Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod

Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod
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  Pilicides regulate pili expression in  E. coli   without affecting the functionalproperties of the pilus rod Veronica A˚berg, a Erik Fa¨llman, b Ove Axner, b Bernt Eric Uhlin, c  Scott J. Hultgren d  and Fredrik Almqvist * a Received 15th September 2006, Accepted 14th December 2006 First published as an Advance Article on the web 19th January 2007  DOI: 10.1039/b613441f  The infectious ability of uropathogenic  Escherichia coli   relies on adhesive fibers, termed pili orfimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that areformed  via  a highly preserved assembly and secretion system called the chaperone-usher pathway.We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt bothtype 1 and P pilus biogenesis in  E. coli  . In this study, we show that the pilicides do not affect thestructure, dynamics or function of the pilus rod. This was demonstrated by first suppressing theexpression of P pili in  E. coli   by pilicide treatment and, next, measuring the biophysical propertiesof the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic forcemicroscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers weredetermined by optical tweezers force measurements on individual pili and were found to be intact.The presented results establish a potential use of pilicides as chemical tools to study importantbiological processes  e.g.  adhesion, pilus biogenesis and the role of pili in infections andbiofilm formation. Introduction A vast number of pathogenic microbes utilize the so-calledchaperone-usher pathway to assemble surface organelles(pili/fimbriae) associated with virulence and biofilm formation.For example, pili on uropathogenic  Escherichia coli   facilitateadherence to host cells leading to urinary tract infections. Amodel for this assembly process has been provided fromextensive studies on pilus biogenesis in uropathogenic  E. coli  ,which commonly express both P and type 1 pili. 1 Interferingwith the function of the chaperone-usher pathway offerspossibilities to gain new insights in this complex assemblymachinery. This strategy to prevent pilus formation alsorepresents an alternative and attractive approach to meet thevital needs to develop new anti-infective agents. 2,3 In brief, pili consist of a number of subunit proteins; PapA,E, F, G, H, and K in the case of P pili, and FimA, F, G, and Hin type 1 pili. The major subunit is PapA in P pili and FimAin type 1 pili. These major subunits are arranged in a  y 1  m mlong, 4 rod-like helical structure 5,6 and the base of the pilusrod is anchored to an outer membrane assembly site contain-ing the usher protein (PapC/FimD). 1 At the top of each rodthere is a flexible tip that includes an adhesin (PapG/FimH)at the distal end. 7,8 The adhesins PapG/FimH contributewith target binding specificity by specific interactions togalabiose on kidney cells, or mannose on bladder cells,respectively. 9,10 Although the assembly process and structures of theseorganelles have been well described, their role in the infectioncascade has not yet been fully elucidated. Pili may facilitate theescape of host defenses allowing bacteria to persist in theurinary tract and cause recurrent infections. 11,12 Moreover,the structure and dynamics of the pilus rod have gained anincreasing interest during the last years and both the shaft andits features are now believed to be important for bindingspecificity and also persistence to host defenses  e.g.  shearforces from body fluids. For example, recent studies show thatthe structure of the shaft itself affects binding specificity, 13,14 asproposed by affecting the conformation of the FimHadhesin. 13 A conformational change in the adhesin of FimHhas also been used to explain the increased binding strength totarget cells that was observed when type 1 piliated  E. coli   weresubjected to shear forces comparable to the ones of bodyfluids. 15 Moreover, the helical arrangement of the repeatedPapA/FimA subunits provides a highly flexible shaft thatcan unfold and refold. 14,16–18 This elongation possibility of the shaft could be beneficial for maintained attachmentunder shear stress. Presumably the role of the shaft and itsdynamic properties is far more sophisticated than what isknown today. 19 It is of great value to be able to study both the propertiesof pili as well as their behavior and impact on importantbiological processes. Most of the present knowledge about theprocess of pilus assembly has been gained from geneticanalyses. Mutants lacking one or more of the componentsare blocked at a particular step and the bacteria may becomecompletely devoid of pili or, alternatively, produce non-functional pili structures rendering the bacteria unable to bindto the specific receptor. However, the genetic alterations maycause additional effects at the level of transcription and/ortranslation in polycistronic operons. a Organic Chemistry, Umea˚ University, SE-90187 Umea˚, Sweden.E-mail:; Fax: +46 90138885;Tel: +46 907866925 b Department of Physics, Umea˚ University, SE-90187 Umea˚, Sweden c Molecular Biology, Umea˚ University, SE-90187 Umea˚, Sweden d  Department of Molecular Microbiology, Washington University in St.Louis School of Medicine, St. Louis, MO 63110, USA PAPER  | Molecular BioSystems 214  |  Mol. BioSyst. , 2007,  3 , 214–218 This journal is    The Royal Society of Chemistry 2007  An alternative approach to genetic manipulation is to utilizesmall synthetic molecules that specifically can interfere with aparticular event in the process. We have earlier reported thatsubstituted 2-pyridones, referred to as pilicides, inhibit pilusbiogenesis in uropathogenic  E. coli  . They have been proposedto inhibit chaperones, thus prohibiting the essential eventwhere the N-terminal of the usher recognizes and binds toincoming chaperone-subunit complexes. 20 Pilicides therebyspecifically block the pilus assembly process and can thuspotentially be used to regulate the amount of pili beingexpressed on the surface of bacteria. Pilicides will complementgenetics and offer a new level of fine-tuning by their ability tomodulate pili production.It has recently become possible to measure the physicalproperties of single pilus shafts, 14,16–19,21,22 which allowed us toinvestigate if the suppressed amounts of pili (formed in thepresence of low doses of pilicide) still displayed intactbiomechanical properties. Preserved pilus properties wouldapprove of the application of pilicides as chemical tools toproduce bacteria with reduced quantities of pili, and still withmaintained pilus function. On the other hand, if structurallydifferent, and possibly weaker and more fragile pili werefound, it would still be interesting with respect to bothadhesion and persistence to shear forces.In this study we compare the properties of P pili assembledin the absence of pilicides to P pili expressed under treatmentof pilicide at different concentrations. This comparisonwas accomplished using optical tweezers force measurementson piliated living bacteria. The degree of piliation wasstudied with hemagglutination (HA) and atomic force micro-scopy (AFM). Results and discussion The P pili-expressing  E. coli   strain HB101/pPAP5 was grownin the presence of two pilicides 20,23–25 ( 1 26,27 and  2 , 25 Fig. 1) atthree different concentrations (1.4, 2.5 and 3.5 mM).The expression of pili was then evaluated in a hemagglutina-tion (HA) assay where the ability of bacteria to agglutinateerythrocytes reflects their level of piliation. The non-piliproducing strain HB101/pBR322 was included as a negativecontrol. Few pili result in a low HA-titer, which could beobserved for pilicide treated bacteria (Table 1). The HA-titerwas altered over an 8-fold range indicating that at higherpilicide concentration there was virtually no specific bindingby the bacteria.Since the HA-titer reflects the average expression of P pili inthe bacterial population, we used AFM to directly monitor theeffect of pilicides on single bacterial cells (Fig. 2). Asanticipated, each individual bacterium had fewer pili attachedto their surface when grown in the presence of pilicide. Inaddition, a dose-dependent level of piliation was observedwhere the highest pilicide concentration resulted in largelynon-piliated bacteria. At the lowest pilicide concentrations ( i.e. 1.4 mM) the bacteria still carried a large number of pili butclose examination revealed that there were fewer pili per cell.The results obtained with P pili are consistent with the onesrecently demonstrated for type 1 pili, 20 indicating that pilicidesprimarily affect the onset of the pilus assembly rather than thepilus elongation/polymerization process, once it is initiated.By Western immunoblot analyses we also assessed theeffects of pilicide treatment on the level of major P pilussubunit proteins. As shown in Fig. 3A, the amount of PapA Fig. 1  Pilicides  1  and  2  inhibit formation of pili in uropathogenic E. coli  . Table 1  HA results with  E.coli   HB101/pPAP5 cultured in thepresence of pilicides  1  and  2 Strain PilicideConcentration/mMHA(slide test) a HA-titer b HB101/pBR322 None — — 2 (1–4)HB101/pPAP5 None — +++ 64 (32–64)HB101/pPAP5  1  3.5 — 4 (4–8)HB101/pPAP5  1  2.5 (+) 16 (8–16)HB101/pPAP5  1  1.4 ++ 16 (8–16)HB101/pPAP5  2  3.5 — 8HB101/pPAP5  2  2.5 — 8HB101/pPAP5  2  1.4 ++ 16 (16–32) a Agglutination was in the range of strong (+++) to undetectable(—).  b Representative HA-titer (the highest dilution that stillprovides agglutination) from two runs with duplicate HA-dilutionseries. The ranges of the four HA-titers are given in parentheses. Fig. 2  AFM images of HB101/pPAP5 treated with different con-centrations of pilicide  1 . Bacterial cells from plate cultures grownin presence of the specified concentrations of pilicide were imagedby AFM as described in the Experimental section. White barrepresents 2.5  m m.  This journal is    The Royal Society of Chemistry 2007  Mol. BioSyst. , 2007,  3 , 214–218  |  215  protein was gradually decreased in response to increasingpilicide concentrations. However, the pilicide treatment didnot seem to affect the level of the usher protein PapC. Forcomparison we analyzed the level of PapA protein in the caseof mutants completely defective in key components in thechaperone-usher pathway. As shown in Fig. 3B, lack of eitherthe chaperone (PapD) or the usher (PapC) led to such adrastic reduction of pili that PapA was not even detected inthe immunoblot assay and consequently such bacteria arecompletely non-piliated. 28,29 Deficiency in PapH on the otherhand, believed to act as an assembly terminating subunit, 30 didnot alter the PapA level. Taken together, our results supportthe suggestion that pilicides affect pilus assembly in such a waythat this molecular machinery can be partially inhibited.To assess the functional properties of pili produced underpilicide treatment at sub-inhibitory concentrations, thebacteria were studied with force measurements using opticaltweezers. The measurements were performed on individual Ppili on living cells according to a high precision procedurethat has previously been developed and described in detailby us. 16,21 In short, a sample consisting of 9  m m diameteractivated polystyrene beads, 3  m m diameter polystyrene beads(coated with the carbohydrate receptor) and diluted bacteria in30  m L PBS, was prepared on a silanized hydrophobiccoverslip (Fig. 4).A free-floating bacterium was trapped by the opticaltweezers with low power ( y 20 mW at the sample) and firmlymounted on the activated large bead resulting in covalentbinding. The large bead was in turn immobilized to the coverslip through a hydrophobic interaction. A small bead wassubsequently trapped by the optical tweezers with normalpower (120 mW at the sample) and brought to a position closeto the bacterium. A force measuring calibration procedure,based upon Brownian motion, was used to calibrate thestiffness of the trap before each new set of measurements. Thesmall bead, to which part of the pilus fiber adhere non-covalently and non-specifically, was then brought close to thebacteria and aligned axiosymmetrically. The data acquisitionwas started and the piezo stage was set in motion to separatethe bacterium from the small bead within the equilibriumspeed of unfolding/refolding,  , 0.1  m m/s. Since multiple piliattachments are common at small separations between thebacterium and the small bead, the separation was continueduntil only one pilus was attached. The pilus was then extendedup into region III (see below) for a complete unfolding andan overstretching of the PapA rod. At a force of 70–80 pN,the direction of the large bead was reversed, allowing thepilus to retract.Measurements on pili expressed by bacteria grown underinfluence of pilicides (Fig. 5, panel B and C) show comparableforce curves to pili expressed by bacteria grown under normalconditions (Fig. 5, panel A). The force response of a pilusunder elongation reflects the physical structure of the pilus.Region I of the force curve,  i.e.  the linear increase of the forcefrom 0–27 pN, which can be seen in panel B and C , srcinatesfrom elastic stretching of the helical structure of the rod.Region II, the plateau at 27 pN, is due to a sequentialunfolding of the layer-to-layer bonds (and thereby of thehelical structure) of the PapA rod. Region III, with itscharacteristic s-shape, is due to an elastic stretching of thelinearized PapA sequence. 16,22 Refolding of the PapA rodfollows the force curve of unfolding except for a small dip Fig. 3  Western immunoblot analysis of Pap pili proteins. Whole cellextracts of strains expressing Pap pili were analyzed using antiserarecognizing the PapA major pilus subunit and the PapC usher protein,respectively. (A) HB101/pPAP5 treated with different concentrationsof pilicide. The relative amount of PapA and PapC, as determined byimaging of the Western blots, is expressed as a ratio. The sample fromthe untreated bacteria was used as a reference and was arbitrarily set to1.0. Strain HB101/pBR322 was used as negative control. (B) Effect of different  pap  operon mutations on the level of PapA protein. Samplesof bacteria were obtained from the following strains: HB101/pPAP30(  papH  ); HB101/pPAP32 (  papC  ); HB101/pPAP37 (  papD ), and HB101/pPAP5 (  pap + ) respectively. A protein present in all samples, and thatwas recognized by antibodies in the anti-PapA antiserum, is indicatedby the asterisk and is shown for comparison as an internal control forequal sample loading. Fig. 4  Measurement procedure adopted from Jass  et al  . 16 (A) Abacterium is mounted onto the 9  m m activated bead attached to thecoverslip. The trapped bead serves both as a surface onto which thebacterial pili bind and as a force indicator in the optical tweezerssystem. (B) The trapped bead is moved close to the bacterium forminga strong bond between the bacterial pili and the bead. (C) A forceis exerted on the pili by moving the coverslip with the large bead.The displacement of the trapped bead is thereby a measure of theexerted force. 216  |  Mol. BioSyst. , 2007,  3 , 214–218 This journal is    The Royal Society of Chemistry 2007  between region II and III and some occasional dips due tomisfoldings.Misfoldings are normally found at refolding and depend on,among other things, the speed of retraction and the number of PapA units. The pili in panel A and B show one misfoldingwhereas the pilus in panel C shows four misfoldings. Thelength of region II reflects the stretched part of the pilus fiberand can be used to calculate the number of pili subunitsinvolved. 22 The curves A and B in Fig. 5 were found to involve y 1100 and  y 800 subunits, respectively, whereas curve Cconsists of 2500 subunits. We thereby attribute the largernumber of misfoldings in curve C to the larger number of subunits being unfolded in that pilus. In each curve less than0.15% of the subunits misfold. The misfoldings are thereforenot caused by the treatment with pilicides.Worth mentioning is that the force measurements on pilicidetreated bacteria compared to measurements on bacteria grownunder normal conditions resulted in more frequent single pilusforce curves. This facilitated the force measurements and alsostrongly indicated that fewer pili were expressed on eachindividual bacterium, which was also shown with AFM.Altogether, the force measurements clearly demonstrate thatalthough the total amount of pili is reduced by the pilicides,the pili that are assembled display intact anchoring, structuraland biomechanical properties. Conclusions Hemagglutination and atomic force microscopy have beenused to verify that pilicides regulate the chaperone-usherpathway in P pili producing  E. coli   in a dose-dependentmanner. Furthermore, optical tweezers force measurementsdemonstrate that pilicides can be applied to suppress theabundance of pili without affecting the biophysical features of the pilus rod. The properties of the function, structure anddynamics of intact pili in cells treated with pilicides remainunchanged, suggesting that these molecules will be importantin the study of biological processes, including the formation of pili and their virulence-associated function. Experimental Bacterial strains and growth conditions The following  E. coli   strains were used: HB101/pPAP5, 31 a clone carrying the wild type  pap  gene cluster from the UPECstrain J96; HB101/pBR322, 31 a vector control; HB101/pPAP32, 29 a  papC   mutant clone; HB101/pPAP37, 29 a  papD mutant clone; HB101/pPAP30, 29 a  papH   mutant clone.Bacterial cells were allowed to grow at 37  u C on TSA-platesor in LB. Bacteria were cultured in the presence of pilicidesas previously described. 23 In case of recombinant bacterialconstructs the media were supplemented with appropriateantibiotics to select for presence of the plasmids. AFM imaging For atomic force microscopy imaging of bacteria whole cellsamples were prepared from over night cultures grown onTSA-plates at 37  u C. The cells were resuspended in 50  m l MQ-water and 5  m l was placed on mica, air-dried and desiccated for1 h. The samples were imaged in air on a Nanoscope IIIa AFM(Digital Instruments) in tapping mode as described before. 32 The images are presented in amplitude mode. Several regionsof each sample were scanned to confirm consistency. Hemagglutination assay Hemagglutination of pilicide treated  E. coli   was determinedaccording to a previously published procedure. 23 Western blot analysis Samples were subjected to SDS-PAGE and were transferred toa PVDF membrane using a semi dry-transfer system (Biorad).The membrane was blocked overnight in 5% milk powder inPBS + 0.1% Tween20 at 4  u C. The primary antibodies wererabbit polyclonal antiserum raised against PapA 33 and thePapC 34 protein. The detection using horse radish peroxidase-conjugated anti-rabbit secondary antibodies was carried outfollowing the instructions provided with the ECL + Kit(Amersham Biosciences). The chemiluminescent bandswere imaged by autoradiography or by using the Fluor-SMultiImager (Bio-Rad). Optical tweezers force measurements The force measurements were performed with an opticaltweezers system and procedures previously described. 16,21 Acknowledgements We are grateful to Monica Persson for technical assistance inatomic force microscopy analyses. This work was supported bythe Swedish Research Council (grants no. 2004-4312 (to FA),2005-4662 (to OA), and 2005-3151 (to BEU)) and performedwithin the Umea˚ Centre for Microbial Research (UCMR). Fig. 5  Force measurements on individual P pili structures showingthe elongation ( A  ) and the refolding ( r ). The unfolding regions(I–III) of each pilus are marked and separated by dashed lines. (A) Atypical force response from an untreated bacterium cultured undernormal growth conditions. Initial multiple pili attachment is denotedwith an asterisk. (B) A typical force response of a bacterium cultured inpresence of 2.5 mM  1 . (C) A representative force response from abacterium cultured in presence of 1.4 mM  2 .  This journal is    The Royal Society of Chemistry 2007  Mol. BioSyst. , 2007,  3 , 214–218  |  217  Economical support for the construction of a force measuringoptical tweezers system from the Kempe foundation and fromMagnus Bergvall’s foundation is acknowledged. The Knut andAlice Wallenberg foundation is acknowledged for financialsupport to FA and for the AFM equipment. References 1 F. G. Sauer, H. Remaut, S. J. Hultgren and G. Waksman, Fiberassembly by the chaperone-usher pathway,  Biochim. Biophys. Acta ,2004,  1694 , 259–267.2 Y. M. Lee, F. Almqvist and S. J. Hultgren, Targeting virulence forantimicrobial chemotherapy,  Curr. Opin. Pharmacol. , 2003,  3 ,513–519.3 M. N. Alekshun and S. B. Levy, The  Escherichia coli   mar locus -Antibiotic resistance and more,  Drug Discovery Today , 2004,  70 ,451–456.4 E. Bullitt and L. 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