Pilicides inhibit the FGL chaperone/usher assisted biogenesis of the Dr fimbrial polyadhesin from uropathogenic Escherichia coli - PubMed (original) (raw)
Pilicides inhibit the FGL chaperone/usher assisted biogenesis of the Dr fimbrial polyadhesin from uropathogenic Escherichia coli
Rafał Piatek et al. BMC Microbiol. 2013.
Abstract
Background: The global spread of bacterial resistance has given rise to a growing interest in new anti-bacterial agents with a new strategy of action. Pilicides are derivatives of ring-fused 2-pyridones which block the formation of the pili/fimbriae crucial to bacterial pathogenesis. They impair by means of a chaperone-usher pathway conserved in the Gram-negative bacteria of adhesive structures biogenesis. Pili/fimbriae of this type belong to two subfamilies, FGS and FGL, which differ in the details of their assembly mechanism. The data published to date have shown that pilicides inhibit biogenesis of type 1 and P pili of the FGS type which are encoded by uropathogenic E. coli strains.
Results: We evaluated the anti-bacterial activity of literature pilicides as blockers of the assembly of a model example of FGL-type adhesive structures--the Dr fimbriae encoded by a dra gene cluster of uropathogenic Escherichia coli strains. In comparison to the strain grown without pilicide, the Dr⁺ bacteria cultivated in the presence of the 3.5 mM concentration of pilicides resulted in a reduction of 75 to 87% in the adherence properties to CHO cells expressing Dr fimbrial DAF receptor protein. Using quantitative assays, we determined the amount of Dr fimbriae in the bacteria cultivated in the presence of 3.5 mM of pilicides to be reduced by 75 to 81%. The inhibition effect of pilicides is concentration dependent, which is a crucial property for their use as potential anti-bacterial agents. The data presented in this article indicate that pilicides in mM concentration effectively inhibit the adherence of Dr⁺ bacteria to the host cells--the crucial, initial step in bacterial pathogenesis.
Conclusions: Structural analysis of the DraB chaperone clearly showed it to be a model of the FGL subfamily of chaperones. This permits us to conclude that analyzed pilicides in mM concentration are effective inhibitors of the assembly of adhesins belonging to the Dr family, and more speculatively, of other FGL-type adhesive organelles. The presented data and those published so far permit to speculate that based on the conservation of chaperone-usher pathway in Gram-negative bacteria , the pilicides are potential anti-bacterial agents with activity against numerous pathogens, the virulence of which is dependent on the adhesive structures of the chaperone-usher type.
Figures
Figure 1
Blocking the adherence of E. coli Dr + strain to CHO-DAF + cells by pilicides. The propensity of bacteria binding to CHO-DAF+ and CHO-DAF- cells was evaluated by staining with Giemsa (magnification x 10 000, Olympus CKX41 microscope). The following bacterial preparations were used in the adherence assays: negative control – E. coli BL21DE3/pACYC184, grown on TSA plates with 5 % DMSO, non-fimbriated strain; positive control - E. coli BL21DE3/pBJN406, grown on TSA plates without pilicide, fully-fimbriated strain; pilicide 1 and pilicide 2 - E. coli BL21DE3/pBJN406 grown on TSA plates in the presence of 0.5, 1.5, 2.5 and 3.5 mM of agents 1 and 2, respectively. In the experiments presented by M and N panels the E. coli BL21DE3/pBJN406 strain was grown in the presence of 3.5 mM of pilicides. The figure presents representative results obtained from three independent experiments. Each experiment was composed from the four-fold repetition for each used bacterial preparation. The bacterial adherence to 40 CHO cells was determined for each repetition. Presented in the figure pilicides 1 and 2 are the literature agents which, at a 3.5 mM concentration, inhibit the assembly of FGS type 1 and P pili.
Figure 2
Blocking of Dr fimbriae-dependent agglutination of human erythrocytes by pilicides. The following bacterial preparations, normalized to OD600, were used in the hemagglutination assays: negative control – E. coli BL21DE3/pACYC184, grown on TSA plates with 5% DMSO, non-fimbriated strain; positive control - E. coli BL21DE3/pBJN406, grown on TSA plates without pilicide, fully-fimbriated strain; chloramphenicol –E. coli BL21DE3/pBJN406, grown on TSA plates without pilicide, the agglutination experiment was performed in the presence of 2 μM of chloramphenicol; pilicide 1 and pilicide 2 - the E. coli BL21DE3/pBJN406 grown on the TSA plates in the presence of 3.5 mM of agents 1 and 2, respectively. HA titer represents two-fold serial dilutions of normalized bacterial suspensions. The initial 1, 2 and final 128 dilutions are not presented. In the case of HA assays with bacteria cultivated in media in the presence of pilicide the black triangles mark the highest dilution which still provides visible agglutination.
Figure 3
Relative determination of Dr fimbriae amount on bacteria treated with pilicides. (A) SDS-PAGE analysis of the fimbrial fractions isolated from the following bacterial cultures: lanes 1,5 - BL21DE3/pBJN406, grown on TSA plates without the pilicide, fully-fimbriated strain; 2,6 - BL21DE3/pACYC184, non-fimbriated strain; 3,7 and 4,8 - BL21DE3/pBJN406, grown in the presence of 3.5 mM of agents 1 and 2, respectively. Before electrophoresis, the samples from 1 to 4 and from 5 to 8 were incubated for 60 min at 100°C and 25°C, respectively. M – the SDS-PAGE LMW Calibration Kit weight standard. Arrow denoted monomeric DraE protein. (B) Western blotting analysis of the fimbrial fractions, performed to confirm the complete depolymerization of Dr fimbriae during sample denaturation. 1,2,3 – the same samples as in lanes 2,1 and 5 in panel B, respectively. (C) SDS-PAGE analysis of fimbrial fractions isolated from E. coli BL21DE3/pBJN406 grown on TSA plates supplemented with different concentrations of pilicide 1 (Pil1) and pilicide 2 (Pil2). Densitometrically evaluated DraE amounts are expressed as the percentage of mean value present relative to bacterial strain cultivated without pilicide, arbitrary set to 100%: 0.5 mM – 92 and 97%; 1.5 mM – 45 and 55%; 2.5 mM – 32 and 25%; 3.5 mM – 25 and 20% for strains grown in the presence of pilicide 1 and 2, respectively. (D) Evaluation of bacteria fimbriation using an ELISA assay with microtitre plates coated with type IV human collagen. The Dr fimbriae exposed on the bacteria adhered to collagen were visualized using anti-Dr antibodies. The following bacterial preparations were used in the assay: 1 - BL21DE3/pACYC184, non-fimbriated strain; 2-5 - BL21DE3/pBJN406, grown in LB medium supplemented with 3.5, 2.5, 1.5 and 0.5 mM of agent 1, respectively; 6 - BL21DE3/pBJN406, grown in LB medium without the pilicide, fully-fimbriated strain. The bars represent the s.d. of the mean of three independent experiments in duplicate.
Figure 4
DraB as a model of the FGL subfamily of chaperones. (A) Characteristic elements of the DraB structure (PDB ID: 4DJM) specific to the FGL chaperones in relation to the PapD (PDB ID: 2WMP), - the representative of the FGS subfamily. The part of β-strand A1 with hydrophobic residues participating in subunit interaction represented in the bonds mode is denoted in red. The fragment of the long N-terminal region of the β-strand A1 characteristic for FGL chaperones observed in the DraB is denoted in yellow. The F1 strand-loop-G1 strand hairpin motif is denoted in green with the alternating hydrophobic residues of the β-strand G1 participating in the DSC reaction denoted in the bonds mode. In the F1-G1 loop of the DraB protein, the conserved residue motif KDW characteristic for FGL chaperones are marked with the bond mode. The disulfide bond binding β-strands F1 and G1 in the DraB structure conserved in the entire FGL subfamily is marked in yellow bond mode. The F1-G1 loop region was modeled using MODELLER v9.2 software. (B) Structural alignment of the usher binding site of DraB (red) and PapD-pilicide (PDB ID: 2J7L) (blue) with denoted hydrophobic patch that includes I93, L32, V56 (PapD) and I110, L56, L32 (DraB) residues forming pilicide (pink) binding motif. At the beginning of the F1-G1 loop the region of two proline residues forming “proline lock” conserved in the family of chaperones is denoted (P111 and P112 in the DraB – yellow; P94 and P95 in the PapD - green).
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