Allelic variation of type 3 fimbrial adhesin MrkD affects the fimbriation and adhesion activity
3.2 Results and discussion
3.2.1 Identification of four novel mrkD alleles
Recently, the incidence of K. pneumoniae meningitis in newborns and adult patients have been reported worldwide (Lee et al., 2003). Since the role of type 3 fimbriae in determining the tissue tropism has been suggested (Hornick et al., 1992;
Schurtz et al., 1994), the presence of a specific type 3 fimbrial adhesin mrkD allele in meningitis isolates was investigated. Using the primers specific to mrkD1p, PCR analysis showed that all the 17 meningitis isolates carry mrkD gene, and four different
mrkD RFLP types were obtained. Each of the PCR products was then cloned and their
sequences determined. The BLASTX (http://www.ncbi.nlm.nih.gov/BLAST/) analysis revealed 4 novel mrkD alleles, designated mrkDV1, mrkDV2, mrkDV3, and
mrkDV4 (under the GenBank accession number AY225462, AY225463, AY225464, and AY225465). Notably, fourteen of the isolates carry mrkDV1 RFLP and others include one each of the variants mrkDV2 (VHm2), mrkDV3 (VHm5) and mrkDV4
(VHm10). This suggests that the K. pneumoniae carrying mrkDV1 RFLP is a prevalent strain. All the isolates carry mrkD gene implying a possible correlation of type 3 fimbriae with the disease. Nevertheless, more isolates are needed to establish the association.
3.2.2 Amino acid sequence analysis
Comparative analysis with sequence of K. pneumoniae MGH78578 (http://genome.wustl.edu/) revealed an identical mrkD except that a G deletion was found in mrkDV1 at the position 355. The nucleotide deletion caused a frame shift and resulted in a truncated protein, of which the pilin domain was replaced with a garbled sequence of 57 amino acid residues at the C-terminus. It is hence the name MrkDV1T
for the truncated form of the adhesin. As shown in Fig. 1, the conserved receptor binding and pilin domains, and the cysteine residues could be identified in each of the MrkD variants. The comparison indicated that MrkDV2 and MrkDV4 share the most identity, which is 88.1%. Less were found between MrkDV2 and MrkDV3 with 79.3%
identity, and MrkDV3 and MrkDV4 with 80.2%. In the receptor domain of MrkDV3, a varied sequence from residues 120 to 140, and an RGD motif of integrin recognition site (Ruoslahti and Pierschbacher, 1987) were identified (Fig. 1). In addition, the residues which have been proposed to facilitate the interaction of MrkD with other fimbrial component (Sebghati and Clegg, 1999), were unique in MrkDV3 (C102 and R200). The D-R-N (residues 68 to 70) of MrkD1P that has been shown to affect the fimbrial activity (Sebghati and Clegg, 1999) appeared to be replaced by different residues in MrkDV2, MrkDV3 and MrkDV4. These implied regulatory roles of the
varied sequences for the fimbriae activity.
3.2.3 Type 3 fimbriae activity of the meningitis isolates
It has been reported that type 3 fimbriae of K. pneumoniae mediate a specific adherence to different kinds of human epithelial cells (Schurtz et al., 1994; Tarkkanen
et al., 1997). To examine influences of the mrkD allelic variation on the fimbrial
adhesive activity, three epithelial cell lines Hep-2, HCT-8, and Int-407 were used. As shown in Fig. 2A, the bacteria VHm5 of mrkDV3 allele exerted a relatively higher level of the cell adhesion activity. While, 14 of the mrkDV1 strains revealed different levels of activity. The subsequent analysis using western blotting hybridization with the anti-MrkA antiserum indicated that the expression of type 3 fimbriae could only be observed in VHm2 (mrkDV2), VHm5 (mrkDV3), and 6 of the mrkDV1 strains (Fig.
2B). These implied that, besides type 3 fimbriae, other factor(s) such as capsular polysaccharide, which has been reported to impede the bacterial adherence to cells (Sahly et al., 2000), is/are involved in determining the cellular adherence activity.
3.2.4 Expression of the recombinant type 3 fimbriae
To rule out the possibility that other factors resided in K. pneumoniae interfere with the activity of type 3 fimbriae, an E. coli type 3 fimbriae display system was
established. The production of type 3 fimbriae on the surface of the recombinant bacteria was confirmed by western blot analysis (Fig. 3). The TEM analysis (Fig. 4) revealed that no fimbriae on the surface of JM109[pGEMT-easy] could be observed.
Only in a small portion, approximately one tenth of the bacteria JM109[pmrkABC], some short and erect fimbriae were found. In addition, several long fimbriae were found on the surface of E. coli JM109[pmrkABCF], suggesting that MrkF, as a minor fimbrial subunit, is able to function as an initiator for the growth of the filament. In the absence of MrkD adhesin, however, the growth of filament could not be properly terminated and hence appeared lengthy. The speculation is supported by the appearance of extremely long and bundle fimbriae on the surface of E. coli JM109[pmrkABCDV1TF], which could be caused by an interaction of the truncated MrkDV1T with the usher protein leading to uncontrollable length of the fimbriae.
Different from the uniform fimbrial pattern observed on JM109[pmrkABCDV2F]
and JM109[pmrkABCDV4F], the fimbriae on the surface of JM109[pmrkABCDV3F]
are entangled and give rise to a meshwork-like morphology. The sequence comparison in Fig. 1 indicated that unique residues of MrkD V3 are probably the determinants in facilitating MrkD interaction with other fimbrial protein for the distinct morphology.
3.2.5 The mrkF gene is a component of mrkABCDF operon
The common structural characteristics of major pilin subunit are (i) two cysteine residues; (ii) a conserved pattern of alternating hydrophobic residues at position 4, 6, and 7 from the COOH terminus; (iii) a penultimate tyrosine; and (iv) a Gly at position 14 from COOH terminus (Girardeau et al., 2000). As shown in Fig. 5, sequence analysis of MrkF revealed a signal peptide using LipoP in ExPASy proteomic tools and all the characteristics of major subunit. In addition, MrkF also possessed sequence motifs that are conserved among fimbrial subunits (Girardeau and Bertin, 1995). The intergenic sequence of 16 bp between mrkD and mrkF also implied mrkF is a component of the mrk operon structure of mrkABCDF. The RT-PCR analysis of
mrk expression in K. pnemoniae NTUH-K2044 revealed a co-expression of mrkA,
mrkD, mrkF, and the intergenic region between mrkD and mrkF (Fig. 6). This
indicated mrkF belongs to mrk operon. The previous study using E. coli display system has shown MrkF played a role in regulating the length of the filament and the fimbrial activity (廖心瑋，民國九十五年). A mrkF deletion mutant is being constructed, and the fimbrial morphology and activity will be compared to that of wild-type bacteria toward understanding the role of MrkF.
3.2.6 Activity assessments of the recombinant fimbriae
As shown in Table 3, the bacteria JM109[pmrkABCDV3F] and JM109[pmrkABCDV4F] expressed approximately 16 HA units, and JM109[pmrkABCDV2F] had less of the activity. Whereas, JM109[pmrkABCDV1TF] as well as the bacteria carrying pGEMT-easy, pmrkABC, or pmrkABCF exhibited no hemagglutination. This suggested that the MrkDV1T truncation alters conformation of the MrkD receptor binding domain and hence no hemagglutination activity could be detected. As shown in Fig. 7A, JM109[pmrkABCDV3F] expressed the highest level of adhesive activity to either of the three cell lines. Allelic variation of MrkD has been shown to affect the binding activity and specificity to collagen (Schurtz et al., 1994).
The Fig. 7B showed that JM109[pmrkABCDV3F] also revealed the strongest binding activity to collagen IV and V, and JM109[pmrkABCDV4F] had a medium level activity. Moreover, the biofilm formation analysis revealed that JM109 [pmrkABCDV3F] retained the highest activity (Fig. 7C). JM109[pmrkABCDV2F] and JM109[pmrkABCDV4F] also exhibited a comparable activity of biofilm formation.
These support the finding that type 3 fimbriae is a major determinant for K.
pneumoniae biofilm formation (Jagnow and Clegg, 2003). As shown in Fig. 1, the sequence comparison indicated that the D-R-N (residues 68 to 70) of MrkD1P and the variation sequence in the receptor domain of MrkDV3 are probably the determinants
the fimbrial activity. Interestingly, an autoaggregation phenotype was observed only for JM109[pmrkABCDV3F] (Fig. 7D) suggesting the meshwork like fimbriae increased the interaction of the bacteria. The alteration of receptor-binding domain of FimH has been shown to affect the autoaggregation (Schembri et al., 2001). It is also likely that the varied sequence in the receptor domain of MrkDV3 also confers the bacteria an autoaggregation property.
Since the number of fimbrial filaments varies among bacterial cells, the above-mentioned activity measurements could only determine the average binding activity between a given bacterial population and the target molecules. To determine precisely the direct interacting force between a fimbria and its target molecules, the optical tweezers was also used to investigate whether the MrkD really plays a role in presenting the highest activity of highest JM109[pmrkABCDV3F]. Therefore, a typical record of the bead’s displacement during the measurement is illustrated in Fig. 8. The adhesive force of a single fimbria, which expressed with each of mrkD alleles, to collagen IV measured using optical tweezers were 2.03 ± 0.03 pN, 3.79 ± 0.12 pN, and 2.87 ± 0.15 pN for mrkDV2, mrkDV3, and mrkDV4, respectively. This further supported the result of collagen binding analysis. It has been reported that interacting force between a single type 1 fimbriae and an α-C-mannoside ligand (Liang et al., 2000) was 1.7 pN. The adhesive force was close to our data of the adhesive force
between type 3 fimbriae and collagen IV, suggesting that type 1 and type 3 fimbriae may be expression in the part of body with same fluid flow rate. According to Stokes’s Law (F=6(pi)RnVc, R is the radius of the sphere, n is the viscosity, and Vc is the velocity through a continuous fluid), it is calculated that 1 pN could support 1 μ m bead to resistant 50 μm/sec flow rate. In addition, the calculated flow rate is about 40 mm/sec in the bladder and about 300 μm/sec in blood capillary. In addition, the data of the adhesive force of a single fimbria of type 3 fimbriae will give more information in the bacterial infection research.
3.2.7 RGD peptide inhibits the adhesion of JM109[pmrkABCDV3F] to HCT-8
It has been reported that the RGD sequence in FHA (Filamentous hemagglutinin) of Bordetella pertussis is involved in the interaction of the bacteria with macrophage (Relman et al., 1990). To determine if the RGD motif in MrkDV3 affects the bacterial adherence to cells, the peptide GRGDSP was added as a competitor in the cell adherence assay. As shown in Table 2, the adhesion of JM109[pmrkABCDV3F] to HCT-8 cell was reduced by the addition of GRGDSP and the inhibition was in a dose-dependent manner. In contrast, no inhibition was observed when GRADSP peptide was added. This supported a role of the RGD sequence in affecting the adhesion activity of the fimbriae. RGD tripeptide, which is present in many adhesive
ECM and cell surface proteins, is recognized by integrins on the cell surface (Ruoslahti, 1996). The RGD sequence of B. pertussis FHA has been demonstrated to interact specifically with α5β1 integrin (Ishibashi et al., 2001). As shown in Table 4, the anti-α5β1 integrin monoclonal antibody was able to inhibit the adhesion of JM109[pmrkABCDV3F] to HCT-8, indicating the presence of an interaction of MrkD
V3 with α5β1 integrin.
Taken together, we have shown in the study that MrkF is able to serve as an initiator for the growth of type 3 fimbriae and mrkF is a component of mrkABCDF operon. In addition, the proper growth of the filament and fimbrial morphology appeared to be MrkD adhesin dependent. Moreover, MrkDV3 may promote the bacterial adhesion to HCT-8 cells through the interaction of its RGD sequence with integrin.