Co-immunoprecipitation was performed according to the procedures described (59). Briefly, 200 μl of the purified fimbriae (100 μg) were incubated with Bovine serum albumin (5 μg/ml), and anti-MrkA antibody (2 μl) and anti MrkF antibody (2 μl) were added respectively to the reactions. After incubation with gentle rocking overnight at 4°C, protein A-Sepharose beads (50 μl) (Amersham) were then added followed by a second incubation for 3 h at 4°C. The protein A-sepharose beads were collected by centrifugation at 6,000 rpm for 3 min, washed three times with 1 ml of 0.1% DOC, and resuspended in 40 μl of protein lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl) and 10 μl of 5 x protein sample buffer. Finally, after incubation at 95°C for 10 min, 20 μl of the immunoprecipitate was analyzed by SDS-PAGE and visualized via western blotting hybrdization with either anti-MrkA or anti-MrkF antibody.
8. Western blotting analysis
As the method described (48), total cell lysates were resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (ImmobilonTM-P, Millipore). Subsequently, the membranes were blocked with 5﹪
skim milk in 1 x phosphate-buffered saline (PBS) at 4℃ for overnight. After washing 3 times with 1 x PBS, the membranes were incubated with a 3000-fold diluted MrkA or MrkF anti-serum at room temperature for 2 h. Followed by incubation with a 10000-fold diluted alkaline phosphatase-conjugated anti-mouse immunoglobulin G at room temperature for 2 h, additional 3 washes were applied and the bound antibodies
were detected by using the chromogenic reagents BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (Nitro blue tetrazolium).
9. Collagen binding assay
Collagen binding assay was carried out as described (39). Briefly, the wells of flat bottom microtiter plates were coated following incubation overnight at 4°C with 0.5 mg/ml of type IV or V collagen diluted in carbonate- bicarbonate buffer, pH 9.6.
Each sample was performed triplicate. Prior to incubation with bacteria, nonspecific binding sites were blocked by incubation for 2 h at 22°C with a 2% (w/v) solution of bovine serum albumin in PBS. Subsequently, 100 μl of bacterial suspensions (1010 bacteria/ml) prepared in phosphate-buffered saline-Tween 20 (PBS-T) (pH 7.4; 0.5 ml Tween 20 in 1 liter of PBS), was added to the wells. Following incubation for 2 h at 22°C with gentle shaking, unattached bacteria were removed by washing three times in PBS-T. For each well, the adherent bacteria were washed out by 0.1% Triton X-100, diluted in optimal concentration, and plated onto LB agar plates for colony formation.
10. Biofilm formation assay
The ability of bacteria to form biofilm in vitro was detected using the microtiter plate assay with a minor modification of the described method (60). One hundred microliters of overnight grown bacteria diluted 1/100 in LB broth were inoculated into each well of a 96-well microtiter dish and incubated at 37°C for 48 h to allow the formation of biofilm. After thorough by washing the dish with water, 150 μl of crystal violet (1%) was added to each well and the plate was incubated for 30 min at room temperature. The plate was then washed with water, the dye was solubilized in 1%
SDS (150 μl/well), and the absorbance at 595 nm was determined. Each biofilm
formation was presented by the average absorbance of three independent experiments.
11. Autoaggregation assay
As described (61), settling profiles were performed on overnight cultures (4 ml) grown in GCAA broth at 37°C. At the beginning, all cultures were shaking vigorously for 20 sec. Samples (10 μl) were taken from the top of the culture at regular time intervals and were spread on microscope slide. After dried by heat, the samples were stained with 1% crystal violet and roasted again. Then, the samples were observed under light microscopy with 100 x object lens.
Results
1. Overexpression and purification of MrkD and MrkB recombinant proteins Our previous studies have shown that sequence variation of MrkD influenced the expression of type 3 fimbriae, which include changes in the activities of collagen binding and biofilm formation, and morphology of the fimbriae. In addition, the recombinant E. coli displaying with type 3 fimbriae of mrkDv3 was found to have the highest level of either collagen binding or biofilm formation activity. As shown in Fig.
1, sequence comparison of MrkDv3 with the other MrkD variants, MrkDv1, MrkDv2, MrkDv4 and MrkDntuh, revealed a variable region from Gly120 to Gln140. It is interestingly to note that RGD residues which are active modulators of cell adhesion are contained in the region. As shown in Fig. 2, a signal peptide sequence in MrkF was observed and a conserved pilin domain of MrkA (34) was found also to be contained in MrkF suggesting that MrkF is a component of type 3 fimbriae.
In order to determine the role of the variation, MrkDv3, MrkDv3NL, MrkDv3N, and MrkDv3NS containing expression plasmids were generated. As shown in Fig. 3, MrkDv3N contained the N-terminal domain of MrkDv3 from residues 1 to 159, and MrkDv3NS contained residues 1 to 120, lacking the variable region Gly120 to Gln140,
while MrkDv3NL contained the region from residues 1 to 183. Total cellular proteins from each of the recombinant bacteria containing the plasmids expressing respectively the proteins, MrkDv3, MrkDv3NL, MrkDv3N, and MrkDv3NS, were collected and analyzed by SDS-PAGE. As shown in Fig. 4A and 4B, the recombinant protein MrkDv3 and MrkDv3NL induced by IPTG, with the final concentration of either 0.1 μg/ml or 0.5 μg/ml, could be observed with the predicted size of 37 kDa (MrkDv3) and 27.9 kDa (MrkDv3NL). However, the proteins formed inclusion bodies. In order to improve the solubility, several methods such as 25℃ induction
(lanes 8-10 in Fig. 4A and 4B) and lower concentration of IPTG (0.1 μg/ml, lanes 2-7 in Fig. 4A and 4B) were tried but failed. Similarly, as shown in Fig 4C and 4D, the IPTG inducible proteins MrkDv3N and MrkDv3NS with the predicted size 25 kDa and 23 kDa, respectively, remained insoluble form.
In case that not enough chaperone proteins are present for properly folding of the overexpressed proteins, the recombinant plasmid pdMrkB-1-pET, containing the MrkB chaperone protein encoding gene, was generated and co-expressed with pdMrkDv3-1-pAC in the recombinant E. coli. In addition, the signal peptides of MrkB and MrkD proteins were both removed to avoid from aggregation in the periplasm. Whatsoever, even the recombinant chaperone proteins appeared to be insoluble (data not shown). Finally, the recombinant protein dMrkDv3N were purified by denaturalization using 6 M urea and were refolded by dialysis against the buffer of 20 mM Tris-HCl containing 20% glycerol. Nevertheless, most of the proteins remained aggregated and only a small amount of protein obtained with a concentration of 54 μg/ml.
2. Analysis of the purified dMrkDv3N in competition assay.
The purified dMrkDv3N, containing the N-terminal 159 amino acids was subsequently added as a competitor in the collagen IV binding assay. As shown in Fig.
5, the purified dMrkDv3N proteins added with either 5.4 μg or 8.1 μg respectively appeared to reduce the binding of E. coli JM109[pMrkABCDv3F] to the type IV collagen. The inhibition was enhanced with the increasing of the recombinant protein dMrkDv3N indicating that the N-terminal 159 amino acids containing MrkDv3 is able to compete for the type IV collagen binding activity of the E. coli JM109[pMrkABCDv3F].
3. MrkF is a component of type 3 fimbriae
As shown in Fig. 6A, a protein band corresponding to the predicted size of MrkA could be observed. After the gel hybridization with anti-MrkF antiserum, a protein band appropriate for 22 kDa, which is the predicted size for MrkF, was detected indicating the specificity of the raised antibody (Fig. 6B).
In order to know whether MrkF is a component of type 3 fimbriae, type 3 fimbriae were purified from JM109[pmrkABCDF] and analyzed by SDS-PAGE and further detected with anti-MrkF and anti-MrkA antibodies. As shown in Fig. 7A and B, the MrkA corresponding band with approximately 23 kDa could be observed in the purified fimbriae from JM109[pmrkABCDF] and JM109[pmrkABCD]. While analyze with anti-MrkF antibody, the detected band of approximately 22 kDa could only be found in the purified fimbriae from JM109[pmrkABCDF] (Fig. 7C).
According to the reported fimbrial organization of several well-known fimbriae, the filament is constructed by subunit association (77). As a component of type 3 fimbriae, MrkF must interact with MrkA in some way to form a fimbria. Using anti-MrkA antibody or anti-MrkF antibody appeared to be able to pull down both MrkA and MrkF from the purified fimbriae of JM109[pmrkABCDF] and JM109 [pmrkABCF] as detected by either anti-MrkA antibody (Fig. 8A) or anti-MrkF antibody (Fig. 8B). In contrast, the pull down lysates from the purified fimbriae of JM109 [pmrkABCD] could only be detected by anti-MrkA antibody.
Subsequently, localization of MrkF on type 3 fimbriae was demonstrated by immuno-electron microscopy (immuno-EM). As shown in Fig. 9, the pili labeled in situ with gold-tagged anti-mouse IgG antibodies against the anti-MrkF antibody revealed the signals at the middle part of the filament suggesting that type 3 fimbriae is consisting of stretches of MrkA interrupted by MrkF at regular intervals.
4. MrkF influences the fimbriation of the recombinant E. coli
Although lacking the mrkD adhesion gene, a few fimbriae could be observed on the surface of the recombinant E. coli HB101[pmrkABCF] (Fig. 10 ) suggesting that MrkF is an initiator for the assembly of the fimbriae. However, in comparing with that of HB101[pmrkABCD], the limited number of fimbriae on the surface of HB101 [pmrkABCF] also suggested a minor role of MrkF in initiation of the fimbriation. As shown in Fig. 10, the recombinant E. coli HB101[pmrkABCDF] exerted more and shorter fimbriae than that of HB101[pmrkABCD] indicating a more efficient fimbriation but a tighter control for the length of the filament. Nevertheless, after being heated at 55℃ for 10 min, an obvious falling off of the MrkF-lacking-fimbriae from the bacterial surface supported the role of MrkF as a controller for the stability of type 3 fimbriae as proposed (34)
5. Assessment of the activity of the recombinant type 3 fimbriae
Type 3 fimbriae had been shown to bind specifically to type IV and type V collagen (62). The collagen binding activity of the recombinant E. coli JM109[pmrkABCDF], JM109[pmrkABCD] and JM109[pGEMT] were hence compared. The recovered bacteria JM109[pmrkABCDF] were more than JM109[pmrkABCD] in either collagen IV binding assay (Fig. 11A) or collagen V binding assay (Fig. 11B), suggesting that lacking of MrkF causes the lower binding activity to collagen.
Biofilm formation activity of type 3 fimbriae has been demonstrated to be mediated by MrkA or MrkD at different conditions (36, 63). As shown in Fig. 12, the recombinant E. coli JM109[pmrkABCDF] appeared to have higher biofilm formation activity than JM109[pmrkABCD] implying that MrkF plays a role in biofilm formation. In addition, the activity of biofilm formation was comparable with
Pseudomonas aeruginosa PAO1, a strong biofilm formation strain, indicated that the recombinant fimbriae produced by E. coli JM109[pmrkABCDF] could help efficiently for the biofilm formation. It has previously been demonstrated that the MrkD adhesin of type 3 fimbriae was not required for an efficient biofilm formation on abiotic plastic surface (35). Notably, the E. coli JM109[pmrkABCF] which expressed few fimbriae with no MrkD appeared to lose the ability to form biofilm (Fig. 12).
Autoaggregation, which is mediated by bacteria surface self-recognizing adhesins or autoaggregative fimbriae, is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria (64, 65, 66, 67). The recombinant E. coli JM109[pmrkABCDF] appeared to show conspicuous autoaggregation after overnight cultured in GCAA medium (Fig. 13). In contrast, no autoaggregative appearance could be observed in JM109[pmrkABCD]. This suggested a functional role of MrkF in autoaggregation. To investigate the possibility, mrkF gene was introduced respectively into the non-autoaggregative strains, JM109[pmrkABCDv1] and JM109[pmrkABCDv2]. As predicted, introduction of the MrkF encoding gene conferred the bacteria JM109[pmrkABCDv1F] and JM109[pmrkABCDv2F] an autoaggregative phenotype (Fig. 13). In addition, the autoaggregative phenomenon of JM109 [pmrkABCDv3] and JM109 [pmrkABCDv4]
could be enhanced after introducing the mrkF gene in the bacteria.
Discussions
Fimbriae are important appendages for bacteria to infect host. The adherene to host epithelial of fimbriae influence the host range and tissue tropism of bacteria. Many features of famous pili such as type 1 pilus, P pilus, in uropathagenic E. coli were well studied, including adherent receptor, structural components, organization, and their function. However, investigation of type 3 fimbriae which is universal expressed in Enterobacteriaceae is still not enough.
For example, the assembly model, adhesin structure, and fimbrial organization are poorly understood.
1. Improvement of the solubility of the recombinant protein
In many cases, the insolubility of the recombinant proteins prevent further assays. In this study, several strategies have been tried but failed. Nevertheless, we intend to solve the problem in the future by construction of the recombinant plasmids for optimal expression in AD494 (DE3) or BL21trxB (DE3), which is thioredoxin reductase-deficient strain often used to maximize soluble protein expression (78). An alternative approach is to fuse the protein with a solubility-enhancing tag such as the Trx●TagTM, GST●TagTM, or Nus●TagTM sequences (68).
2. Localization of MrkF
So far, there is only one report to suggest the role of MrkF in stabilizing the structure of type 3 fimbriae (34). While analysis of MrkF sequence, a typical signal peptide may found and the sequence alignment with MrkA showed a conserved pilin domain (Fig. 2), implying that MrkF is also a component of type 3 fimbriae. As shown in Fig. 8, co-immunoprecipitation assay demonstrated an
interaction of MrkA and MrkF which supports further the association of MrkF with MrkA on the fimbriae. Moreover, TEM of immuno-gold labeled fimbriae showed that the labeled MrkF appeared to be inserted to the fimbrial shaft at regular intervals. During the assembly of a pilus, the order of subunit on fimbriae is decided by the affinity between subunit-chaperone complexes and usher (75).
As shown in the Fig. 9, many MrkA proteins on the fimbriae appeared to be interrupted by a few MrkF suggesting that MrkA-chaperone complexes have higher affinity to usher than the MrkF-chaperone complexes.
3. Functional role of MrkF
The TEM analysis indicated that all the recombinant E. coli were fimbriated except HB101[pmrkABC]. The fact that adhedsin is an initiator for the assembly of fimbriae has been reported (46, 69). Interestingly, the bacteria HB101[pmrkABCF], lacking the MrkD adhesin as an initiator, appeared to be fimbriated (Fig. 10) suggesting a role of MrkF in initiating the fimbriation.
Whatsoever, the bacteria HB101[pmrkABCDF] exhibiting more fimbriae on the surface than HB101[pmrkABCD] supported the role of MrkF in controlling the stability of fimbriae.
In order to colonize surfaces, most bacteria grow as organized biofilm communities (70). Adherence to non-biological surfaces constitutes the first step in biofilm development (13). The type 3 fimbrial major subunit MrkA has been reported to facilitate biofilm formation on abiotic surface (13). The bacteria JM109[pmrkABCDF] having higher biofilm formation activity than JM109[pmrkABCD] could be contributed to the increasing amount of type 3 fimbriae presented on the bacteria surface. On the other hand, the lacking of MrkF in JM109[pmrkABCD] could result in lower level of fimbriation thereby less
MrkA was available to help for biofilm formation.
The attachment of bacteria to a surface often results in the proliferation into complex microcolony structure. A number of factors including antigen 43 (Ag43) (73), curli (74) and type 1 fimbriae (60) have been implicated in microcolony formation and autoaggregation in E. coli. In particularly, type 1 fimbriae have been shown to confer bacterial autoaggregation and enhance biofilm formation on abiotic surfaces. In K. pneumoniae, similar to type 1 fimbiae, type 3 fimbriae appeared to mediate the biofilm formation (13). Aggregation and microcolony formation often prelude to biofilm formation and hence the apparent autoaggregation of the recombinant E. coli JM109[pmrkABCDF] led to a high level of biofilm formation activity. As shown in Fig. 13, the recombinant E. coli JM109[ABCDF], but not JM109[mrkABCD], appeared to autoaggregate in GCAA medium. Moreover, the autoaggregative phenomenon of the recombinant E. coli could be induced by transforming the bacteria with the plasmid carrying a mrkF gene, suggesting a role of MrkF in autoaggregative phenotype. It is likely that lacking of MrkF alters the structure of the 3 fimbriae and hence the decreasing activity of autoaggregation. Overall, this study indicated that MrkF is a component of type 3 fimbriae and a role of MrkF in initiating the fimbriation was also suggested.
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