肺炎链球菌生物膜细胞基质观察

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms Mirian Domenech,1,3 Ernesto García,1,3 Alicia Prieto2 and Miriam Moscoso1,3* 1Departamento de Microbiología Molecular y Biología de las Infecciones, and 2Biología Medioambiental, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. 3CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain. Summary Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA–protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lys- ozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (posi- tions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA–LytC protein com- plexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluoro- phores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1,4) and Glc- NAc(1,4) (in its deacetylated form) in the pneumo- coccal biofilm was confirmed by GC-MS techniques. Introduction The growth and dispersal of microbes, whether patho- genic or environmental, commonly involves the produc- tion of biofilms; indeed, they are thought to be present in the vast majority of bacterial infections (Wolcott and Ehrlich, 2008). A biofilm is defined as a thin layer of microorganisms adhered to the surface of an organic or inorganic substrate embedded in an extracellular matrix (Costerton et al., 1995). This matrix consists of a mixture of biopolymers (extracellular polymeric substances or EPS) synthesized largely by the biofilm-producing micro- organisms themselves. Although EPSs such as proteins, DNA and polysaccharides are common to most biofilms, matrix composition can vary greatly depending on the bacterial species involved and environmental conditions (Flemming and Wingender, 2010). The human pathogen Streptococcus pneumoniae (or pneumococcus) is a leading cause of pneumonia, menin- gitis and bloodstream infections in the elderly, and one of the main pathogens responsible for middle ear infections in children. It is carried asymptomatically in the nasophar- ynx of many healthy adults, and in as many as 20–40% of healthy children (colonization begins shortly after birth) (Weiser, 2010). Recent reports have shown the in vivo formation of S. pneumoniae biofilms on adenoid and mucosal epithelial tissues in children with recurrent or chronic ear infections (for a recent review, see Domenech et al., 2012). Using low-temperature scanning electron microscopy (LTSEM) techniques, our group detected a biofilm matrix containing an intercellular, fibre-like material that linked the pneumococcal cells to one another and to the glass substrate on which they were grown (Moscoso et al., 2006). The presence of extracellular proteins in this matrix was inferred from the biofilm-disaggregating activ- ity of proteolytic enzymes (Moscoso et al., 2006). Several authors have reported extracellular DNA (eDNA) to be an important EPS in pneumococcal biofilms, based on the dramatic disappearance and inhibited for- mation of these films following treatment with DNase (Moscoso et al., 2006; Hall-Stoodley et al., 2008; Carrolo et al., 2010; Trappetti et al., 2011). However, other authors have been unable to confirm this (Muñoz-Elías et al., 2008). As well as being a structural element of the matrix, other roles have been ascribed to eDNA in differ- ent biofilms (Fuxman Bass et al., 2011), including: (i) Received 7 May, 2012; accepted 21 July, 2012. *For correspondence. E-mail mmoscoso@cib.csic.es; Tel. (+34) 918 373 112; Fax (+34) 915 360 432. bs_bs_banner Environmental Microbiology (2013) 15(2), 502–516 doi:10.1111/j.1462-2920.2012.02853.x © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd acting as an adhesin in the initial phase of biofilm forma- tion (attachment to the substrate) and contributing to increased mucus viscosity; (ii) being involved in horizontal gene transfer, thus contributing to the spread of antibiotic resistance; and (iii) attenuating inflammatory mediator production and macrophage invasion. Extracellular RNA seems not be a critical biofilm element (Domenech et al., 2012). Controversy exists over whether extracellular polysac- charide is a component of the S. pneumoniae biofilm matrix. To our knowledge, only two studies have sug- gested this to be the case. In one, wheat germ agglutinin (WGA) was found to stain the biofilm formed by a clinical pneumococcal isolate of an undefined capsular type (Donlan et al., 2004). In the other, a cocktail of five lectins of different specificity (including WGA) stained the bio- films formed by strains of six different capsular types (Hall-Stoodley et al., 2008). Using encapsulated S. pneu- moniae to demonstrate the existence of a polysaccharide among the EPS is, however, problematic since lectins also bind the capsular polysaccharide, as demonstrated more than 30 years ago (Ebisu et al., 1977). However, Hall-Stoodley and colleagues (2008) reported the biofilm formed by the non-encapsulated R6 strain to bind lectins, suggesting the presence of a non-capsular carbohydrate EPS. Unfortunately, the use of a lectin mixture precluded any further identification of that putative polysaccharide. Glycosylated proteins may also be represented among the EPSs. Protein glycosylation is a common post- translational modification in eukaryotes, but recently it has become clear that it also occurs in archaea and bacteria (Larkin and Imperiali, 2011). Many glycoproteins act as virulence factors in medically important bacterial patho- gens, such as the giant (4776 amino acid residues) pneu- mococcal serine-rich repeat protein (PsrP; SP_1772), which is synthesized by accessory genes (including several novel glycosyltransferases) present only in certain clonal lineages of S. pneumoniae (e.g. the TIGR4 strain but not the R6 strain) (Tettelin et al., 2001; Löfling et al., 2011). The PsrP protein, which is encoded by the psrP gene located in an accessory region, acts as an adhesin and is involved in biofilm formation, apparently by promot- ing the formation of large bacterial aggregates (Sanchez et al., 2010). A number of genes putatively encoding gly- cosyltransferases lie close to psrP. Recent studies have shown the first step in the O-linked glycosylation of PsrP to be the transfer of N-acetylglucosamine (GlcNAc) resi- dues to the polypeptide, a reaction catalysed by the GtfA/B complex (Wu et al., 2010); the second might be the addition of glucose (Glc) to the GlcNAc-modified PsrP in a reaction catalysed by SP_1768, as recently demonstrated for the protein Nss, an SP_1768 orthologue of Strepto- coccus parasanguinis (Zhou et al., 2010). Taken together, these data indicate that, when working with biofilms syn- thesized by strains encoding PsrP, such as the common TIGR4 strain, the glycosylation of this protein may con- found attempts to identify possible polysaccharides among the EPS. The present work provides substantial evidence that the biofilm formed by S. pneumoniae R6 contains a matrix made up of eDNA, protein and polysaccharide components. Results Visualization of the pneumococcal biofilm matrix Despite possible problems of sample shrinkage, LTSEM has a number of advantages compared with conventional scanning electron microscopy. For this type of analysis the latter would require the preparation of samples by either critical point drying or freeze-drying. These tech- niques would cause the almost complete disappearance of EPS from the matrix. In contrast, LTSEM preparations involve the use of techniques (see Experimental proce- dures) that allow the structural water of biofilms to be retained, preserving the natural fabric of the matrix, including the EPS. Moreover, no chemical fixation is necessary in LTSEM, eliminating this source of arte- facts (Sutton et al., 1994). LTSEM examination of the ultrastructure of S. pneumoniae biofilms previously revealed the participating cells to be interconnected by small, thin filaments (Moscoso et al., 2006). The present, improved electron micrographs of pneumococcal biofilms growing on polystyrene microtitre dishes confirm the pres- ence of these intercellular filaments; indeed, they revealed a conspicuous net-like matrix linking the cells (Fig. 1). DNA as a component of EPS The detection of eDNA (using specific, fluorescent, double-stranded DNA staining) in S. pneumoniae biofilms grown for 1 to 8 days in a complex medium has been previously reported (Hall-Stoodley et al., 2008). However, the use of stains such as propidium iodide, ethidium bromide, DAPI (4′,6-diamidino-2-phenylindole) and SYTO for visualizing this eDNA requires the sensitivity of CLSM be strongly increased. Consequently, good quality images are difficult to obtain (Allesen-Holm et al., 2006). Recently, DDAO [7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2- one)] was shown suitable for selectively targeting the eDNA of biofilms due to its molecular size (which does not allow the stains to penetrate intact cell membranes) and because of its enhanced fluorescent properties, which make it easy to detect by CLSM (Dominiak et al., 2011). Figure 2 shows the presence of abundant eDNA in a 14-hour-old biofilm formed by S. pneumoniae strain R6 in C medium, detected by in situ staining with DDAO Composition of the pneumococcal biofilm matrix 503 © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 502–516 and SYTO 9. Although most of the eDNA seems to be cell-associated, discrete areas of the biofilm that stained with DDAO but not with SYTO 9 (and vice versa) were found when the preparations were observed at higher magnification (Fig. 2D). A DDAO-stained planktonic R6 control culture showed no fluorescence (data not shown). Evidence of protein–DNA interactions Earlier work showed that pneumococcal biofilm integrity is severely impaired by treatment with proteases during or after biofilm formation (Moscoso et al., 2006; Muñoz- Elías et al., 2008). It is thus conceivable that extracel- lular and/or surface-attached proteins form part of the EPS matrix. In fact, several surface-attached proteins belonging to the family of the choline-binding proteins (CBPs) (López and García, 2004), namely LytA N-acetylmuramoyl-L- alanine amidase (EC 3.5.1.28; NAM-amidase), LytB glu- cosaminidase, LytC lysozyme (EC 3.2.1.17; muramidase), CbpA (PspC) adhesin, PcpA putative adhesin and PspA (pneumococcal surface protein A), are somehow involved in biofilm formation since mutants that cannot produce just one of these proteins are less able to produce biofilms (Moscoso et al., 2006). No such reduction is observed, however, in Pce phosphocholinesterase or CbpD putative NAM-amidase/endopeptidase mutants. Reduced biofilm- formation capacity has also been confirmed for lytC mutants in two independent studies (Muñoz-Elías et al., 2008; Domenech, 2012). Although no experimental evi- dence is available, it is conceivable that one or more of these (and/or other) surface proteins may interact with some of the EPS (e.g. eDNA). This hypothesis is here confirmed. When different CBPs were incubated with DNAs of different origin, strong binding between DNA and LytC was shown (by electrophoresis). When purified LytC was incubated with chromosomal DNAs obtained from bacteria with different G + C contents [from 28% (Clostrid- ium acetobutylicum) to 73% (Micrococcus roseus)], the protein was capable of binding to all DNAs irrespective of their base composition (Fig. 3A). Moreover, the LytC–DNA binding reaction took place independently of factors such as DNA size, strandedness or topological state. However, binding was completely abolished by the addition of 10 mM EDTA (but not with 10 mM EGTA). Furthermore, the addi- Fig. 1. LTSEM image of a S. pneumoniae R6 biofilm formed on the surface of a glass coverslip. Filamentous material (indicated by arrows) links the pneumococcal cells to one another. On the right, a magnification of the area indicated by the white square is shown. The reticular nature of the intercellular matrix can be observed. Fig. 2. CLSM evidence of eDNA in pneumococcal biofilms. A biofilm of the S. pneumoniae strain R6 was stained with a combination of SYTO 9 (A, green) and DDAO (B, blue). Image (C) is a merger of the two channels. Image (D) is an enlarged vision of the area marked with a square in (C). Arrows indicate labelling with DDAO alone. Scale bars = 25 mm. 504 M. Domenech, E. García, A. Prieto and M. Moscoso © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 502–516 tion of 10 mM MgCl2 to an EDTA-inhibited complex restored complex formation. This indicates the require- ment of Mg2+ ions for LytC–DNA complex formation (Fig. 3B). Interestingly, LytC-bound DNA appears to remain intact since it migrates to its expected position in gels after its removal from the protein–DNA complex with proteinase K. In addition, although the binding reaction was inhibited at NaCl concentrations above 50 mM, and the LytC–DNA interaction was fully susceptible to SDS attack (final concentration 1%), the complex could not be broken by the addition of 1.5 M NaCl, 2% choline chloride, or both (results not shown). Control experiments per- formed under varying experimental conditions showed the optimal conditions for the formation of LytC–DNA com- plexes to involve a pH of < 9.5 (optimal pH 6–7.5), tem- peratures of above 16°C (preferably at 37°C), an LytC : DNA (w/w) ratio of �3 (optimum when ª25), and a reaction time of at least 1 min (optimal incubation time 1 h) (Fig. S1). It should be noted that formation of LytC–DNA led to no detectable inhibition of the catalytic activity of the muramidase when tested against radioactively labelled pneumococcal cell walls (results not shown). Fig. 3. Formation of LytC–DNA complexes. A. Agarose gel (0.7%) electrophoresis of LytC bound to various chromosomal DNAs (0.1 mg). The G + C content (%) of each DNA is indicated in parentheses. The binding reactions were performed over 1 h at 37°C in 10 mM Tris-HCl buffer (pH 7.0). A_CIB, Azoarcus sp. CIB; Eco, E. coli; Aca, A. calcoaceticus; Spn, S. pneumoniae; Cac, Clostridium acetobutylicum; Mro, Micrococcus roseus. The symbols ‘+’ and ‘-’ indicate, respectively, that the incubation mixture included, or did not include, LytC (2.6 mg). S, BstEII-digested l DNA. B. Binding of LytC to different kinds of DNA, i.e. fragments of HindIII-digested DNA (0.3 mg); the covalently closed circular form of pGL30 (40 ng); pGL30 (40 ng) and pUC19 (40 ng) linearized with SalI or EcoRI respectively; small size fragments of the replicative form (RF) of fX174 digested with HaeIII (160 ng); single-stranded (ss) M13mp18 DNA (40 ng). The symbols ‘+’ and ‘-’ indicate, respectively, that the incubation mixture included, or did not include, LytC (2.6 mg). In some cases (PK), the binding mixture was digested with proteinase K (100 mg ml–1; 15 min, 37°C) after the protein–DNA complex was formed and then analysed by agarose gel electrophoresis. In one experiment (E), the binding reaction (1 h, 37°C, in Tris-HCl buffer, pH 7.0) was performed in the presence of 10 mM EDTA. C. Various bacterial-encoded (LytA, LytB, LytC, Pce and CbpF) and phage-encoded (Cpl-1, Skl and Pal) purified CBPs (López and García, 2004) (1 mg each) were incubated with pGL30 (40 ng) and analysed in 0.7% agarose gels. Cpl-7 is a muramidase of phage origin that does not belong to the CBP family of proteins (García et al., 1990). BSA, bovine serum albumin; HEWL, hen-egg white lysozyme. D. Inhibitory effect of CbpF on LytC–DNA binding. Plasmid pGL30 (40 ng) was incubated under standard conditions with LytC (1 mg) or LytC plus CbpF (0.5–2 mg). C, control pGL30. E. Electron micrographs of pGL30 (left panel) or pGL30-LytC complex (right panel). Scale bars = 200 nm. F. LytC (1 mg) and the indicated peptides (1 and 5 mg) were incubated with pGL30 (40 ng) and analysed in 0.7% agarose gels. Composition of the pneumococcal biofilm matrix 505 © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 502–516 Although LytC appears to form the strongest DNA– protein complexes, LytB and CbpF were also associated with perceptible delays in DNA migration in agarose gels (Fig. 3C). These three proteins may therefore be included in the currently growing list of moonlighting pro- teins (Henderson and Martin, 2011). Whereas LytB, a chain-dispersing enzyme involved in cell separation (De las Rivas et al., 2002), is known to be involved in biofilm formation (see above), CbpF, an inhibitor of the LytC autolytic activity (Molina et al., 2009), had not been pre- viously examined in this respect. Interestingly, when CbpF was present in sufficient quantity, the LytC–DNA binding reaction was remarkably inhibited (Fig. 3D). Electron micrographs of the LytC–DNA complexes con- firmed the existence of protein–DNA aggregates (Fig. 3E), which explains their abnormal migration in agarose gels. It has been shown that LytC is released to the super- natant of planktonic pneumococcal cultures in relatively large amounts (Eldholm et al., 2009). This appears to be also true for S. pneumoniae biofilms. When biofilms were incubated with anti-LytC serum, labelling of both the cell surfaces and the intercellular matrix could be revealed (Fig. 4B). In addition, colocalization of LytC and eDNA (stained with DDAO) was observed in the intercellular spaces (Fig. 4B and D). The formation of LytC–DNA com- plexes has an ionic basis (in agreement with the depend- ence of optimal complex formation at a neutral or acidic pH), and it is strong enough to withstand high ionic strength environments (i.e. 1.5 M NaCl; see above). Fig. 4. CLSM evidence of LytC–eDNA complexes in pneumococcal biofilms. A biofilm of S. pneumoniae R6 was stained with a combination of SYTO 9 (A, green), anti-LytC serum followed by Alexa Fluor 647-labelled goat anti-rabbit IgG (B, red) and DDAO (C, blue). Image (D) is a merger of the three channels. Image (E) is an enlarged vision of the area marked with a rectangle in (D). Arrows indicate intercellular areas labelled with anti-LytC and DDAO. Scale bars = 30 mm. 506 M. Domenech, E. García, A. Prieto and M. Moscoso © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 15, 502–516 However, the mature form of LytC (468 amino acid resi- dues) has a net negative charge (61 Glu + Asp versus 53 Arg + Lys; predicted pI 6.1) at physiological pH, which contrasts with that shown by other cationic polymers such as polylysine and polyarginine, or indeed positively charged proteins such as avidin and human and hen egg-white (HEW) lysozymes (pI 10.7), all of which are well known to interact with DNA (Morpurgo et al., 2004). Surface plasmon resonance and circular dichroism spec- troscopy were used to study the interactions between plasmid pUC19 and positively charged peptides derived from the human lysozyme. The dissociation constants of the whole protein and peptides derived from it fell in a r