Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, USA1
University of Maryland School of Medicine2 and Research Service3, Veterans Administration Medical Center, Baltimore, MD 21201, USA
Author for correspondence: Robert Belas. Tel: +1 410 234 8876. Fax: +1 410 234 8896. e-mail: belas{at}umbi.umd.edu
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ABSTRACT |
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Keywords: quorum sensing, LuxS, swarming, virulence, urinary tract infection
Abbreviations: AHL, N-acylhomoserine lactone; AI, autoinducer; UTI, urinary tract infection
The GenBank accession number for the sequence reported in this paper is AY044337.
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INTRODUCTION |
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Stickler and his collaborators (McLean et al., 1997 ; Stickler & Hughes, 1999
; Stickler et al., 1998
) have demonstrated the importance of P. mirabilis biofilms in the formation of struvite crystals during UTI. When grown on nutrient agar, P. mirabilis also forms a similar biofilm (or bacterial colony) with a unique pattern of development. These colonies frequently develop a concentric bulls-eye pattern (Fig. 1
), underscoring the cyclic nature of this motile behaviour. As shown in Fig. 1
, each cycle of biofilm development may be divided into four parts: (i) swarmer cell differentiation, (ii) the lag period prior to active movement, (iii) swarming colony migration, and (iv) consolidation (where the cells stop moving and de-differentiate to swimmer cell morphology). The bulls-eye pattern is itself mirrored in the activity of a set of proteins that are also expressed coordinately with the cycles of swarming. Included in this group of swarmer-cell-dependent proteins are a set of virulence factors, including flagellin, the ZapA protease, urease and haemolysin (Allison et al., 1992
). It has been postulated, based in part on the evidence of co-ordinate expression of virulence factors during cellular differentiation, that the swarmer cell and swarming behaviour may be involved in UTI and pathogenesis (Allison et al., 1992
, 1993
; Chippendale et al., 1994
).
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Quorum sensing is a common mechanism that acts to control bacterial physiology by regulating gene expression in response to population density. Quorum sensing relies on the accumulation of small extracellular signalling molecules, referred to as autoinducers, to modulate the transcription of target genes and operons. Autoinducer 1 (AI-1) quorum systems, using N-acylhomoserine lactone (AHL) derivatives as the signalling molecules, have been uncovered in many Gram-negative bacteria (Fuqua et al., 1996 ; Swift et al., 1996
). The genes under the influence of AHL quorum sensing regulation include those encoding virulence factors (Parkins et al., 2001
; Passador et al., 1993
; Pirhonen et al., 1993
; Tang et al., 1996
; Telford et al., 1998
), as well as those responsible for biofilm formation (Davies et al., 1998
; De Kievit et al., 2001
; Eberl et al., 1996
). However, despite extensive analysis, AHL AIs do not appear to play a major role in P. mirabilis swarming colony biofilm formation (Belas et al., 1998
).
Quorum sensing can be achieved through molecules other than AHL. Bassler and co-workers (Bassler, 1999 ; Surette & Bassler, 1998
; Surette et al., 1999
) have described an alternative quorum sensing mechanism that uses an autoinducer molecule, referred to as autoinducer 2 or AI-2, that appears to be highly conserved in both Gram-negative and Gram-positive bacteria (Schauder & Bassler, 2001
). In V. harveyi, the membrane protein LuxQ, which is a homologue of P. mirabilis RsbA, acts as the receptor of AI-2. The gene luxS is crucial for AI-2 activity, and luxS orthologues have been found in many bacteria (Bassler, 1999
; Surette et al., 1999
). The molecular identity of AI-2 has been recently reported as a derivative of furanone (Schauder et al., 2001
).
With the exception of its role in the regulation of V. harveyi luminescence, the function of AI-2 remains enigmatic for most of the bacterial species found to possess luxS orthologues. For example, many pathogenic bacteria possess genes with homology to luxS, leading to speculation that AI-2 may function to regulate aspects of bacterial virulence and pathogenicity. Recent reports have tested this hypothesis using enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) (Sperandio et al., 1999 ), Helicobacter pylori (Forsyth & Cover, 2000
; Joyce et al., 2000
), Shigella flexneri (Day & Maurelli, 2001
) and Porphyromonas gingivalis (Chung et al., 2001
; Frias et al., 2001
). The strongest connection between AI-2 activity and pathogenicity has been seen in E. coli O157:H7, where AI-2 controls the expression of type III secretion gene transcription and protein secretion (Sperandio et al., 1999
). AI-2 activity may also function to control a gene involved in haemin acquisition in P. gingivalis (Chung et al., 2001
) and could, through this interaction, be involved in P. gingivalis pathogenicity. However, a luxS mutation in S. flexneri had no effect on virulence (Day & Maurelli, 2001
) and a similar mutation in H. pylori had no effect on the expression of any known virulence factor (Joyce et al., 2000
). Thus, greater knowledge of the role of AI-2 in the regulation of virulence and biofilm formation is needed to understand the function of AI-2 quorum sensing regulation in bacterial pathogenicity.
We are interested in understanding the genetic mechanisms controlling the development of P. mirabilis biofilms, the swarming colony pattern, and how swarming behaviour enhances the uropathogenicity of these bacteria. We hypothesized that AI-2 quorum sensing may influence both P. mirabilis biofilm and colony pattern formation, as well as virulence. In this report, we show the luxS-dependent synthesis of AI-2 by P. mirabilis and the coordinate expression of AI-2 during swarming migration, and compare the phenotype and virulence of mutants defective in AI-2 production to those of the wild-type.
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METHODS |
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A nonpolar null mutation in luxSPM was constructed by first removing the plasmid-borne EcoRI site in pRS104, resulting in pRS107 (Table 1). A 847 bp SmaI fragment containing the aphA-3 gene from pUC18K (Menard et al., 1993
) was inserted at the EcoRI site internal to luxSPM by blunt-end ligation to Klenow-treated plasmid. The resulting plasmid was named pRS110. Plasmid pRS110 was digested with SacI and KpnI, and a 2·5 kb fragment containing luxS'::aphA::'luxS was ligated to pGP704 (Miller & Mekalanos, 1988
) also digested with SacI and KpnI, resulting in pRS112. This plasmid was transformed into E. coli SM10
pir and conjugally transferred to P. mirabilis BB2000 by filter mating (Belas et al., 1991a
). Mutation of the chromosomal copy of luxSPM was confirmed by antibiotic resistance spectrum (KmR ApS), PCR amplification of luxSPM locus from the putative luxS strain, and measurement of AI-2 activity using the V. harveyi luminescence assay. Several colonies that lacked AI-2 resulting from the introduction of luxS'::aphA::'luxS were found. A representative was chosen and named RS601.
Nucleotide sequencing and analysis.
Double-stranded DNA was used as a template for nucleotide sequencing by the recommended procedures of the Prism Ready Reaction Dye Deoxy Termination Kit (Applied Biosystems) in conjunction with Taq polymerase and a model 373A DNA sequencer (Applied Biosystems). Nucleotide and deduced amino acid sequences were analysed with Vector NTI V5.0 software (Informax) and the BLAST family of programs (Altschul et al., 1990 , 1997
; Gish & States, 1993
; Worley et al., 1995
). Phylogenetic trees of P. mirabilis LuxS relatedness were constructed by aligning the LuxS deduced amino acid sequences obtained from complete and partial genome databases using CLUSTAL W V1.75 [http://www.es.embnet.org/Doc/phylodendron/clustal-form.html] (Thompson et al., 1994
) and arranged with the computer program Phylodendron [http://iubio.bio.indiana.edu/soft/molbio/java/apps/trees/].
The following nucleotide sequences and accession numbers were used in generating LuxS dendrograms: Escherichia coli MG1655, U00096; Salmonella typhimurium LT, WUGSC_99287; Salmonella paratyphi A, WUGSC_32027; Salmonella typhi CT18, WUGSC_573; Klebsiella pneumoniae, Contig1032; Yersinia pestis strain CO-92, Sanger_632; Vibrio harveyi, AF120098; Vibrio cholerae chromosome I, AE003852; Shewanella putrefaciens, sputre_7833; Neisseria meningitidis serogroup B strain M, AE002098; Neisseria gonorrhoeae, OUACGT_485; Haemophilus influenzae Rd, U32731; Pasteurella multocida PM70, AE004439; Actinobacillus actinomycetemcomitans, OUACGT_714; Campylobacter jejuni, AL111168; Deinococcus radiodurans R1, AE000513; Clostridium perfringens, AB028629; Streptococcus mutans, UOKNOR_1309; Streptococcus pneumoniae, S.pneumoniae_3836; Streptococcus equi, sequi_Contig243; Streptococcus pyogenes strain SF370, AE004092; Enterococcus faecalis unfinished, gef_11360; Clostridium difficile strain 630 (epidemic type X) unfinished, Sanger Centre unassigned; Staphylococcus epidermidis, TIGR_1282; Staphylococcus aureus, Sanger_1280_3; Helicobacter pylori 26695, AE000511; Bacillus subtilis, AL009126; Bacillus anthracis, TIGR_1392; Bacillus halodurans C-125, BA000004; Borrelia burgdorferi, AE000783; Clostridium acetobutylicum, C.aceto_gnl; Porphyromonas gingivalis W83, P.gingivalis_GPG.con; Haemophilus ducreyi strain 35000HP, HTSC_730.
Preparation of cell-free conditioned media for AI-2 assays.
Conditioned media from broth cultures were prepared using a modification of the protocol described by Sperandio et al. (1999) . Overnight cultures grown in LB at 37 °C were diluted 1:100 in fresh LB and cultured in a shaking water bath at 37 °C. When the cultures reached an OD600 of 0·3, the bacteria were again diluted 1:100 in fresh LB and incubated in a shaking water bath at 37 °C until an OD600 of 1·2 was reached (about 12 h incubation). The bacteria were removed by centrifugation (14000 g, 5 min, 20 °C) and the supernatant filter-sterilized (0·2 µm pore-size cellulose acetate filter; Millipore). Aliquots of the conditioned media were kept on ice to be used immediately in AI-2 assays or stored at -20 °C until needed.
Production of AI-2 during P. mirabilis swarming migration and behaviour on L agar medium was assessed as follows. P. mirabilis overnight cultures grown in LB were prepared for swarming behaviour assays as described by Belas et al. (1998) , in which a 5 µl inoculum of about 2·5x106 cells was dispensed on the L agar surface, incubated at 37 °C, and assayed for swarming motility. To determine AI-2 activity during swarming migration and colony formation, a cylinder of agar 1 cm in diameter and 1 cm in height from the outer edge of the developing swarming colony was removed at 1 h intervals using a sterile brass cork borer (Fisher Scientific). Colony-forming units (c.f.u.) were determined in parallel by removing the cells from the surface of the agar core with vigorous mixing, followed by growth of serial dilutions of the suspension on L agar. AI-2 activity was also measured at discrete points within a preformed 5 h old swarming colony that represents one cycle of swarming behaviour. In this case, agar cores were removed from the point of inoculation, a point one-third of the way to the periphery, a point two-thirds of the distance to the periphery, and at the periphery of the colony. Each agar plug with overlying bacteria was frozen at -20 °C for 18 h and then thawed rapidly by addition of 300 µl AB medium. Insoluble agar and bacterial cells were removed by centrifugation and filtration as described above, and the supernatant assayed immediately or stored at -20 °C. This freeze-squeeze process resulted in 800900 µl fluid.
V. harveyi luminescence assay for AI-2.
The presence of AI-2 in the conditioned media was assayed by using the V. harveyi BB170 (luxN::Tn5) reporter strain, which responds only to AI-2 (Surette & Bassler, 1998 ). The luminescence assays were performed as described by Surette & Bassler (1998)
, using an EG&G Berthold Microlumat LB 96P luminometer. The data are reported as relative light units (RLUs) or as the fold stimulation of light emission by V. harveyi BB170 compared to the RLU values obtained from the corresponding DH5
(AI-2-) negative control.
Murine UTI virulence assay.
A modification (Johnson et al., 1987 ) of the well-established mouse model of ascending UTI (Hagberg et al., 1983
) was used to measure P. mirabilis virulence. Female CBA/J (Harlan SpragueDawley) were transurethrally challenged with about 3x107 c.f.u. of bacteria per mouse. After 7 days, the mice were killed, and bacteria recovered from urine, bladder and kidneys were enumerated on LSW- agar plates containing the appropriate antibiotics. The range of detection in this assay is 102109 c.f.u. (ml urine)-1 or c.f.u. (g tissue)-1 (Li et al., 1999
).
Statistical methods.
Mean numbers of c.f.u. ml-1 or g-1 from cultures of urine or tissue homogenates were compared by the MannWhitney and non-parametric ANOVA tests.
Materials and reagents.
All reagents were of the highest purity available. Components of bacteriological media were purchased from Difco. Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs, Boehringer Mannheim Biochemicals, Promega or Qiagen and were used according to the suppliers recommendations.
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RESULTS AND DISCUSSION |
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A luxS orthologue is responsible for AI-2 activity in P. mirabilis
The luxS gene is essential for AI-2 production in many bacteria, prompting us to determine whether P. mirabilis genomic DNA contained a luxS orthologue. Southern blots of P. mirabilis genomic DNA separately digested with five restriction endonucleases were hybridized to radioactively labelled pRSluxSEC containing a PCR fragment of E. coli luxS. The analysis of these blots indicated that EcoRI digestion resulted in a 1·6 kb DNA fragment that hybridized to luxSEC. An EcoRI digest of genomic DNA was ligated to pBluescript SK(+) and transformed into E. coli DH5, a strain lacking AI-2 activity (Surette et al., 1999
). The resulting recombinant colonies were screened for AI-2 activity using the V. harveyi BB170 AI-2 assay. Five of the cell-free conditioned media supernatants from the recombinant E. coli produced AI-2 activity equal to or in excess of conditioned media from DH5
harbouring pRSluxSEC (Table 2
). Restriction mapping of the plasmid carried by these bacteria revealed that each of these recombinant E. coli harboured the same foreign DNA, presumably containing luxSPM, and indicating that luxSPM is functional in E. coli. A single representative plasmid, pRS104 (Table 1
), was used for further work.
The nucleotide sequence of the EcoRI fragment of P. mirabilis genomic DNA inserted in pRS104 was determined. The analysis of the 1598 bp locus is shown in Fig. 4(a). Three ORFs were found, one of which is highly homologous to other bacterial luxS deduced amino acid sequences (Fig. 4b
). The homology of ORF2 to other LuxS sequences is greatest with LuxS of E. coli (67% identity) and Salmonella enterica serovar Typhimurium (65% identity). We refer to this ORF as luxS and use luxSPM to distinguish it from other luxS genes. As shown in Fig. 4(a)
, two partial ORFs were also discovered on either side of luxSPM. To the left of luxS, as drawn in Fig. 4(a)
, is an ORF whose deduced amino acid sequence is homologous to E. coli gshA, encoding
-glutamylcysteine synthetase, and to the right of luxS is an ORF whose deduced amino acid sequence has partial homology to a series of proteins involved in ion or antibiotic transport. The data presented in Fig. 4(a, b)
show that the P. mirabilis luxS locus is similar in its arrangement of genes to the luxS locus of E. coli and S. enterica serovar Typhimurium. These data also demonstrate that luxSPM is homologous to the luxS genes of other bacteria (Fig. 4b
). The latter point is further strengthened by the ability of luxSPM to complement the luxS defect in DH5
, thereby producing AI-2 activity in the recombinant strain.
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As anticipated, RS601 did not produce detectable AI-2 activity (Table 2), when assayed using the V. harveyi BB170 sensor. We compared RS601 to the parent strain (BB2000) in swimming motility and chemotaxis behaviour, and swarming motility and behaviour. Swimming speed and chemotaxis were measured microscopically as well as during growth in Mot agar. When compared to the wild-type bacteria, RS601 showed no differences in either swimming speed or chemotaxis behaviour. An analysis of swarmer cell morphology, movement and biofilm formation was conducted. Swarmer cell differentiation, the timing of the beginning of swarming migration and the swarming colony pattern formation of RS601 were also indistinguishable from those of the wild-type. Other phenotypes, such as growth rate and the production of urease, protease and haemolysin, were also unaffected by the luxS mutation. We conclude that the luxS mutation does not lead to an overt change in P. mirabilis motility or biofilm phenotypes as determined by this set of assays.
AI-2 activity does not affect P. mirabilis virulence during UTI
It may be that defects in AI-2 synthesis are not apparent when the bacteria are grown in nutrient-rich conditions, but are manifested under conditions that challenge the survival of the cells. To determine if loss of AI-2 activity affected P. mirabilis virulence, we compared the uropathogenicity of RS601 to that of wild-type P. mirabilis, using a modification (Johnson et al., 1987 ) of the mouse model of ascending UTI (Hagberg et al., 1983
). This mouse model has been successfully used by others to assess the uropathogenicity of E. coli (Johnson et al., 1993a
) and Providencia stuartii (Johnson et al., 1987
), and by our group to assess uropathogenicity of P. mirabilis mutants defective in flagellin synthesis (Mobley et al., 1996
) and strains defective in the production of an extracellular metalloprotease (Walker et al. , 1999
).
Fig. 5 shows the colonization of mouse urine, bladder and kidneys 7 days after transurethral challenge with either RS601 or wild-type P. mirabilis. Ten mice were used per bacterial strain. Two mice infected with wild-type P. mirabilis died from stone blockage on day 6 and were not used in the analysis. A pairwise comparison of the mean c.f.u. for each tissue (indicated by the horizontal line in each column in Fig. 5
) suggests a noticeable increase in the survival of the AI-2-deficient mutant in each tissue. The trend towards increased survival in the luxS strain appears in all samples (urine, bladder and kidney) examined. The P values (>0·05), however, do not reflect a statistical significance to these differences. Thus, a luxS mutant is not attenuated in virulence, suggesting that AI-2 quorum signalling is not required for P. mirabilis UTI.
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ACKNOWLEDGEMENTS |
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Received 17 August 2001;
revised 6 November 2001;
accepted 12 November 2001.