The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression

Microbial Pathogenesis 46 (2009) 243–252

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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath

The SfaXII protein from newborn meningitis E. coli is involved in regulation of motility and type 1 fimbriae expression Annika E. Sjo¨stro¨m a, Carlos Balsalobre a,1, Levente Emo¨dy b, f, Benita Westerlund-Wikstro¨m c, Jo¨rg Hacker d, e, Bernt Eric Uhlin a, * a

Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden (MIMS) , Umeå University, S-90187 Umeå, Sweden Department of Medical Microbiology and Immunology, University Medical School, Szigeti ut 12, H-7624 Pecs, Hungary General Microbiology, Faculty of Biosciences, P.O. Box 56, FIN-00014 University of Helsinki, Finland d Robert-Koch-Institut, Nordufer 20, D-13353 Berlin, Germany e ¨ r Molekulare Infektionsbiologie, University of Wu ¨ rzburg, Ro ¨ntgenring 11, D-97070 Wu ¨ rzburg, Germany Institut fu f Veterinary Medical Research Institute, Hungarian Academy of Sciences, Hungaria krt. 21, 1143 Budapest, Hungary b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 July 2008 Received in revised form 16 January 2009 Accepted 22 January 2009 Available online 4 February 2009

The genomes of pathogenic Escherichia coli may contain several different fimbrial operons. How bacteria regulate and coordinate the choice of fimbrial expression under different circumstances remains largely unanswered. In this report we have investigated the role of the sfaXII gene associated to the SfaII fimbrial determinant in the E. coli isolate IHE3034. sfaXII belongs to a subfamily of genes, the 17 kDa genes, located near different fimbrial operons in uropathogenic and newborn meningitis E. coli (NMEC) strains. Using the NMEC isolate IHE3034 and non-pathogenic E. coli strains we found that the sfaXII gene had an inhibitory effect on type 1 fimbriae expression. Down-regulation of type 1 fimbriae was exerted at transcriptional level both by inhibiting expression from the fimA promoter and by reducing the frequency of OFF-to-ON switching. The effect of sfaXII on expression of the recombinase FimB that catalyzes OFF-toON switching might explain the described reduction in percentage of ON cells. Moreover, expression of the sfaXII gene strongly influenced motility and flagella production of the NMEC isolate IHE3034. We propose that the sfaXII gene, and presumably other members in the 17 kDa gene family, may play a role in the control of virulence related gene expression in pathogenic E. coli. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: NMEC Regulation S fimbriae Type 1 fimbriae Motility

1. Introduction Escherichia coli is the most abundant aerobic commensal bacterium in the human fecal flora. Nevertheless, it is a frequent cause of extraintestinal infections including sepsis, urinary-tract infections (UTI) and newborn meningitis [1]. Generally, the first step in an infection process is adhesin-mediated tissue recognition by the bacterium and its adherence to specific receptors on the surface of eukaryotic cells. E. coli isolates from UTI and newborn meningitis commonly express S, P, and type 1 fimbrial adhesins that specifically recognize a-Sialyl-2,3-galactose [2], Gal(a1-4)bGal [3], and a-D-mannoside [4] receptors, respectively. The genes for the biogenesis of these fimbrial adhesins are organized in large polycistronic clusters termed the sfa, pap, and fim determinants,

* Corresponding author. Tel.: þ46 90 7856731; fax: þ46 90 772630. E-mail address: [email protected] (B.E. Uhlin). 1 Present address: Departament de Microbiologia, Facultat de Biologia, Universitat de Barcelona, Avgda Diagonal 645, 08028 Barcelona, Spain. 0882-4010/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2009.01.007

respectively. Expression of the three fimbriae is tightly regulated by environmental conditions and is subjected to phase variation. In the case of S and P fimbriae, phase variation depends on the Damdependent methylation pattern of two GATC sites located in the promoter region [5,6]. Phase variation of the fim determinant depends on the inversion of a 314 bp chromosomal region containing the fimA promoter and that is flanked by two 9 bp inverted repeats [7]. This inversion process is catalyzed by the FimB and FimE site-specific recombinases [8]. Type 1 fimbriae frequently occur among the Enterobacteriaceae family [9] and it is shown that expression of type 1 fimbriae enhances the virulence of uropathogenic E. coli (UPEC) by mediating adherence and bacterial invasion of bladder epithelial cells [10]. There are evidences that regulatory cross-talk may occur between different fimbrial determinants. Morschha¨user et al. [11] found that expression of the S fimbriae (SfaI) determinant by the uropathogenic strain 536 was stimulated by specific regulators encoded in the P-related fimbriae (Prf) determinant. Moreover, using the uropathogenic strain J96 it was found that expression from the pap determinant correlated with down-regulated

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2. Results

expression of type 1 fimbriae. This was shown to occur by some direct inhibition of recombination in combination with altered expression of both FimB and FimE recombinases and the regulatory cross-talk was mediated by the PapB protein encoded in the pap operon [12,13]. Another important feature for bacterial virulence is the ability to move. The most common system of bacterial movement is mediated by the production of flagella. A large number of genes and operons (grouped in early, middle and late genes) are involved in the biosynthesis of the flagella (reviewed by Chilcott and Hughes [14]). The activating complex encoded from the flhDC operon (early genes) tightly regulates expression of the flagella structural genes (late gene products). The fliC gene encoding for the flagellin subunit that builds up the filamentous structure is one of the last genes expressed in the flagellum synthesis cascade. The expression of the flhDC operon is tightly regulated attending to environmental cues by the action of several global regulators. However, the involvement of specific regulators in the control of flhDC expression should also be considered. The sfaXII gene (accession number AJ492821) belongs to the 17 kDa gene family encoding putative MarR-like regulatory proteins. As the other genes in this family, the sfaXII gene is situated downstream of the adhesin coding gene in a fimbrial operon. As recently revealed by transcriptional analyses the sfaXII gene is also part of the main sfaII fimbrial operon [15]. Two promoters give rise to two transcripts of the gene, one short only covering the sfaXII gene and one long covering the whole main sfaII operon (sfaBII– sfaXII). In this report the role of sfaXII in the expression of NMEC virulence factors has been studied. We found that expression of sfaXII down-regulated expression of type 1 fimbriae by affecting the DNA inversion process. Moreover, it was observed that expression of the sfaXII gene affected bacterial motility. While the present work was under review for publication, it was reported that the highly homologous PapX protein represses motility [16]. Efficient expression of the S fimbriae would require motionless bacteria as it appears contradictory to promote simultaneously attachment and movement. Moreover, the cross-talk between S fimbriae and type 1 fimbriae here described, confirm the observation that bacterial cells preferentially express one unique adhesin fimbrial system at the time [17]. In this report we propose that SfaXII, a protein encoded in an open reading frame located downstream of the structural genes of the fimbrial operon, mediates cross-talk between S fimbriae and type 1 fimbriae and motility.

2.1. Creation of sfaXII mutants in the newborn meningitis isolate IHE3034 To try to elucidate the function of the SfaXII protein, different mutants of the sfaXII gene were constructed in the strain IHE3034. A KmR gene insertion mutant in position þ51 (between codon 17 and 18) and an in-frame deletion mutant lacking 73% of the coding sequence were transferred to the chromosome of the IHE3034 strain by homologous recombination giving the strains AES1 and AES4, respectively (see Materials and methods). As expected, no PCR amplification of the sfaXII gene was obtained in the AES4 derivative (Fig. 1B). It was also shown that the primers used could amplify homologous 17 kDa genes from the prs, pap, and foc fimbrial systems (i.e. prsX, papX, and focX) (Fig. 1B). Therefore, these results corroborated the conclusion that the strain IHE3034 only has one copy of the 17 kDa gene that thereby was eliminated in the DsfaXII derivative. 2.2. Effect of sfaXII mutations on virulence determinants in IHE3034 The potential role of the sfaXII gene in the expression of the S fimbriae and other virulence traits was investigated by comparing wt and mutant derivatives of the strain IHE3034. The observations are summarized in Table 1. Using polyclonal antiserum recognizing the SfaII pilin protein, agglutination tests were performed with the wt and mutant strains (Table 1). No apparent effect of the sfaXII mutations on sfaII fimbriae expression was observed. These results are consistent with observations made by immunofluorescence studies with the IHE3034 and AES1 strains (our unpublished data). The strain IHE3034 has, in addition to S fimbriae, the potential to express other fimbrial adhesins: type 1 and Mat [1,18,19]. In order to assess if the sfaXII mutations might affect the adhesion properties of the bacteria we performed agglutination assays with mammalian blood and yeast cells. As shown in Table 1, both mannose-sensitive hemagglutination (MSHA) and yeast agglutination (MSYA) were clearly increased in the case of the sfaXII mutant strains when compared to the wt. This result suggested that the mutation of the sfaXII gene affected type 1 fimbriae expression. Transcomplementation of strains AES1 and AES4 with the plasmid pAES1 was performed and, by providing the wt sfaXII locus, the MHSA and MSYA were restored to wt levels. Mannose-resistant hemagglutination (MRHA), as result of adhesion due to adhesins others than

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Fig. 1. Schematic overview of the sfaII fimbrial operons and PCR analysis of different 17 kDa genes. A. Schematic drawing showing the genetic organization of the sfaII fimbrial gene cluster. B. Internal sfaXII primers (17Apa1 and 17Apa6) were used for PCR amplification to verify the presence of 17 kDa genes. IHE3034 – wt, sfaXþ II (lane 2); AES4 – sfaXII deletion mutant (lane 3); pAZZ50, sfaII clone (lane 4); pPAP601, prs clone (lane 5); pDHU1, papX clone (lane 6); and pBSN50, foc clone (lane 7). The three Mw standard bands (lane 1) represent 500, 300, and 200 bp, respectively.

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Table 1 Virulence factors investigated in this study.

IHE3034 (wt)

AES1 (sfaXIITkan)

AES4 (DsfaXII)

AES1/pAES1 (sfaXþ II )

AES4/pAES1 (sfaXþ II )

þ () () þ þ þ

þ þ þ þ þ þ

þ þ þ þ þ þ

NDg () () ND ND ND

ND () () ND ND ND

a

Detection of SfaII fimbriae by agglutination test using polyclonal SfaII antiserum. Mannose-sensitive hemagglutination using human blood (þ agglutination, () weak or no agglutination). c Mannose-sensitive yeast agglutination (þ agglutination, () weak or no agglutination). d Detection of the K1 antigen by agglutination test with beads coated with antibodies against K1 antigen. e Detection of the O18 antigen by agglutination test using polyclonal anti O18 serum. f Bacterial resistance to 50% vol/vol human serum (þ, no significant reduction of cfu/ml after 3 h serum exposure). g ND, not determined. b

80 AES14/pBB2-1/pBR322 AES14/pBB2-1/pAES1

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0,01 8

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2.3. Effect of SfaXII on fimA expression in IHE3034 derivatives

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AES1/pAES1

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AES1/pBR322

10 IHE3034/pAES1

Percent ON- cells

The result in the preceding section suggested that expression of type 1 fimbriae might by affected by the SfaXII protein. To confirm this finding and determine at which level such regulation might occur, transcriptional studies were performed. The single copy plasmid pBB2-1, carrying a fimATlacZYA fusion in pPR274, was used to monitor transcriptional fim expression during growth in LB at 37  C [20,21]. We used a lac derivative of the IHE3034 sfaXII mutant strain, AES14, carrying the plasmid pBB2-1 and either the þ vector control pBR322 (sfaX II ) or pAES1 (sfaXII ). As shown in Fig. 2A, the strains had the same growth profiles irrespective of expressing the sfaXII gene or not. However noticeable differences were observed when the level of expression of type 1 fimbriae was monitored. The fimA expression was lower in all growth phases when the sfaXII gene was present. The linear increase of fimA expression continues upon entry into stationary phase in the absence of the SfaXII protein. However, in the presence of the SfaXII there was a clear decrease in the slope of the transcriptional profile at the beginning of stationary phase evidences an SfaXII-mediated repression of fimA transcriptional expression. No b-galactosidase activity was detected when samples of the strain AES14 with the vector control pPR274 were measured (data not shown). Similar tests were performed with the lac derivatives of the IHE3034  (sfaXþ II ) and AES1 (sfaXII ) strains, AES6 and AES14 respectively, carrying only the fimATlacZYA fusion plasmid pBB2-1. These strains showed the same growth profile but, despite the lack of sfaXII in AES14, we could not detect any significant difference in the b-galactosidase activity during any growth phase when comparing with the sfaXII wt strain (data not shown). Transcriptional expression of type 1 fimbriae is subject to phase variation by recombination of the invertible DNA fragment containing the main promoter of the fim determinant (fimA promoter)

0 4

2

IHE3034/pBR322

type 1 fimbriae, was tested using blood from different sources. A weak MRHA was observed only when bovine blood was used. However, no significant differences were obtained in the agglutination tests comparing the MRHA of the wt and the sfaXII mutant derivatives (data not shown). Phenotypical determination of K1 capsule production, O18 antigen expression, and resistance to serum was performed and no significant differences between the strains were found (Table 1).

-galactosidase activity (______) (Miller Units)

SfaII fimbriaea MSHAb MSYAc K1 antigend O18 agglutinatione Serum resistancef

10

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OD600 (……)

Assay

245

Fig. 2. Effect of sfaXII on expression of type 1 fimbriae in derivatives of IHE3034. A. fim expression was monitored by measuring the b-galactosidase activity from the fimATlacZYA fusion plasmid pBB2-1 in the strains AES14/pBB2-1/pBR322 (sfaX II / p(fimATlacZYA)/vector; filled squares) and AES14/pBB2-1/pAES1 (sfaX II /p(fimATlacZYA)/p(sfaXII); empty squares). The cultures were grown in LB at 37  C and the b-galactosidase activity (solid line) and OD600 (dotted line) were plotted. Data represent an average of two independent experiments and vertical bars represent the standard deviation (SD). B. The percentage of ON cells was determined by PCR analysis of samples from overnight, statically grown, cultures. Upper panel: The PCR-amplified fim switch region digested with HinfI of the different strains. Lower panel: Quantification of the intensities of the upper panel bands. IHE3034/pBR322 (wt/vector; black  bar) IHE3034/pAES1 (wt/p(sfaXþ II ); grey bar), AES1/pBR322 (sfaXII /vector; black-white þ bar), and AES1/pAES1 (sfaX II /p(sfaXII ); grey-white bar). The diagram represents results from two independent experiments.

[7]. Therefore, expression is dependent both on the percentage of cells in the population that are expressing the fim determinant and the level of expression from those cells. To investigate if the sfaXII gene was affecting the fim transcriptional expression in IHE3034 by

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altering the phase variation control, the percentage of ON cells (expressing the fim operon) was determined using a PCR-based approach (see Materials and methods). Results exemplifying the effect of SfaXII on the percentage of ON cells obtained with derivatives from a highly fimbriated IHE3034 stock are shown in Fig. 2B.

indicator plates (see Materials and methods). Corroborating the results obtained with the strain IHE3034, the presence of sfaXII drastically decreased the percentage of fimA-expressing cells in K12 derivative strains (Fig. 3C).

2.4. Effect of SfaXII on the type 1 fimbriae phase variation

2.5. Effect of SfaXII on the expression of the FimB and FimE recombinases

To further study the effect of the sfaXII gene on type 1 fimbriae expression a strain derived from E. coli K-12, strain AAEC198A, carrying a chromosomal fimATlacZYA fusion was used. Since this strain is lacking 17 kDa family genes the effect of SfaXII on the fim expression was tested by introducing plasmid pBR322 and its derivative pAES1 containing the sfaXII gene. Transcriptional expression was monitored by determination of the b-galactosidase activity from bacterial cultures in same conditions as earlier described. Similarly to what was found with strain IHE3034, the presence of pAES1 significantly down regulated the transcriptional expression of fimA (Fig. 3A) and a two-fold decrease in the fimA transcriptional expression was detected when the sfaXII gene was expressed. Studies were then performed to discriminate between the possible effects of the SfaXII on either the phase variation or the activity of the main promoter of the fim determinant. The fimApromoter activity was tested by using strain AAEC374A. This derivative of AAEC198A has the invertible DNA fragment in ‘‘locked-ON’’ orientation and is deficient in phase variation control because it carries mutations in the fimB fimE genes. Therefore, alterations in the b-galactosidase activity monitored from this strain would reflect primarily alterations in the transcriptional activity from the fimA promoter. The presence of the sfaXII gene (plasmid pAES1) resulted in a 15–20% decrease in transcription from the fimA promoter when compared to the vector control strain (Fig. 3B). While seemingly significant, the observed difference in the level of fimA expression in the presence of the sfaXII gene in strain AAEC374A could not fully explain the down-regulation observed with the strain AAEC198A. Therefore, the possible effect on the switching process that controls the fim determinant phase variation was considered. To test this, the percentage of fimAexpressing cells in the population of the AAEC198A strain carrying either the plasmid pAES1 or pBR322 was determined using

The sfaXII-mediated reduction in the percentage of fimAexpressing cells could be the consequence of either a stimulation of the ON-to-OFF switch or a repression of the OFF-to-ON switch. In E. coli, the inversion process responsible for the phase variation of type 1 fimbriae expression is catalyzed by two site-specific recombinases, FimE and FimB, that mainly catalyze the switch from ON-to-OFF and OFF-to-ON, respectively [8,22]. To elucidate whether the sfaXII-mediated effect on the fim switch was due to induced alterations of expression of these enzymes, expression of the genes encoding for the two recombinases was monitored. The strains AAEC200 and BGEC088 have lacZYA fusions with fimE at transcriptional and translational levels, respectively, and strains AAEC261A and BGEC056 have lacZYA fusions with fimB at transcriptional and translational levels, respectively. All these strains were transformed with either the pAES1 plasmid or the vector control pBR322. As seen in Fig. 4, the presence of pAES1 did not significantly change the expression of fimE, as monitored by using both the transcriptional (AAEC200, fimETlacZYA) and translational (BGEC088, FimE-LacZ) fusion strains. However, a 30% reduction in the expression of fimB was detected with both the transcriptional (AAEC261A, fimBTlacZYA) and translational (BGEC056, FimB-LacZ) fusion strains expressing sfaXII. The described effect of SfaXII on FimB would suggest that the decreased expression of this recombinase would cause a reduction in OFF-to-ON switching and consequently the observed reduction in percentage of ON cells. To verify this hypothesis OFF-to-ON switching was monitored upon controlled induction of the sfaXII gene expression. The sfaXII gene was cloned under the control of the ParaBAD arabinose inducible promoter resulting in plasmid pASS5. Using a non-fimA expressing colony (OFF colony) as inoculum, fim expression was followed upon induction of the sfaXII gene by addition of 0.0035% arabinose (this arabinose concentration gave a similar level of reduction of fimA expression as when we used the

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(locked on) Fig. 3. Effect of the sfaXII gene on the expression of type 1 fimbriae in E. coli K-12. The cultures were grown in LB at 37  C to mid-log phase. A. fimA expression was monitored by measuring the b-galactosidase activity of strain AAEC198A carrying either pBR322 (black bars) or pAES1 (sfaXþ II ; grey bars). B. fimA expression was monitored by measuring the bgalactosidase activity of strain AAEC374A carrying either pBR322 (black bars) or pAES1 (sfaXþ II ; grey bars). C. The percentage of fimA-expressing cells was calculated in cultures from AAEC198A/pBR322 and AAEC198A/pAES1. The data represent the average of three independent experiments and vertical bars represent SD. An asterisk indicates a significant difference according to Student’s t-test.

1.5

P=0.10

P=0.0003**

pBR322 pAES1(SfaXII)

247

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AAEC198A/pASS5 non-induced sfaXII

AAEC198A/pASS5 induced sfaXII

P=0.0008**

AAEC200 fimE::lacZ

BGEC088 FimE-LacZ

AAEC261A fimB::lacZ

BGEC056 FimB-LacZ

Fig. 4. Effect of the sfaXII gene on the expression of the FimB and FimE recombinases. Strains AAEC200 (fimETlacZ), BGEC088 (FimE-LacZ), AAEC261A (fimBTlacZ), and BGEC056 (FimB-LacZ) carrying either the pBR322 vector (black bars) or the pAES1  plasmid (sfaXþ II ; grey bars) were grown in LB at 37 C and samples were taken at midlog phase for determination of the b-galactosidase activity. The data represent the average of three independent experiments and vertical bars represent SD. An asterisk indicates a significant difference according to Student’s t-test.

2 1.5 1 0.5 0.4 generations 0

Our preliminary tests of possible effects of sfaXII on motility of the bacteria on low agar plates suggested some difference between the clinical isolate IHE3034 and the sfaXII mutant (AES1 and AES4) strains upon prolonged incubation (Fig. 6A, and data not shown). Furthermore, when the wt sfaXII gene was introduced in trans on the medium copy vector construct pAES1 both the mutant and the wt strains showed reduced motility (Fig. 6B). No differences were observed in the growth rate of the different strains when grown in liquid medium that could explain the decreased spreading on plates (data not shown). These results suggested that the expression of SfaXII might affect the function or expression of the flagella.

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OFF-ON switching frequency (per cell per generation)

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0.04 0.02 0

2.6. Effect of SfaXII on flagellum production

0.01

0

ON cells

plasmid pAES1 with sfaXII under control of its proximal native promoter; data not shown). An increase in the expression of fimA was found in the non-induced culture, whereas reduced expression was detected when sfaXII was expressed (Fig. 5A). Furthermore, when the percentage of ON cells was quantified a clear increase in fimA-expressing cells was found in the case of the non-induced cultures in comparison with the sfaXII induced cultures (Fig. 5B, left panel). The percentages of fimA-expressing cells at two time points after sfaXII induction were plotted in Fig. 5B (right panel). In the non-sfaXII induced culture the population of ON cells increased up to 40% between the 0.4 and 1.5 generation time points. However, the sfaXII induced culture had a proportion of ON cells after 1.5 generations not significantly different (P ¼ 0.91) from that at the 0.4 generations time point. These results suggested that sfaXII expressing cells had reduced probability of switching from an OFF- to ON-direction and consequently displayed a decrease in the percentage of fimA-expressing cells. Therefore, the frequency of the OFF-to-ON switching after induction of sfaXII expression in AAEC198A/pASS5 was calculated, and the result highlighted a significant decrease after 1.5 generations (Fig. 5C). This corroborates the above presented results on the percentage of ON cells (Fig. 5B) and the fimA expression (Fig. 5A). Altogether, our data confirmed that expression of SfaXII could affect type 1 fimbriae expression by decreasing the FimB-mediated ON switch. In summary, it can be concluded that expression of the sfaXII gene has a repressing effect on type 1 fimbriae expression in both E. coli K-12 strains and the E. coli clinical IHE3034 derivatives. Our results indicate that the expression of sfaXII influenced the expression of type 1 fimbriae by affecting both the fimA-promoter activity and, to a greater extent, the FimB-mediated OFF-to-ON switching.

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¨ m et al. / Microbial Pathogenesis 46 (2009) 243–252 A.E. Sjo¨stro

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Generations Fig. 5. Effect of induced sfaXII expression on the OFF-to-ON switch. The sfaXII gene was placed under the control of the arabinose inducible ParaBAD promoter in the plasmid pASS5. Non-induced cultures are denoted in black and the induced in grey. A. An OFFculture of AAEC198A/pASS5 grown overnight at 42  C was used to inoculate fresh minimal MOPS medium supplemented with glycerol either in presence or absence of arabinose (0.0035 %). The cultures were incubated at 37  C and b-galactosidase activities (squares) and OD600 (triangles) plotted. B. At the same time points as in A, the percentage of fimA-expressing cells was calculated (left panel). In the right panel the values of fimA-expressing cells after 0.4 and 1.5 generation times growing either in presence or absence of SfaXII are shown. C. The calculated frequency to switch from OFF orientation to ON orientation. All data represent the average values of three independent experiments and vertical bars represent SD. An asterisk indicates a significant difference according to Student’s t-test.

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Growth zone diameter (mm)

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in the whole cell extracts (Fig. 6C, lanes 1 & 3). However, the immunoblot analyses in case of SfaXII over expression supported the results from the motility assay by showing a more than 15-fold reduction in level of flagella subunits in the pAES1 carrying strains (Fig. 6C). These results were further verified by atomic force microscopy (AFM) analysis of the AES1/pBR322 and AES1/pAES1 cells. No flagella were observed in samples of bacterial cells carrying the pAES1 plasmid whereas we could detect flagella in the samples of the vector control strain (data not shown).

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FliC

Fig. 6. Effect of sfaXII expression on flagella expression. The effect on motility of strains growing on LB medium containing 0.3 % agar at 37  C. A. The diameter of spreading of the strains IHE3034 (wt; black square), AES1 (sfaX II ; light grey square), and AES4 (sfaX II ; grey square) was measured at different time points after inoculation. B. Insert: þ  Cells of the strains AES1/pAES1 (sfaX II /p(sfaXII )) and AES1/pBR322 (sfaXII /vector) after 8.5 h growth. Diagram: The diameter of spreading of the strains IHE3034/pBR322 (wt/vector; black square), IHE3034/pAES1 (wt/p(sfaXþ II ); black triangle), AES1/pBR322  þ (sfaX II /vector; grey square), and AES1/pAES1 (sfaXII /p(sfaXII ); grey triangle) was measured at different time points after inoculation. C. The effect on flagella synthesis. Strains 1: IHE3034/pBR322 (wt/vector), 2: IHE3034/pAES1 (wt/p(sfaXþ II )), 3: AES1/  þ  pBR322 (sfaX II /vector), and 4: AES1/pAES1 (sfaXII /p(sfaXII )) were grown in LB at 37 C to early stationary phase and samples were prepared to examine the levels of FliC protein by Western blot analysis.

Further studies of the effect of the SfaXII protein on flagella expression of strains IHE3034/pBR322, IHE3034/pAES1, AES1/ pBR322, and AES1/pAES1 grown in liquid medium were performed. Samples were taken at early stationary phase, when flagella are found to be most abundant, and expression of the FliC flagella subunit protein was examined by Western blotting using H7 flagella antiserum. When comparing the SfaXII defective strain with wildtype we did not detect any significant difference in FliC amount

In this study we showed that the sfaXII gene, present in strains with the ability to express S fimbriae, negatively affects the expression of type 1 fimbriae and flagella production. The sfaXII gene was recently shown to be part of the major sfaII fimbrial operon [15]. Here we demonstrate that sfaXII is a new component also in the regulatory cross-talk between fimbrial genetic determinants. The sfaXII gene codes for a 17 kDa protein with partial homology to the MarR family of regulatory proteins found in many human bacterial pathogens (reviewed by Alekshun and Levy [23]). There are increasing evidences that such proteins play a role in regulation of motility. The mrp gene cluster in Proteus mirabilis, encoding for the Proteus-like MR/P fimbriae, includes the mrpJ gene coding for a protein that SfaXII shows amino acid sequence similarity to [24]. Similarly to what we have observed in the case of sfaXII, elevated expression of MrpJ from a plasmid clone caused repression of flagella production and impaired motility in P. mirabilis. Moreover, Li et al. [24] showed that ectopic over expression of the protein denoted PapX (to which SfaXII has 96% amino acid identity) from the E. coli strain CFT073 had a repressing effect on motility in P. mirabilis. Recent investigations with the CFT073 strain revealed that motility of the E. coli strain was affected upon papX expression [16]. Furthermore, the results indicated that the PapX protein caused inhibition of CFT073 motility by repressing transcription of motility-associated genes, including the genes for the FlhDC master regulator. Considering the high homology between the SfaXII and PapX proteins, a common mode of action would seem likely in their effect on the flagella and motility in the IHE3034 and CFT073 strains. Similarly, the HosA protein from Enteropathogenic E. coli (EPEC) has been shown to cause impaired motility when over expressed [25]. In contrast to the SfaXII mutant, the HosA mutant showed decreased motility on low agar plates. Such seemingly contradictory results could have different alternative explanations: i) the percentage of flagella expressing cells is reduced but the flagellated ones are more motile; ii) flagellated cells express shorter, but more effective flagella; or iii) the number of flagella/cell is reduced but the remaining flagella are hyper motile. Nevertheless, the decreased motility seen when the sfaXII/hosA/papX genes were over expressed, and the reduction in flagella subunit expression, suggests that these regulatory genes are causing down-regulation of the flagella synthesis. Dissection of the initially observed increase in mannose-sensitive agglutination detected in SfaXII-deficient IHE3034 derivatives made us conclude that the pathogen specific regulator SfaXII that apparently can be co-expressed with the S fimbriae [15] caused a relevant decrease in type 1 fimbriae transcriptional expression. This was evidently occurring by a decrease both in the percentage of type 1 fimbriated cells and in the number of fimbriae present in such cells. Notably, such regulation is observed both in the pathogenic IHE3034 and in K-12 derivatives, indicating that the involvement of SfaXII in the control of type 1 fimbriae transcription does not strictly require any other factor specific from pathogenic strains.

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Our data shows that SfaXII affect the expression of the FimB site-specific recombinase and consequently the switching from the OFF-to-ON orientation. Further studies will be required to determine whether the role of SfaXII on transcriptional activity from the fimA promoter is direct or indirect. We should have in mind that the fimA promoter and inverted repeats of the fim invertible element are very close together, and transcription from the fimA promoter and recombination are probably mutually exclusive [26]. Factors that inhibit fimA transcription might therefore also inhibit FimB recombination. It should also be mentioned that genes (e.g. ipuA, ipuB and ipbA) encoding additional recombinases potentially involved in fimA recombination have been discovered in some pathogenic E. coli but lacking in the E. coli K-12 strains [27]. While we do not know if they are present and play some role also in the strain IHE3034, our findings with SfaXII in E. coli K-12 derivatives suggest that fimB is the main target in this case. Interestingly, preliminary immunofluorescence studies revealed that also the degree of S-fimbriae expression was different and it was inversely related to the type 1 fimbriae expression (our unpublished data). In short, the subculture with higher percentage of type 1 fimbriated cells had lower percentage of Sfimbriated cells and vice versa. These findings are reminiscent to the observation reported by Snyder et al. [28] that type 1 fimbriation and Pap fimbriation in case of a UPEC isolate appeared inversely coordinated. The sfaXII gene itself is evidently subject to strong repression by the nucleoid protein H–NS and its low level expression is growth phase dependent with higher levels observed at the transition from late logarithmic phase to stationary phase [15]. At present it is not known what condition or situation may cause relief from the H–NS repression and result in full expression from the sfaXII gene. However, as was evident from our present studies, when it was expressed by sfaXII plasmid clones in each cell of a bacterial population, the SfaXII protein can cause distinct phenotypic changes of the bacteria leading to altered host interaction at the single cell level. It is also feasible that such regulatory effects are transient and may occur in a subpopulation of the bacteria in their natural niches. A schematic representation of how the H–NS controlled SfaXII levels may affect expression of type 1 fimbriae and motility/flagella and a summary of the SfaXII repressing effects on type 1 fimbriae are shown in Fig. 7.

A

H-NS

249

In summary, our findings demonstrate that the MarR-like proteins, here represented by the sfaXII gene product, affects not only flagella production and motility, but also expression of at least one other E. coli fimbrial determinant, the commonly occurring type 1 gene cluster. Our findings with the sfaXII mutants thereby reveal a novel role of these regulatory proteins in the control of phase switching of the mannose-binding type 1 fimbriae. Furthermore, the recent demonstration that the sfaXII gene is part of the main S fimbriae operon implies that the SfaXII protein is mediating a regulatory coupling between the S and type 1 fimbrial genes. Our data, together with previously reported findings [25,24,28] suggest that genes coding for the 17 kDa-like proteins were acquired by the cells presumably by horizontal transfer since they are located within genomic islands/pathogenicity islands downstream of pathogenicity-related fimbrial systems. We suggest that the role of the sfaXII gene, and presumably it can be a common role of the 17 kDa genes, may be to act as a coordinating component in the regulation of virulence factors contributing to the pathogenicity of UPEC and NMEC strains. 4. Materials and methods 4.1. Bacterial strains, plasmids, and growth conditions The E. coli strains and plasmids used in the present work are described in Table 2. Unless otherwise stated, the strains were grown at 37  C in either Luria–Bertani (LB) broth with vigorous shaking or on tryptone yeast (TYS) agar. When necessary, antibiotics were added at following concentrations: carbenicillin – 50 mg ml1, kanamycin – 50 mg ml1 and chloramphenicol – 12 mg ml1. 4.2. Cloning and recombinant DNA techniques Molecular genetic manipulations were performed essentially as described by Sambrook et al. [29]. The sfaXII gene and 666 bp upstream of the gene were PCR-amplified from plasmid pAZZ50 using the primers SfaII-1 (50 -GCGGATCCGCTGAGTGTCAATATTTCC-30 ) and SfaII-3 (50 -CGGTCGACGGATCAGCATCACTAGG-30 ) that contain additional BamHI and SalI restriction sites (indicated in bold letters), respectively. The PCR product was cloned into the vector pUC18 resulting in the plasmid pASS01. The sfaXII gene from pAZZ50 was also PCR-amplified using primers SfaII-2 (5-CCGAATTCAGTAATATGGAG-30 ) and SfaII-3 (50 -CGGTCGACGGATCAGCATCACTAGG-30 ) that contain additional EcoRI and SalI restriction sites (indicated in bold letters), respectively and cloned under the control of the inducible ParaBAD promoter into pBAD30, resulting in plasmid pASS5.

SfaXII

4.3. Construction of chromosomal sfaXII gene mutants Type 1 fimbriae

Motility/flagella

B fimB

fimE

fimA ON

SfaXII Fig. 7. A. Schematic representation of how the H-NS controlled SfaXII levels may affect expression of type 1 fimbriae and motility/flagella. B. Summary of the SfaXII repressing effects on type 1 fimbriae.

The sfaXII mutants were obtained using plasmid pASS01 and the QuikChangeÔ Site-Directed Mutagenesis kit (Stratagene) following the manufacturer’s instructions. Plasmid pASS02 was constructed by introducing an ApaI restriction site 17 codons downstream from the start codon of the sfaXII gene using the primers 17Apa1 (50 -CAGACACCAAAAAAACAAGGGCCCGGTATATCCTTTCGGG-30 ) and 17Apa2 (50 -CCCGAAAGGATATACCGGGCCCTTGTTTTTTTGGTGTCTG30 ) (ApaI sites indicated in bold letters). Using primers 17Apa5 (50 -CTGAAAGAAATCTGGGGCCCTCTGACCCATGATGAACAGG-30 ) and 17Apa6 (50 -CCTGTTCATCATGGGTCAGAGGGCCCCAGATTTCTTTCAG30 ) (ApaI sites indicated in bold letters) and the plasmid pASS02 an additional ApaI restriction site was introduced 138 codons downstream from the start codon of the sfaXII gene. The resulting plasmid was subsequently digested with ApaI and re-ligated to generate

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250 Table 2 E. coli strains and plasmids used in this study. Strain/plasmid

Description/relevant characteristics

Reference/source [35] [35]

AAEC200 AAEC261A BGEC056 BGEC088 IHE3034 AES1 AES4 AES6 AES14

MG1655 DlacZYA fimA-lacZYA MG1655 DlacZYA fimA-lacZYA fimB-am6 fimE-am18 (switch locked on) MG1655 DlacZYA fimE-lacZYA MG1655 DlacZYA fimB-lacZYA MG1655 fimB-lacZYA FimB-LacZ MG1655 Dlac fimE-lacYA FimE-LacZ NMEC clinical isolate, O18K1:H7 IHE3034 sfaXIITkan, KmR IHE3034 DsfaXII IHE3034 DlacZ AES1 DlacZ, KmR

Plasmid pBR322 pUC18 pK03 pUC4KAPA pBAD30 pAZZ50 pPAP601 pDHUl

Cloning vector, CbR, TcR Cloning vector, CbR, lacZa, ori ColE1 Suicide vector, ori M13, sacB, repATS, CmR CbR, KmR , ori ColEl CbR, ori ColE1, inducible ParaBAD promoter pBR322, CbR, IHE3034 sfaII determinant pACYC184, CmR, J96 prs determinant pACYC184, papX from J96 pap determinant

pBSN50 pASSOl pASS02 pASSl pASS4 pASS5 pAESl pPR274 pBB2-l

pJB8, cosmid clone from J96, foc determinant pUC18, sfaXII gene from sfaII operon pASS01, sfaXIITApaI site pUC18, sfaXIITkan pUC18, DsfaXn pBAD30, sfaXII gene from sfaII operon pBR322, sfaXII gene from sfaII operon Mini-F replicon, CmR pPR274, fimA: .lacZYA

Strain AAEC198A AAEC374A

[35] [35] I.C. Blomfield I.C. Blomfield [1] This study This study [15] This study [36] [37] [30] Pharmacia Biotec [38] [39] [40] This laboratory/ unpublished data [41] This study This study This study This study This study [15] [42] [21]

pASS4 that contains a 365 bp in-frame deletion allele of the sfaXII gene. To obtain an sfaXII gene insertion mutant, the kanamycin resistance (KmR) cassette from pUCKAPA plasmid was used. To facilitate subsequent genetic manipulations, the SalI sites present in the KmR cassette was removed by digestion of pUCKAPA with SalI, Klenow filling at the sticky ends and subsequent ligation. The modified KmR cassette was then cloned into the ApaI site of plasmid pASS02, resulting in plasmid pASS1. The BamHI–SalI fragments from both pASS1 and pASS4 plasmids were cloned into the suicide vector pKO3. Both mutant alleles of the sfaXII gene were transferred to the chromosome of the NMEC clinical isolate IHE3034 by homologous recombination [30], resulting in the strains AES1 (sfaXIITkan) and AES4 (DsfaXII). An sfaXII, lacZ double mutant was obtained using the strain AES1, crossover PCR technique and homologous recombination as previously described [30]. The strain was denoted AES14. 4.4. SfaII fimbriae agglutination assay Bacterial cultures grown statically overnight in LB medium at 37  C were adjusted to an optical density at 600 nm (OD600) of 1.0 and mixed 1:1 (vol/vol) with polyclonal SfaII antiserum on a glass slide.

4-day intervals with 0.2, 0.5, 1.0, and twice 2.0 ml of boiled cells adjusted to 109 cells ml1 in phosphate-buffered saline. Four days after the last inoculation, rabbits were sacrificed; sera were collected and stored in aliquots at 20  C until use. For evaluation of the presence of O18 antigen, boiled cells of E. coli IHE3034 strain and its derivatives were adjusted to a density of 109 cells ml1 in saline and added (0.05 ml) to serial dilutions of the immune serum prepared in saline (0.5 ml). The reaction was measured after overnight incubation at 37  C. The highest dilution of the antiserum eliciting a clearcut aggregation of bacterial cells was taken as the endpoint of the reaction. Saline without antiserum served as control. 4.7. Serum bactericidal test The serum resistance assay was essentially performed as previously described by Nagy et al. [31]. Briefly, bacteria were grown overnight in LB medium, washed in saline, and diluted to 106 CFU ml1. The bacterial suspension was mixed with an equal volume of human serum and incubated at 37  C in microtiter plates. Samples were taken at 0, 1, 2, and 3 h and viable cell counts determined. The assays were performed both with normal and heat-inactivated (56  C for 30 min) serum. 4.8. Agglutination assays Bacteria grown in either liquid or on solid media were harvested, washed, and the cell suspension adjusted to an OD600 of 1.0 in phosphate-buffered saline (PBS). Agglutination of erythrocytes was performed by mixing 1:1 (vol/vol) the bacterial suspensions with 8% blood suspension in PBS. Yeast agglutination to detect adhesion due to type 1 fimbriae expression was performed with a suspension of Saccharomyces cerevisiae in PBS adjusted to an OD600 of 1. Mannose-sensitivity was tested by adding 3% a-methylD-mannoside (wt/vol) to both blood and yeast suspensions. 4.9. PCR based assays for phase switch orientation analysis Bacteria from plate were inoculated in 3 ml LB to an OD600 of 0.005 and incubated statically at 37  C ON. The ON culture was used to inoculate 3 ml fresh medium to an OD600 of 0.005 and incubated statically at 37  C ON. This was repeated once, in all three ON incubations. The analysis was performed as earlier described [12,32]. In short, PCR was run using Fim switch region specific primers and ON cultures diluted to an OD of 0.17. The resulting fragments were digested with HinfI followed by separation on 6.5% TBE-acrylamide gel. Detection and quantification of bands were performed using the ChemiDoc XRS system with the QuantityOne analysis software, BioRad. 4.10. Determination of the percentage of fimA-expressing cells (ON cells) on indicator plates

The presence of K1 antigen was determined by the Wellcogen Neisseria meningitidis B/E. coli K1 rapid latex test (Murex Biotech Limited) according to the manufacturer’s instructions.

The bacterial cultures were grown to mid-log phase, serially diluted in saline, and plated onto 42  C pre-warmed minimalMOPS plates supplemented with 10 mM thiamine, 0.4% (wt/vol) glucose and 40 mg X-gal ml1. The plates were incubated at 42  C where the fimA-expressing cells gave rise to blue colonies and nonfimA-expressing cells gave rise to white colonies. Data is presented as the percentage of blue colonies in relation to the total number of colonies.

4.6. O18 agglutination assay

4.11. FimB-promoted OFF-to-ON switch

For preparation of O18 specific antiserum the E. coli O18, 764/2 strain (Wu¨rzburg, Germany) was used. Rabbits were immunized at

To quantify the fimB-promoted OFF-to-ON switch a non-fimA expressing colony was selected from a minimal-MOPS plate

4.5. K1 antigen assay

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(white colony, see above) and inoculated in minimal MOPS medium supplemented with 10 mM thiamine and 0.4% glycerol and aerobically grown overnight on a shaker at 42  C to minimize the phase fim-switching [33]. This culture was used to inoculate fresh minimal MOPS medium to an OD600 of 0.012 either in the absence or presence of 0.0035% arabinose and incubated at 37  C with vigorous shaking. Hourly samples were serially diluted in saline and spread onto 42  C pre-warmed minimal-MOPS plates and incubated at 42  C for 24 h. Colonies were counted and the percentage of fimA expressing colonies (blue colonies) calculated. The probability of switching OFF-to-ON was calculated by the formula [33]:

pðswitchÞ ¼ 1 

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi x n 1 ; 100

where n ¼ number of generations, x ¼ percentage of ON cells. 4.12. Western blotting Bacteria were pelleted by centrifugation and resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Identical amounts were subsequently subjected to SDS-PAGE (15% polyacrylamide concentration) and thereafter transferred to a polyvinylidene fluoride (PVDF) microporus membrane. To detect expression of flagella, polyclonal rabbit antiserum directed against purified flagella of serotype H7 was used as primary antibody [34,18]. Further visualization was carried out using the ECLþ method described by the manufacturer (Amersham Biosciences). Detection of bands was performed using the ChemiDoc XRS system, BioRad. 4.13. Motility assay Overnight cultures were diluted in LB and adjusted to an OD60 of 1.0. A drop (4 ml) was put on solid LB medium containing 0.3% agar and incubated at 37  C. Growth was monitored by size measurements of the diameter of the spreading bacteria. 4.14. Statistics Statistical analyses are made using the t-test provided by the Microsoft Office Excel 2003 program. Acknowledgements We gratefully acknowledge I.C. Blomfield for the many fim-lac fusion strains and thank B. Schwan (La Crosse University, Wisconsin) for providing us with the plasmids pPR274 and pBB2-1. This work was carried out in the frame of the European Virtual Institute for Functional Genomics of Bacterial Pathogens (CEE LSHB-CT-2005-512061) and the ERA-NET project ‘‘Deciphering the intersection of commensal and extraintestinal pathogenic E. coli’’ and was supported by grants from the Swedish Research Council, the European Graduate College (‘‘Gene Regulation in and by Microbial Pathogens’’ of the German Research Council (DFG)), the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Hungarian Research Foundations (OTKA 62092, ETT 333/2006), the Faculty of Medicine, Umeå University, and it was performed within the Umeå Centre for Microbial Research (UCMR). C. Balsalobre was recipient of a travel grant from the Federation of European Microbiological Societies (FEMS).

251

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