1 Departamento de Química, Bioquímica y Biología Molecular, Universidad Cardenal Herrera-CEU, 46113 Moncada, Valencia, Spain
2 Instituto de Investigaciones Citológicas, FVIB, 46010 Valencia, Spain
3 Mikrobielle Genetik, Universität Tübingen, D-72076 Tübingen, Germany
4 Instituto de Agrobiotecnología y Recursos Naturales, CSIC-Universidad Pública de Navarra, 31006 Pamplona, Navarra, Spain
5 Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Náquera-Moncada Km 4,5, 46113 Moncada, Valencia, Spain
Correspondence
José R. Penadés
jpenades{at}ivia.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the bap sequences reported in this paper are DQ008303 (S. chromogenes), DQ008304 (S. xylosus), DQ008305 (S. simulans), DQ008306 (S. epidermidis) and DQ008307 (S. hyicus).
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INTRODUCTION |
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Even though more researchers have realized the importance of CNS intramammary infections, the virulence factors of CNS remain poorly understood. The virulence factors of bovine or ovine staphylococci have been studied most extensively in S. aureus. In this species, many chronic infections are associated with bacterial growth in the form of adherent colonies surrounded by a large exopolysaccharide matrix, constituting a biofilm (Cucarella et al., 2004). Given their aggregate size, biofilms are not susceptible to macrophage phagocytosis, and they become resistant to certain antibiotics (Amorena et al., 1999
; Cucarella et al., 2004
).
The implication of biofilms in chronic infections has triggered an increasing interest in the characterization of genes involved in biofilm formation (Caiazza & O'Toole, 2003; Gotz, 2002
; Lim et al., 2004
; Tormo et al., 2005
; Valle et al., 2003
). In a previous study, we identified a surface protein (Bap, for Biofilm associated protein) implicated in S. aureus biofilm formation (Cucarella et al., 2001
). Interestingly, the bap gene is contained in a mobile pathogenicity island (Ubeda et al., 2003
), and so far it has only been found in bovine mastitis isolates (Cucarella et al., 2001
). The presence of Bap significantly increased the ability of organisms to colonize and persist in the bovine mammary gland in vivo. At the same time, Bap-positive isolates were less susceptible to antibiotic treatments when forming biofilms in vitro (Cucarella et al., 2004
). Analysis of the structural bap gene revealed the existence of alternative forms of the Bap protein, which contain a different number of repeats, in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection (Cucarella et al., 2004
). Altogether, these results demonstrate that the presence of Bap in the bovine intramammary gland may facilitate a biofilm formation that is connected with the persistence of S. aureus. Interestingly, although Bap was previously identified in bovine mastitis S. aureus isolates, a similar protein called Bhp (Bap homologue protein; AY028618) is present in human strains of S. epidermidis, where it may have a function similar to that of Bap.
The purpose of this study was to investigate whether CNS from mastitis are able to form biofilms in vitro, and, if so, whether biofilm formation is mediated via the Bap protein. Not only did we find that the function of the Bap protein is conserved between S. aureus and S. epidermidis, but also that the bap gene is present in several other Staphylococcus species, implying that the primary attachment, as well as the cellcell adhesion function mediated by Bap, may be important virulence factors for several species of this genus.
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METHODS |
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Biofilm formation.
Biofilm formation on abiotic surfaces was quantified essentially as described elsewhere (Heilmann et al., 1996a), with the following modifications. Staphylococci were grown overnight at 37 °C in TSB. The culture was diluted at 1 : 40, in TSB-glucose, and 200 µl of this cell suspension was used per well to inoculate sterile 96-well polystyrene microtitre plates (Iwaki). After 18 h incubation at 43 °C, the wells were gently washed three times with 200 µl sterile PBS, air-dried in an inverted position, and stained with 0·1 % safranin for 30 s. The wells were rinsed again, and the absorbance was determined at 490 nm (Micro-ELISA Autoreader Elx800; Bio-tek Instruments). Each assay was performed in triplicate in five separate experiments.
DNA manipulations.
Routine DNA manipulations were performed using standard procedures (Sambrook et al., 1989). Plasmid DNA from E. coli and staphylococci was purified with a Genelute Plasmid Miniprep Kit (Sigma) according to the manufacturer's protocol, except that the staphylococcal cells were lysed with lysostaphin (12·5 µg ml1; Sigma) at 37 °C for 1 h before plasmid purification. Plasmids were introduced into staphylococcal strains by protoplast transformation, using a previously described method (Götz & Schumaker, 1987
; Götz et al., 1983
). For Southern blot hybridization, staphylococcal chromosomal DNA was extracted using a Genelute Bacterial Genomic DNA Kit (Sigma) according to the manufacturer's protocol, except that the bacterial cells were lysed by lysostaphin (12·5 µg ml1) at 37 °C for 1 h before DNA purification. Chromosomal DNA digested with HindIII was analysed by agarose gel electrophoresis. Gels were blotted onto nylon membranes (Hybond-N 0·45 µm pore-size filters; Amersham Life Sciences) using standard methods (Ausubel et al., 1990
; Sambrook et al., 1989
). The probes corresponding to both the ica operon and the bap gene were generated by PCR using the following oligonucleotides. The ica primers (ica-A, 5'-GCCTTATTTATTGACAGTCGCTACGAA-3'; and ica-B, 5'-CGTGTGCTTTAAGCCATTGAAT-3') were designed to amplify part of the icaA and icaB genes of the icaADBC locus (U43366). Primers sasp-6m (5'-CCCTATATCGAAGGTGTAGAATTGCAC-3') and sasp-7c (5'-GCTGTTGAAGTTAATACTGTACCTGC-3') were used to amplify the bap gene (AF288402). The PCR products of the amplified bap and ica genes were used as DNA probes. Labelling of the probes and DNA hybridization were performed according to the protocol supplied with the PCR-DIG DNA-labelling and chemiluminescent detection kit (Roche).
Cloning of the bap-encoding genetic element from CNS.
Initial PCR analysis using specific primers (sasp-6m and sasp-7c) for the bap gene of S. aureus amplified a 0·97 kb fragment from different species of CNS. These different reaction products were directly cloned into the TOPO-PCR system (Invitrogen), and then sequenced. An outward-directed PCR was performed using a Vectorette II kit (Sigma-Genosys), according to the manufacturer's instructions, to obtain the flanking region to these initial sequences. This system is used to amplify regions of unknown DNA sequence flanking a region of known DNA sequence. Briefly, the target DNA was digested with an appropriate restriction enzyme. Vectorette units were ligated onto the ends of the cleaved target DNA. PCR amplification was carried out with one primer directed to the known sequence (custom primer), and with the other primer specific for the Vectorette unit (Vectorette primer). The amplified products were then cloned into the TOPO-PCR system (Invitrogen), and they were then either sequenced or used as probes in Southern hybridization experiments.
To verify that the deduced sequence of bap represented the native gene without any additions or deletions while manipulating the various clones, sequence information was verified by PCR amplification of select regions of the genes and restriction mapping.
PCR.
PCR was performed with a Techne Progene thermocycler in a volume of 25 µl, using DyNAzyme EXT as recommended by the manufacturer (Finnzymes). Cycling times were regulated according to the properties of the primer pairs.
DNA sequencing and computer analysis.
The nucleotide sequence was determined by the dideoxy chain-termination method, using an ABI 377 model automatic sequencer (PE Biosystems) at the IBMCP-UPV DNA Sequencing Service (Valencia, Spain). Nested deletions were generated (Erase-a-base system; Promega) for C-repeat sequencing. Similarity searches were carried out using the BLAST 2.0 program (Altschul et al., 1997) on the NCBI server. The cloned sequence was compared against GenBank and the publicly available S. aureus genome sequences (TIGR, The University of Oklahoma, and The Wellcome Trust Sanger Institute).
Disruption of bap.
For disruption of bap in the bap-positive biofilm-forming isolate S. epidermidis C533, an internal 1280 bp PCR fragment within the N-terminal region of the bap gene amplified with primers bap-6mP (5'-AAACTGCAGAACAACCAGACAAATCATC-3') and sasp-3cB (5'-GGGGGATCCCCAACCTCGTCAATGGTTAAGTCAGC-3') was cloned into the PstI/BamHI sites of the shuttle vector pBT2 (Brückner, 1997), and the resulting plasmid (pJP15) was introduced into S. epidermidis C533 by protoplast transformation (Götz & Schumaker, 1987
; Götz et al., 1983
). After transformation, transformed bacteria were incubated for 16 h at 32 °C on TSB with chloramphenicol. Subsequently, tenfold serial dilutions of this culture in sterile TSB were plated on TSA with chloramphenicol, and incubated for 24 h at 43·5 °C. After overnight incubation, colonies were analysed for disruption of the bap gene by PCR with primers bapepi-1mK (5'-CTCTACACAAGTGATTCGGTACCTATC-3') and pBT2-1m (5'-TACCCCAGGCGTTTAAGGGC-3'), and the results were confirmed by Southern blot analysis. JP39 represents the insertional bap-mutant strain used in this study.
Complementation studies.
To prove that the biofilm-deficient phenotype of the mutants was due to the disruption of bap, S. aureus Newman and S. epidermidis Tü3298 were complemented with plasmid pCU1 (Augustin et al., 1992) or pJP16. Plasmid pJP16 carries a PCR-amplified fragment from the wild-type S. epidermidis C533 strain, including the bap gene under the control of its own promoter cloned in pCU1 (Augustin et al., 1992
). Primers chrom-6mB (5'-CGCGGATCCTGTTATTTTCATCTATTGGG-3') and bapori-2cS (5'-ACGCGTCGACCCACCTTTGTAAGTGGAGAGCC-3') were used to amplify the bap gene from S. epidermidis C533. Plasmids pCU1 and pJP16 were transformed into staphylococci via protoplast transformation (Götz & Schumaker, 1987
; Götz et al., 1983
) or electroporation (Cucarella et al., 2001
). Phage 85 was used to transduce the plasmids from RN4220 to the Newman strain. Stable expression of Bap was analysed in total bacterial extracts by Western blot analysis of proteins run on SDS-PAGE gels.
Sequence-analysis programs.
The predicted amino acid sequence of Bap was analysed with the PrositeScan program (Combet et al., 2000) on the web server http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html, in order to identify potential functional domains.
Western blot analysis.
The Bap immunoblotting assay was performed as previously described (Cucarella et al., 2002). Briefly, staphylococcal cells from a stationary-phase culture were suspended to an OD600 of 40 in 100 mM PBS containing 5 mM EDTA and 1 mM PMSF. Cells were centrifuged, and suspended in 1 ml digestion buffer [50 mM Tris/HCl pH 7·5, 20 mM MgCl2 and 30 % raffinose (Sigma)]. To each 1 ml sample, 60 µl protease inhibitors (Complete cocktail; Roche), and 60 µl of a 2 mg ml1 solution of lysostaphin (Sigma) were then added, and the suspension was incubated in a 37 °C water bath for 30 min. Protoplasts were sedimented by centrifugation at 6000 g, and the supernatant fraction, which contained the wall-associated proteins, was analysed by SDS-PAGE (10 % separation gel, 4·5 % stacking gel).
For Western blot analysis, protein extracts were prepared and analysed by SDS-PAGE as described above, and blotted onto an Immobilon-P membrane (Millipore). Anti-Bap serum (Cucarella et al., 2001) was diluted 1 : 2500 with Tris-buffered saline (TBS; 50 mM Tris/HCl, pH 7·5, 150 mM NaCl) and immuno-absorbed with 5 % skimmed milk. Alkaline-phosphatase-conjugated protein A (Sigma), diluted 1 : 10 000 in TBS/5 % skimmed milk, was used, and the subsequent chemiluminescence reaction (CSPD; Roche) was recorded.
Statistical analysis.
A two-tailed Student's t test was used to determine the differences in biofilm formation between the groups. Differences were considered statistically significant when P was <0·05.
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RESULTS |
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Bap orthologues induce biofilm formation in the absence of PIA/PNAG exopolysaccharide
The biofilm formation process of S. epidermidis has been related to the products of the chromosomal intercellular adhesion (ica) operon. To determine the necessity of the icaADBC operon in the biofilm formation process of the bap-positive CNS isolates, chromosomal DNA from these isolates was hybridized by Southern blot with an ica-specific gene probe. Interestingly, the results revealed the absence of the icaADBC operon in all the bap-positive CNS analysed (Fig. 6). Dot-blot experiments using anti-PIA/PNAG polyclonal antibodies were carried out to confirm the absence of PIA/PNAG-related exopolysaccharides in the CNS isolates, except in S. xylosus (data not shown). These results suggest that the Bap protein is able to induce biofilm formation on abiotic surfaces in the absence of PIA/PNAG exopolysaccharide.
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The bap genes from the different Staphylococcus species are not carried by SaPIbov2
In a previous study, we demonstrated that the bap gene of S. aureus is carried in a putative composite transposon inserted in SaPIbov2, a pathogenicity island of S. aureus (Ubeda et al., 2003). The putative transposon harboured the bap gene, along with genes encoding an ABC transporter and a transposase. We hypothesized that the bap gene might have used the transposon and/or the pathogenicity island mobility to spread among staphylococcal species. To answer this question, we cloned and sequenced the flanking regions of each of the bap genes. Interestingly, a sequence analysis of these flanking regions showed that the bap genes were not contained in the transposon or in the previously described SaPIbov2 pathogenicity island (data not shown). These results indicate that the presence of bap gene in SaPIbov2 is restricted to S. aureus isolates, and that it is not the mechanism for horizontal transfer amongst CNS species.
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DISCUSSION |
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The prevalence of the bap gene itself among mastitis-related isolates analysed was found to change depending on the herd and geographical area. Remarkably, Bap can be found (usually in a small proportion) among mastitis isolates of different species, yet it is absent from the human Staphylococcus spp. tested so far (our unpublished results). This difference between the human and ruminant mastitis isolates suggests that these strains are not clonally related, and that specific host-dependent pathogenic factors may have evolved independently in both humans and ruminants. This diversity between isolates from different hosts corroborates the results of Herron et al. (2002), who, in a preliminary analysis of the genome of a common clone of bovine S. aureus, showed the presence of numerous genes and sequences that differentiate this bovine isolate from previously characterized human S. aureus strains. These differences indicate that a rational and effective strategy to control intramammary infections caused by bovine-specific isolates may be advantageous.
We have demonstrated in a previous study that calcium modulates Bap-dependent multicellular behaviour in S. aureus (Arrizubieta et al., 2004). We found that adding millimolar amounts of calcium to the growth media inhibited intercellular adhesion and biofilm formation of the Bap-positive strain V329. Addition of manganese, but not magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, virtually no effect of calcium or chelating agents was observed in the aggregation of the Bap-deficient strain m556. Site-directed mutagenesis of two putative EF-hand domains resulted in a mutant strain capable of biofilm formation, but the biofilm of which was not inhibited by calcium. In summary, our results indicated that Bap binds calcium with a low affinity, and that calcium binding renders the protein non-competent for biofilm formation and intercellular adhesion. Interestingly, a motif search of the amino acid sequence of the different Bap proteins using the PROSITE PS00018 definition revealed the presence of these EF-hand domains in the Bap proteins from CNS strains. The presence of the functional EF-hands in the Bap proteins, and the fact that calcium inhibition of Bap-mediated aggregation takes place in vitro at concentrations similar to those found in milk serum, support the possibility of this inhibition being physiologically relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.
Bap is the prototype of a new family of surface proteins involved in biofilm formation
The biofilm structure may depend on the nature of the molecules involved in biofilm formation. BLAST searches (Altschul et al., 1997) for sequence homologues to Bap showed the existence of a novel family of proteins, previously named BAP (Biofilm Associated Proteins; Cucarella et al., 2004
), which are important for biofilm formation in both Gram-positive and Gram-negative bacteria. Members of this family have been described in S. aureus (Bap; Cucarella et al., 2001
), CNS (Bap; this study), S. epidermidis (Bhp; our unpublished results), Enterococcus faecalis (Esp; Shankar et al., 1999
; Toledo-Arana et al., 2001
), Burkholderia cepacia (Bap; Huber et al., 2002
), Pseudomonas putida (mus20; Espinosa-Urgel et al., 2000
) and Salmonella typhimurium (Stm2689; McClelland et al., 2001
). All members of the Bap family share the following characteristics: (i) a high molecular mass; (ii) a signal sequence for extracellular secretion; and (iii) a core domain of repeats, the number of which varies among different isolates (Shankar et al., 1999
) and throughout the course of an infection (Cucarella et al., 2004
). Interestingly enough, a variation in the number of repeats in the analysed proteins affected neither the functionality of the protein nor its biofilm formation capacity. These phase-switching differences in the repeat numbers could be related to an evasion of the immune response, as observed in the structurally related alpha C protein (Madoff et al., 1996
), which undergoes antigenic variation.
Although proteins belonging to this family have been involved in biofilm formation, their presence is not absolutely necessary for the process. In this context, S. aureus and S. epidermidis are fully capable of forming biofilms in the absence of the bap gene, as mediated by the icaADBC operon (Cramton et al., 1999; Heilmann et al., 1996b
), the hla gene (Caiazza & O'Toole, 2003
), or the expression of a 190 kDa protein regulated by the rbf gene (Lim et al., 2004
). Additionally, Cramton et al. (1999)
demonstrated the presence of the icaADBC operon in other staphylococcal species, suggesting that the cellcell adhesion mediating the icaADBC operon is highly conserved within this genus. In the same way, others, as well as ourselves, have described that the biofilm formation process of Ent. faecalis could be independent of Esp (Kristich et al., 2004
; Toledo-Arana et al., 2001
). However, it is important to remark that both staphylococci and enterococci can also form biofilms that are exclusively mediated by either the Bap or Esp expression, respectively (Cucarella et al., 2001
; Toledo-Arana et al., 2001
). Interestingly, the expression of the Bap protein in Ent. faecalis enhanced its biofilm-formation capacity (A. Toledo-Arana, unpublished results), confirming the relationship between structure and function between these two proteins. In conclusion, these results suggest that the biofilm formation process in these species is a very complex process mediated by different genes.
Is bap transferred between staphylococcal species by horizontal transfer mechanisms?
Horizontal gene transfer is an important source of change in bacteria. In a previous study we described SaPIbov2 (Staphylococcal pathogenicity island bovine 2), which encodes the biofilm-associated protein Bap (Ubeda et al., 2003). SaPIbov2 has an extensive similarity to previously described pathogenicity islands of S. aureus. The main difference is that the toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element carrying the bap gene, and also genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element, and it can integrate site-specifically and RecA-independently at a chromosomal att site depending of Sip, an integrase present in SaPIbov2. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. We studied the possibility that transduction can facilitate transfer by taking the high identity between the bap genes present in the different staphylococci into account. Interestingly enough, Richard P. Novick (personal communication) has demonstrated that SaPI1, the prototypical pathogenicity island of S. aureus, can be transduced from S. aureus to CNS. Our preliminary results have shown that SaPIbov2 can also be transduced at a high frequency from S. aureus to CNS. This mechanism could explain the high identity observed between the bap genes, with the exception of the bap gene from S. xylosus. However, analysis of the flanking sequences to the bap genes present in the CNS led to the rejection of this hypothesis, since the sequences are completely different amongst themselves, and also in comparison with those present in SaPIbov2 (unpublished results). However, it is important to remark that it is possible to identify sequences related to transposases and recombinases near the bap gene in our best-characterized bap-positive CNS species, S. epidermidis (our unpublished results). Additionally, similar structures in members of the Bap family proteins have been described for esp, which is flanked by insertion elements (IS) carried into a pathogenicity island of Ent. faecalis (Shankar et al., 2002
). The roles that these transposon-related sequences and pathogenicity islands play in the mechanism implied in the dissemination of the bap gene between staphylococcal species are unknown. We believe that the answers to these questions will clarify the intriguing links between mobile pathogenicity islands, DNA exchange, and the biofilm formation ability of bacteria.
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ACKNOWLEDGEMENTS |
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Received 4 January 2005;
revised 20 April 2005;
accepted 22 April 2005.
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