation was dispersed (final concentration 10 mg/ml) in 0.8% NaCl/0.05% NaN3/0.1 M Tris-HCl (pH 7) and digested with nucleases (50 mg/ml of DNase II type V and RNase A [Sigma Chemical Co., St. Louis, Mo.]) for 18 h at 37°C. Proteinase K was added (50 mg/ml [E. Merck, Darmstadt, Germany]), and the mixture was incubated for 1 h at 55°C and for 24 h at room temperature. The proteinase K digestion was repeated twice and the polysaccharides were precipitated as described above. The pellet was recovered by centrifugation and dissolved in distilled water. LPS was removed by ultracentrifugation (105000 × g, 16 h, 4°C) and samples were freeze-dried. The enzymatic treatment and ultracentrifugation steps were repeated once. This CPS preparation was repurified by the method described by Hirschfeld and co-workers . This method is widely used to remove proteins from polysaccharide preparations. SDS-PAGE-resolved preparations were transferred to PVDF membrane which was stained with colloidal gold to visualize proteins . No trace of contaminant proteins was found (data not shown). CPS was quantified by determining the concentration of uronic acid in the samples, using a modified carbazole assay  as described by Rahn and Whitfield . LPS presence was determined by measuring the 3-deoxy-d-manno-2-octulosonic acid (Kdo) content by the thiobarbituric acid method modified to correct interference due to deoxysugars . Kdo content was less than 0.07%.
Mammalian cell culture and bacterial infection
Monolayers of human lung carcinoma cells (A549, ATCC CCL185) derived from type II pneumocytes were grown to confluence as described before . Cells were serum starved for 18 h before infection. Overnight-grown bacteria were subcultured and grown to exponential phase, harvested by centrifugation (20 min/2700 × g) and resuspended in PBS. The inoculum for the infection was prepared in Earle's buffered salt solution (EBSS), pH 7.4. A549 cells (80–90% confluent) seeded on glass coverslips in 24-well tissue culture plates were subsequently infected with K. pneumoniae strains at a multiplicity of infection (MOI) ranging from 100:1 to 1000:1 and centrifuged for 4 min at 200 × g at 22°C. Infected plates were then incubated for 2 to 5 h at 37°C/5% CO2 in a humidified incubator. For adhesion assays, cells were washed five times with 1 ml phosphate-buffered saline (PBS) pH 7.4 after 2 h of infection and lysed with 0.5%-Triton in PBS. Serial dilutions of the lysates in PBS were plated on LB plates for quantification of viable bacteria. Experiments were carried out in triplicate in three independent occasions and results are expressed as % adhesion = 100 × (n° of bacteria recovered from well/initial n° of bacteria added). Where indicated, bacteria were UV killed by exposure to 1 joule for 3 min in a BIO-LINK BLX crosslinker (Vilber Lourmat).
Cell monolayers were fixed in 3.7% paraformaldehyde in PBS. Rhodamine (RRX)-conjugated phalloidin (Molecular Probes) diluted 1:200 in 10% horse serum/0.1% saponin in PBS was used to stain the actin cytoskeleton. Coverslips were washed twice in PBS containing 0.1% saponin, once in PBS, and incubated for 30 min with phalloidin-RRX. The coverslips were then washed twice in 0.1% saponin in PBS, once in PBS and once in H2O, mounted in Aqua-Poly/Mount (Polysciences) and analysed with a Leica CTR6000 fluorescence microscope.
Analysis of host cell DNA integrity after K. pneumoniaeinfection
A549 cells were infected with K. pneumoniae strains at MOI of 500:1 in tissue culture plates. 6 h post-infection, cells (~2.5 × 106) from 2 wells were collected in PBS by scraping and lysed in 600 μl cold lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.1% SDS). Proteinase K (100 μg/ml) was added and samples were incubated for 3 h at 55°C. Samples were cooled to 22°C and incubated with 20 μg/ml RNase (DNase-free) for 20 min at 37°C. 200 μl 5 M potassium acetate were added and samples were centrifuged (13000 × rpm, 22°C, 1 min). DNA present in the supernatants was precipitated with isopropanol, washed in 70% ethanol and dissolved in sterile water. DNA integrity was analysed by staining with ethidium bromide after resolving the samples by gel-electrophoresis in 1% agarose in TAE.
Cell cytotoxicity and viability assays
A549 cells (cultured in either 24- or 96-well plates) were infected with K. pneumoniae strains (MOI 500:1 or 1000:1, 5 h). Lactate dehydrogenase (LDH) release was measured using a commercial kit (CytoTox 96, Promega). Per cent cytotoxicity was calculated as: (OD490 sample - OD490 medium)/(OD490 max - OD490 medium)*100. OD490 max was obtained with the provided lysis positive control. Measure of formazan production from reduction of MTS tetrazolium by metabolically active cells was performed using cells cultured in 96-well plates. Formazan production (% viability) was measured using a kit (CellTiter 96 AQueous One, Promega) and calculated as: OD490 sample/OD490 max*100. OD490 max was obtained from a monolayer of non-infected cells. Ethidium bromide is taken up by host cells when cytoplasmic membrane integrity is lost, staining nuclei red when visualised by fluorescence microscopy. Cells were cultured on coverslips in 24-well plates and infected as described above (MOI 500:1, 5 h). 15 min before the end of the infection, culture medium was removed and wells were washed with 1 ml PBS. Cells were stained for 10 min with 250 μl of 6 TM ethidium bromide prepared in PBS, washed three times with 1 ml PBS, fixed with 3.7% paraformaldehyde in PBS, and mounted for immunofluorescence analysis as described above. Cytotoxicity (red nuclei) was quantified by counting a minimum of 100 cells in three independent experiments.
Mouse pneumonia model
Overnight-grown bacteria were subcultured and grown to exponential phase. Bacteria were centrifuged (2500 × g, 20 min, 22°C), resuspended in PBS and adjusted to 5 × 106 colony-forming units (c.f.u.)/ml. Five to seven-week-old female C57BI/6j mice were anaesthetized by i.p. injection with a mixture containing ketamine (100 mg/ml) and xylazine (10 mg/ml). 20 μl of bacterial suspension were inoculated intranasally in 4 × 5 μl aliquots. 48 or 72 h post-infection the mice were sacrificed by cervical dislocation and trachea, spleen and liver were dissected, weighed and homogenized in 1 ml PBS. Serial dilutions of the homogenates in PBS were plated on LB agar to determine c.f.u. per gram of tissue.
Statistical analyses were performed with Prism4 for PC (GraphPad Software) using the analysis of variance (ANOVA) or the two-sample t test or, when the requirements were not met, by the Mann-Whitney U test. P < 0.05 was considered statistically significant.
K. pneumoniaeinduces a cytotoxic effect in lung epithelial cells
Next, we asked whether live bacteria are necessary to induce cell rounding. The bacterial inoculum was killed by UV radiation and used to infect cells (MOI 500:1, 4 h). Under these conditions, strain 52145 did not induce cell rounding (Fig. 1C, upper). In order to corroborate this observation, a mock infection was carried out, i.e. same infection conditions as before, but in a tissue culture well without cells. After 4 h, the bacterial suspension was UV irradiated and used to infect a confluent cell monolayer for 4 h. Cell rounding was not observed (Fig. 1C, middle). In addition, the strain 52145-triggered cytotoxic effect was not induced by primed bacteria-free conditioned medium, since A549 monolayers remained intact after 4 h of exposure to bacteria-free medium obtained from previously infected cells (MOI 500:1, 4 h) (Fig. 1C, lower). Taken together, these findings demonstrate that K. pneumoniae strain 52145 induces a cytotoxic effect through a process requiring the presence of live bacteria.
K. pneumoniae-induced cytotoxicity is dependent on the presence of CPS
In summary, these findings indicate that K. pneumoniae alters host cell viability in a process dependent on the presence of CPS.
Correlation between K. pneumoniae-induced cell cytotoxicity and virulence
Therefore, although cytotoxicity is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent, suggesting that additional bacterial factors could be involved in virulence, or that the cytotoxic effect is necessary, but not sufficient, for virulence.
In this study, we show that K. pneumoniae triggers a cytotoxic effect upon infection of human lung epithelial cells. This process requires the presence of capsulated live bacteria through the time of infection. To the best of our knowledge, there are no studies reporting that K. pneumoniae might exert a cytotoxic effect on airway epithelial cells. Our results could point to the underlying mechanism behind the early findings reported by Straus et al., [5, 24] which indicated that K. pneumoniae expressing CPS induces extensive lung tissue damage.
A number of bacterial pathogens induce cytotoxicity in eukaryotic cells, which is frequently dependent on an active type III secretion system (T3SS). For example, enteropathogenic Escherichia coli induces detachment of infected epithelial cells from the substratum and injects the T3SS effector Cif into cells, which induces a cytopathic effect [25, 26]. Bordetella bronchiseptica's necrotic effect on epithelial cells is dependent on the T3SS effector BopB , and also Pseudomonas aeruginosa promotes T3SS-dependent cytotoxicity towards eukaryotic cells [28, 29]. Yet, K. pneumoniae-induced cytotoxicity does not seem to be related to a T3SS, given that in silico analysis of the so far sequenced K. pneumoniae genomes does not identify any T3SS components. Furthermore, PCR analysis using degenerated primers to amplify lcrD homologues present in all known T3SS were negative in all our Klebsiella strains. Recently, it has been shown that P. aeruginosa and enterotoxigenic E. coli deliver toxins directly into host cell cytoplasm using outer membrane vesicles [30, 31]. It is likely that K. pneumoniae also produces outer membrane vesicles. In fact, the extracellular toxic complex described by Straus [5, 24] could be considered a preparation of outer membrane vesicles. It is then tempting to speculate that outer membrane vesicles could be associated with K. pneumoniae cytotoxicity described in our study. Future studies will aim to address this possibility. On the other hand, our results clearly establish that CPS is necessary for the induction of cytotoxicity. CPS is a virulence factor for several pathogens, including Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b and E. coli K1 [32–34]. Of note, no previous reports link the presence of CPS to cytotoxicity. However, just the presence of CPS is not sufficient for K. pneumoniae-induced cytotoxicity because capsulated UV-killed bacteria or purified CPS did not induce this effect. Given the limited current knowledge about K. pneumoniae virulence factors, we can only speculate on the nature of bacterial factor(s) that, together with CPS, could promote cytotoxicity in the host. Signature-tagged mutagenesis approaches have identified several virulence factors [35, 36], but none of them resemble those triggering the cytotoxicity by other bacterial pathogens.
All K. pneumoniae clinical isolates are capsulated, inferring the importance of CPS for virulence. Likewise, CPS is necessary for virulence in an in vivo pneumonia model [15, 35] and for Klebsiella-induced cytotoxicity (this work). However, our data indicate that CPS-dependent cytoxicity is necessary but not sufficient for Klebsiella virulence because strains 43816 and 1850 are less virulent than strain 52145 and the three of them trigger cytotoxicity. This could be explained by differences in the amount of CPS expressed by these strains, although strain 43816 is also considered to be heavily capsulated. The absence of complete correlation between in vitro and in vivo studies has been previously described for other K. pneumoniae isolates. Struve et al., showed that CPS expression reduced K. pneumoniae adhesion to gut and bladder epithelium, when compared to a noncapsulated mutant. However, the presence/absence of CPS had no effect on the colonisation of the gastrointestinal tract, but did play a role in colonisation of the urinary tract . On the other hand, it has been recently postulated that there is an association between CPS serotype, virulence in mice and humans, and frequency of isolation in clinical settings . However, the bacterial strains tested in this study express CPS belonging to serotypes considered to have high potential of causing disease , and strains 52145 and 43816 express the same CPS serotype. Nevertheless, Klebsiella infections should be looked at as the outcome of specific interactions between pathogen and host cells. Indeed, factors on both pathogen and host sides may be involved in the progression of the infection. In this context, it is widely accepted that host innate immunity plays a key role to clear K. pneumoniae infections. Therefore, differences among strains in the resistance to complement and/or to antimicrobial peptides mediated killing may account for differences in virulence [11, 15, 39]. In addition, a wealth of evidence clearly indicates the importance of the inflammatory responses in clearing K. pneumoniae infection and have provided substantial evidence for the protective role of a Th1-mediated response [40–42]. Thus, differences in the induction of inflammatory responses among strains may also underline in vivo behavior. In summary the available data support the notion that CPS-dependent cytotoxicity, together with other bacterially triggered events, is required for virulence. Further studies will attempt to elucidate these novel virulence mechanisms, which may differ among capsulated strains, in order to achieve a comprehensive understanding of K. pneumoniae pathogenesis.
This study allocates a novel role to K. pneumoniae capsule, i.e. the induction of cytotoxicity during the infection of lung epithelial cells. This effect, which has been analysed by using four different approaches, is not capsule serotype dependent, does require the presence of live bacteria, and does not seem to be directly related to bacterial adhesion. Host cell cytotoxicity could be associated with virulence. However, strains expressing different capsule levels were not equally virulent, suggesting that additional bacterial elements could be involved in Klebsiella virulence.
Salary support to V.C. from Govern Balear is gratefully acknowledged. J.G. is a recipient of a Contrato de Investigador "Miguel Servet" from Instituto de Salud Carlos III. This work has been funded by grants from FIS (CP05/00027 to J.G. and PI06/1629 to J.A.B.). Ciberes is an initiative from Instituto de Salud Carlos III, Spain. The authors sincerely thank Dr. Christian Frank for critical reading of the manuscript.
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