Listeria monocytogenes bile salt hydrolase is a PrfA-regulated virulence factor involved in the intestinal and hepatic phases of listeriosis
PrfA-regulated L. monocytogenes virulence factorinvolved in the intestinal and hepatic phases of listeriosis.
Introduction
Bile salts are end-products of cholesterol metabolism inmammals. Bile salts are mostly synthesized in hepato-cytes, where >95% are conjugated to glycine or taurinebefore transport into the biliary canalicular system of theliver and/or storage in the gall bladder. In response to theingestion of food, bile salts are released into the duode-num helping fat digestion and absorption. In addition totheir role in digestion, bile salts have antimicrobial prop-erties. Bile salts are amphipathic molecules, which candegrade lipid-containing bacterial and viral membranes.Some resident enteric microflora and intestinal pathogenshave evolved mechanisms to resist the detergent actionof bile salts by synthesis of porins, transport proteins,efflux pumps or lipopolysaccharide (LPS) (Gunn, 2000).Others have developed the capacity to transform bile saltsby modification of the steroid ring or hydrolysis of the conjugated bile salts. The latter deconjugation reaction is catalysed by bile salt hydrolase (BSH), also called con-jugated bile acid hydrolase (CBAH). This cytoplasmicenzyme is produced by many Gram-positive and Gram-negative commensal bacteria of the resident entericmicroflora, including strains of genera such as Bac-teroides (Stellwag and Hylemon, 1976; Kawamoto et al.,1989), Clostridium (Gopal-Srivastava and Hylemon,1988; Coleman and Hudson, 1995) and Enterococcus(Kobashi et al., 1978; Franz et al., 2001). It has also beencharacterized in lactic acid bacteria such as Bifidobac-terium (Grill et al., 1995; 2000a) and Lactobacillus (Elkinsand Savage, 1998; Grill et al., 2000b; Moser and Savage,2001), some strains of which are part of the human entericmicroflora and/or are used in the dairy industry as probi-otics, owing to their safety for humans when administeredorally and their beneficial effects on human health(Schiffrin and Blum, 2001). BSH has not been describedin enteropathogens, such as Salmonella, Shigella,Yersinia or Campylobacter. BSHs are believed to protectcommensal bacteria from bile salt toxicity and to con-tribute to bacterial survival and intestinal colonization (De Boever et al., 2000; Grill et al., 2000a,b).
Molecular Microbiology (2002) 45(4), 1095–1106
Olivier Dussurget,1 Didier Cabanes,1 Pierre Dehoux,1
Marc Lecuit,1 the European Listeria GenomeConsortium,† Carmen Buchrieser,2 Philippe Glaser2
and Pascale Cossart1*1Unité des Interactions Bactéries-Cellules, and2Laboratoire de Génomique des MicroorganismesPathogènes, Institut Pasteur, 28 rue du Dr Roux, 75724Paris Cedex 15, France.
Summary
Listeria monocytogenes is a bacterial pathogencausing severe food-borne infections in humans andanimals. It can sense and adapt to a variety of harshmicroenvironments outside as well as inside the host.Once ingested by a mammalian host, the bacterialpathogen reaches the intestinal lumen, where itencounters bile salts which, in addition to their rolein digestion, have antimicrobial activity. Comparisonof the L. monocytogenes and Listeria innocuagenomes has revealed the presence of an L. monocytogenes-specific putative gene encoding abile salt hydrolase (BSH). Here, we show that the bshgene encodes a functional intracellular enzyme in all pathogenic Listeria species. The bsh gene is positively regulated by PrfA, the transcriptional activator of known L. monocytogenes virulencegenes. Moreover, BSH activity increases at lowoxygen concentration. Deletion of bsh results indecreased resistance to bile in vitro, reduced bacterial faecal carriage after oral infection of theguinea-pigs, reduced virulence and liver colonizationafter intravenous inoculation of mice. Taken together,these results demonstrate that BSH is a novel
Accepted 15 May, 2002. *For correspondence. [email protected]; Tel. (+33) 1 45 68 88 41; Fax (+33) 1 45 68 8706. †P. Glaser, L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero, P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Chetouani, E. Couve, A. de Daruvar, P. Dehoux, E. Domann, G. Dominguez-Bernal, E. Duchaud, L. Durant, O. Dussurget, K.-D. Entian, H. Fsihi, F. Garcia-Del Portillo, P. Garrido,L. Gautier, W. Goebel, N. Gomez-Lopez, T. Hain, J. Hauf, D. Jackson,L.-M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G. Kurapkat, E. Madueno, A. Maitournam, J. Mata Vicente, E. Ng, H. Nedjari, G. Nordsiek, S. Novella, B. de Pablos, J.-C. Perez-Diaz, R. Purcell,B. Remmel, M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J.-A.Vazquez-Boland, H. Voss, J. Wehland and P. Cossart.
Here, we demonstrate that BSH of the enteropathogenListeria monocytogenes is a virulence factor involved inbacterial survival in the intestinal lumen and liver, two hostenvironments in which L. monocytogenes has to resist theantibacterial effects of bile salts. As described for previ-ously identified L. monocytogenes virulence genes suchas inlA, hly and actA, bsh is positively regulated by thetranscriptional activator PrfA. BSH activity is increased atlow oxygen tension, a condition that is encountered afterbacterial ingestion. Moreover, we show that the bsh geneand BSH activity are present in all human pathogenic Listeria species, thus establishing an epidemiological link between Listeria resistance to bile salts and its abilityto colonize and infect humans.
Results
The bsh gene of L. monocytogenes does not have an orthologue in the non-pathogenic Listeria species L. innocua
We have compared the genomes of the pathogen L.monocytogenes and the non-pathogenic species Listeriainnocua in order to identify putative genes involved in L.monocytogenes virulence (Glaser et al., 2001). A total of270 DNA fragments that are absent from the L. innocuagenome were identified, including a 975 bp open readingframe (ORF), the putative gene product of which displayed 67% identity (219/324 amino acids) to Lacto-bacillus plantarum BSH (EC 3.5.1.24). The genomic orga-nization of the region surrounding the L. monocytogenesbsh gene was identical to that of L. innocua (Fig. 1). In L. monocytogenes, the bsh gene is preceded by thegroES–groEL operon and followed by an ORF ofunknown function, Lmo 2066, both genes being in the
same orientation. In L. innocua, the groES–groEL operonis directly upstream from the orthologue of Lmo 2066, theORF 3530.1. The sizes of these three sequences, 285,1629 and 300 bp, respectively, are identical in the twospecies. Their G+C content (38–41%) was also con-served in the two species. In contrast, the bsh gene hasa lower G+C content, i.e. 36% (Fig. 1).
The bsh gene encodes an active enzyme in L. monocytogenes
The expression of the bsh gene during exponential growthof L. monocytogenes in brain–heart infusion (BHI) at 37∞Cwas assessed by reverse transcription polymerase chainreaction (RT-PCR), which revealed that bsh is transcribedin these conditions (data not shown). BSH activity wasdemonstrated by plating L. monocytogenes onto MRSmedium supplemented with 0.5% glycodeoxycholic acid(MRS-GDCA). According to the principle of the assaydeveloped by Dashkevicz and Feighner (1989), bacteriaproducing BSH deconjugate GDCA and release freedeoxycholate acid (DCA), which precipitates in the acidicmedium. In contrast to L. innocua, incubation of L. mono-cytogenes on MRS-GDCA plates at 37∞C resulted in theproduction of a white halo of precipitated free bile acidsaround colonies, thus demonstrating the presence of BSHactivity (Fig. 2B).
In order to determine whether the bsh gene wasresponsible for the BSH activity and to study the role ofthe enzyme, an isogenic BSH-deficient mutant was con-structed in L. monocytogenes by gene replacement (seeExperimental procedures). Southern blot analysis con-firmed the replacement of the bsh gene (Fig. 2A). To verifythe absence of any BSH activity in the mutant strain,
Fig. 1. Genomic organization of the bsh gene region. Data from the complete genome sequences of L. monocytogenes and L. innocua wereused to draw this map approximately to scale. Arrows indicate the orientation of the genes. Hairpins depict putative terminators. The blacktriangle shows the PrfA box. The G+C percentage is indicated below each gene name. The open reading frames Lmo 2066 and 3530.1 areorthologues.
Listeria monocytogenes bile salt hydrolase 1097
L. monocytogenes EGDe and L. monocytogenes EGDeDbsh were patched onto MRS-GDCA. Only L. monocyto-genes EGDe showed BSH activity (Fig. 2B), demonstrat-ing unambiguously that the BSH activity resulted solelyfrom the bsh gene, in agreement with the absence of aclose paralogous gene in the L. monocytogenes genome.Indeed, the gene product of the ORF Lmo0446 has beenannotated as a putative bile salt hydrolase, but shows42% homology to Bacillus subtilis penicillin amidase andonly weak homologies to the Clostridium perfringens BSH(36%) and L. monocytogenes BSH (31%). Complemen-tation of the mutant strain with the bsh gene under thecontrol of its promoter restored BSH activity (Fig. 2B). Thewild-type, mutant and complemented strains had a similarmorphology, as assessed by light microscopy. They pro-duced similar colonies on BHI agar medium, and theirgrowth rates were similar in BHI at 37∞C. Heterologousexpression of bsh in L. innocua also resulted in BSH activity (Fig. 2B).
The bsh gene is specific to pathogenic Listeria
Strains from the six Listeria species and 12 L. monocy-togenes serovars were screened for BSH activity onMRS-GDCA. The two pathogenic strains, L. monocyto-genes and Listeria ivanovii, as well as Listeria seeligeri,exhibited BSH activity (Fig. 3). The non-pathogenicspecies, L. innocua, Listeria welshimeri and Listeria grayi,did not produce any precipitation zone. All L. monocyto-genes serovars except serovar 4e, which is not found in
mammals, had BSH activity (Fig. 3). DNA hybridizationcarried out on 91 additional Listeria strains confirmed theactivity data except for the BSH-negative L. monocyto-genes serovar 4e, in which a sequence homologous tobsh was nevertheless detected (M. Doumith, personalcommunication).
The bsh gene is positively regulated by PrfA
The bsh transcriptional start point was determined byprimer extension using RNA extracted from L. monocyto-genes growing exponentially in BHI at 37∞C (see Ex-perimental procedures). The transcriptional start point islocated 103 nucleotides upstream of the bsh start codon(Fig. 4A). A sequence similar to the Escherichia coli s70
consensus -10 region was identified upstream of the tran-scriptional start point. The E. coli -35 consensus TTGACAhexamer was lacking from the bsh promoter. A perfectpalindromic sequence, TTAAAAATTTTTAA, was found133 bp upstream of the bsh start codon. The sequencehad two mismatches when compared with the consensusPrfA box found in the listeriolysin gene regulatory region(Table 1). Thus, the transcript starts 30 bp downstreamfrom the PrfA box, and the PrfA box is located in the -41region, a position that is conserved in most PrfA-regulatedpromoters (Table 1, Fig. 4B).
The role of PrfA in the transcription of the bsh gene wasassessed by RT-PCR. cDNA synthesis and PCR wereperformed using bsh-specific oligonucleotides and RNAsthat were isolated from L. monocytogenes and L. mono-
Fig. 2. Disruption of the bsh gene.A. Chromosomal DNAs from L. monocytogenes EGDe and the L. monocytogenes EGDe Dbsh were digested with AccI and analysed bySouthern blotting with a 32P-labelled probe corresponding to the bsh gene. The 1.4 kb fragment containing the bsh gene is indicated by anarrow.B. L. monocytogenes EGDe and L. monocytogenes EGDe Dbsh, L. innocua and these strains transformed with a plasmid carrying the bshgene under the control of its own promoter were patched onto MRS-GDCA medium. Strains expressing the bsh gene produced a halo ofprecipitated DCA.
cytogenes DprfA during exponential growth in BHI at37∞C. As shown in Fig. 5A, the levels of bsh mRNA werelower in the DprfA strain, demonstrating that PrfA regu-lates BSH transcription. Northern blots confirmed thathigher amounts of the 1.1 kb specific bsh transcript weredetected in the RNAs from the wild-type strain comparedwith the RNAs from the DprfA strain (data not shown).
In order to confirm the role of PrfA on BSH synthesis,the hydrolase activities of L. monocytogenes and L.
monocytogenes DprfA were compared using the MRSagar plate assay. The BSH activity of the DprfA strain wasmuch lower than that of the wild-type strain, as revealedby the difference in halo size (Fig. 5B).
BSH activity is increased by hypoxia
As host tissues in which L. monocytogenes faces bilesalts have oxygen tensions lower than atmospheric air,
Fig. 3. Detection of BSH activity in Listeria ssp. Strains of each Listeria species (grey bars) and L. monocytogenes serovars (white bars) werepatched onto MRS-GDCA medium, and the diameter of the halo was measured. Lmo, L. monocytogenes; Liv, L. ivanovii; Lse, L. seeligeri; Lin, L. innocua; Lwe, L. welshimeri; Lgr, L. grayi.
Fig. 4. Identification of the bsh gene promoter.A. Determination of the 5¢-terminus of the bsh transcript. RNA was prepared from a logarithmic culture of L. monocytogenes EGDe growing inBHI broth at 37∞C. The transcriptional start point (TSP, +1) shown in the right lane was determined by primer extension. The +1 is indicated byan arrow, and the corresponding nucleotide is boxed.B. Schematic organization of the bsh promoter. The putative -10 and -35 PrfA boxes of the bsh promoter are underlined, as well as apossible ribosome binding site (RBS) and the start codon. The +1 is indicated by an arrow.
Listeria monocytogenes bile salt hydrolase 1099
we hypothesized that BSH activity could be increased by hypoxia. L. monocytogenes was patched onto MRS-GDCA and incubated at 37∞C in jars under ª 45 mmHgoxygen tension. Hydrolase activity detected on theseplates was compared with that on plates incubated atatmospheric oxygen tension (159 mmHg). L. monocyto-genes deconjugated higher amounts of GDCA inmicroaerophilic conditions than in aerobic conditions (Fig. 6A). These results indicate that L. monocytogenesBSH activity increases in response to a decrease in thelevel of external oxygen.
To determine whether the higher BSH activity was theresult of increased levels of the bsh messenger RNAs,Northern blots were performed using the bsh gene as aprobe. The probe was hybridized to RNAs that were iso-lated from L. monocytogenes grown exponentially at 159mmHg or 45 mmHg oxygen tension in BHI at 37∞C.Similar amounts of the 1.1 kb bsh transcript were detectedin the RNA from cultures exposed to 159 mmHg and 45 mmHg oxygen tension (Fig. 6B), suggesting thatoxygen acts on BSH at the post-transcriptional level.
Although the transcription of the bsh gene was notaltered by hypoxia, we were interested to test the puta-tive effect of oxygen on PrfA expression. Whole-cellextracts of L. monocytogenes growing exponentially at159 mmHg or 45 mmHg oxygen tension in BHI at 37∞Cwere analysed by Western blot using anti-L. monocyto-
genes PrfA polyclonal antibodies. Comparable amountsof PrfA were detected in both cell extracts (data notshown). Thus, hypoxia does not contribute to increasedexpression of prfA.
BSH is involved in resistance to bile in vitro
In order to study the possible role of BSH in resistance tobile toxicity, the minimal inhibitory concentrations (MICs)of porcine bile and purified bile salts were determined forL. monocytogenes EGDe and L. monocytogenes EGDeDbsh. Both bile and purified bile salt MICs for the mutantwere twofold lower than that of the wild-type strain, 0.08%and 0.15% respectively.
BSH activity contributes to L. monocytogenes survivalwithin the intestinal lumen
To study the possible role of BSH in the intestinal phaseof listeriosis, i.e. in the earliest stage of the infectiousprocess, the persistence of the BSH-deficient strain wasstudied and compared with the parental strain or the com-plemented strain. To this end, oral infection with either theBSH-deficient strain or the L. monocytogenes parentalstrain was performed in guinea pigs, a species in whichL. monocytogenes oral inoculation results in systemicdisease (Lecuit et al., 2001). Quantification of stool
Fig. 5. Effect of PrfA on L. monocytogenesbsh gene and BSH activity.A. Effect of PrfA on the expression of the bshgene. The bsh gene transcription wasanalysed by RT-PCR using RNA preparedfrom logarithmic cultures of L. monocytogenesEGDe and L. monocytogenes EGDe DprfAgrowing in BHI broth at 37∞C. The 0.9 kbfragments correspond to the bsh RT-PCRproduct, and the 0.8 kb fragments correspondto rrn RT-PCR control product.B. Effect of PrfA on BSH activity. L.monocytogenes EGDe and L. monocytogenesEGDe DprfA were patched onto MRS-GDCAmedium, and plates were exposed to 45mmHg oxygen tension at 37∞C.
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Table 1. Comparison of the promoter sequences regulated by the PrfA activator.
Gene Function PrfA box D1 D2
hly Pore-forming toxin C A T T A A C A T T T G T T A A C G 33 131plcA Phosphatidylinositol-specific phospholipase C C G T T A A C A A A T G T T A A T G 32 24mpl Zinc-metalloprotease processing the phospholipase C, PlcB A A T T A A C A A A T G T A A A A G 32 150actA Surface protein responsible for actin-based motility G A T T A A C A A A T G T T A G A G 32 148inlA Invasin responsible for the crossing of the intestinal barrier G G A T A A C A T A A G T T A A T T 31 397prfA Transcriptional activator of virulence genes A G C T A A C A A T T G T T G T T A 30 31bsh Bile salt hydrolase A T T T A A A A A T T T T T A A A G 30 103
Palindromes are indicated by arrows. Distances between palindromes and the +1 (D1) and between the +1 and the start codon (D2) areindicated in nucleotides.
microorganisms after intragastric injection of a sublethalinoculum (ª 1010 bacteria) into Hartley guinea pigs wasperformed for L. monocytogenes EGDe, L. monocyto-genes EGDe Dbsh and L. monocytogenes EGDe Dbshcomplemented with the bsh gene. For the wild-type strainin our experimental conditions, the number of bacteria instools was 107 after 24 h and decreased only slightly withtime. Bacterial counts of the bsh mutant were reduced by4–5 log at 48 h after infection compared with the wild-typeand complemented strains and continued to decreasewith time (Fig. 7). These results demonstrate that BSHplays a role in L. monocytogenes persistence within thegastrointestinal tract. In agreement with our data, prelim-inary results show that, when L. innocua or L. monocyto-genes are transformed with a plasmid carrying the bshgene, there is a gain of function, i.e. intestinal multiplica-tion is increased by ª 1 log compared with the parentalstrains.
BSH activity is involved in L. monocytogenes virulenceafter intravenous inoculation
BSH is a cytosolic enzyme, and it is unlikely that it couldplay a role in crossing the intestinal barrier. To study therole of BSH in the infectious steps following this step andto bypass the intestinal intraluminal effect of BSH, intra-venous infection with either the BSH-deficient strain or its parental counterpart was performed in mice, the bestcharacterized model so far to study the systemic phaseof listeriosis. LD50 values after intravenous injection intoBALB/c mice were determined for L. monocytogenesEGDe and L. monocytogenes EGDe Dbsh. The LD50
obtained for the mutant was two- to threefold higher thanthat of the wild-type strain (3 ¥ 104 and 8 ¥ 103 cfu respec-tively). The significance of this attenuation in the BSHmutant was studied further by comparing over a timecourse the infection level of liver, spleen and brain of miceinfected intravenously with either the mutant and the wild-type bacteria. An unambiguous difference between thetwo strains could be observed 2 days after inoculation in the liver and spleen, but only by the third day after
inoculation in the brain (Fig. 8). These results suggest thatBSH contributes to the survival of L. monocytogenes intarget organs other than the intestine, particularly in theliver filter, which bacteria have to cross and in which bilesalt concentration is high.
Discussion
This study describes the existence and role of BSH activ-ity in pathogenic Listeria species. BSH is a novel type ofvirulence factor that allows bacterial survival within thehost, in the intestinal lumen and in the liver tissue, thusplaying a key role in the intestinal and hepatic phase oflisteriosis. It is the first description of a BSH produced bya pathogenic bacterial species that is not considered asa member of the normal resident enteric microflora. Thebsh gene was discovered by comparing the L. monocy-togenes genome with that of the non-pathogenic species
Fig. 7. Effect of BSH on persistence in the guinea piggastrointestinal tract. L. monocytogenes EGDe (black), L.monocytogenes EGDe Dbsh (white) and L. monocytogenes EGDeDbsh complemented (grey) were inoculated intragastrically ingroups of three guinea pigs with ª 1010 cfu. Listeria growth wasfollowed in the stools at 24, 48 and 72 h.
0.5-1.0-
2.0-
A B
159 mmHg 45 mmHg kb 45 m
mH
g
159 m
mH
g
Fig. 6. Effect of oxygen tension on L.monocytogenes BSH.A. Effect of hypoxia on BSH activity. L.monocytogenes EGDe was patched ontoMRS-GDCA medium, and plates wereexposed to 159 mmHg or 45 mmHg oxygentension at 37∞C.B. Effect of hypoxia on the expression of thebsh gene. The bsh gene transcript wasanalysed by Northern blot using RNAprepared from logarithmic cultures of L.monocytogenes EGDe growing at 159 mmHgor 45 mmHg oxygen tension in BHI broth at37∞C. The 1.1 kb transcript corresponding tothe bsh transcript is indicated by an arrow.
Listeria monocytogenes bile salt hydrolase 1101
showing 67% identity. In addition to the sequence homol-ogy, the G+C percentage of the L. plantarum bsh gene isclosest to that of L. monocytogenes after Lactobacillusacidophilus, 34% and 36% respectively. Lactobacilli,along with Bifidobacterium and Lactococcus, are non-pathogenic bacteria that are part of the human residentmicroflora and are referred to as lactic acid bacteria.Some strains are probiotics used in many dairy productsbecause they are believed to be safe and could be
Fig. 8. Effect of L. monocytogenes BSH onvirulence in mice. L. monocytogenes EGDe(black) and the L. monocytogenes EGDeDbsh (white) were inoculated intravenously ingroups of four BALB/c mice with ª 104 cfu.Bacterial growth was followed in the spleen,the liver and the brain at 24, 48 and 72 h.
L. innocua and searching for genes that were absent fromthe sequence of the latter species. It is not clear how andwhy L. monocytogenes acquired the bsh gene. The G+Ccontent of L. monocytogenes bsh, 36%, is lower thanthose of neighbouring genes, and it may have beenacquired from low-G+C content bacteria. Indeed, whenthe BSH amino acid sequence of Listeria was comparedwith bacterial BSHs from public databases, Lactobacillusplantarum was the closest relative of L. monocytogenes,
beneficial to human health, possibly by stimulation of theimmune system (Miettinen et al., 1996). At several stagesof their life cycle, L. monocytogenes and Lactobacillishare the same microenvironments, e.g. not only do theyhave a similar intestinal tropism, but they also grow ondecaying vegetation, in food and in silage. It has beenproposed that the intestine could be a reservoir for L.monocytogenes, as asymptomatic faecal carriage of L.monocytogenes in healthy adults is estimated at between2% and 10% (Schlech, 2000), thus placing Listeria at theborder between pathogen and commensal microorgan-isms. It is possible that Lactobacilli or other bacteria fromthe normal enteric microflora and L. monocytogenesmight be able to exchange genetic material. The L. mono-cytogenes bsh gene could have resulted from one ofthese exchanges. In agreement with this hypothesis,putative competence genes have been detected in the L. monocytogenes genome (Glaser et al., 2001). Theextremely conserved genomic organization of the regionsurrounding the bsh locus in L. monocytogenes and L.innocua suggests that L. innocua has lost the bsh gene,in agreement with the current opinion that L. innocua isderived from L. monocytogenes (Chakraborty et al., 2000;Glaser et al., 2001).
BSH was shown to be produced as an active enzymeby all assayed strains pathogenic for mammals. Indeed,L. ivanovii and all L. monocytogenes serovars, exceptserovar 4e, produced the enzyme. However, a sequenceorthologous to the L. monocytogenes EGDe bsh genewas detected in the serovar 4e, in which it might not betranscribed or might encode a non-functional enzyme. Itis of interest to note that strains of serovar 4e are mostlyfound in amphibians (Weber et al., 1995) and that BSHactivity may not be necessary for Listeria to infect or col-onize these hosts, as they have been shown to synthe-size atypical bile salts, such as varanic acid, bile alcoholsulphates and unconjugated bile salts (Yoshii et al., 1994;Une et al., 1996). Interestingly, the only non-pathogenicListeria that produced a BSH was L. seeligeri. Although ithas been implicated in one case of human meningitis(Rocourt et al., 1986), this species is experimentally non-pathogenic. It is closely related to pathogenic Listeria, as it is haemolytic and possesses the prfA–plcA–hlympl–actA–plcB virulence gene cluster in its genome, butthis cluster is slightly modified (Gouin et al., 1994;Chakraborty et al., 2000). Expression of these virulencegenes might be too low in this species to confer patho-genicity (Gouin et al., 1994).
The observation that the BSH was only produced bystrains that also expressed the virulence gene regulatorPrfA led us to investigate the role of PrfA in the expres-sion of BSH. The transcriptional start point was mapped,and a PrfA box was identified in the -35 region, a distancethat is conserved in the PrfA-dependent promoters. PrfA-
dependent promoters are related to class II CRP-depen-dent promoters. They have a -35 region that is not similarto the E. coli consensus and to which the positive regu-lator binds (Raibaud and Schwartz, 1984; Sheehan et al.,1995; Niu et al., 1996; Bockmann et al., 2000). The BSHPrfA box is a perfect palindrome, albeit with 10 of the 12nucleotides of the PrfA-binding consensus sequence con-served. Sequence data were completed by a functionalanalysis showing that the transcription of the bsh genewas positively regulated by PrfA. Not only was BSH pro-duction controlled by PrfA, but its activity was dependenton the oxygen tension, as hypoxia increased its hydrolyticactivity. A similar observation has been made in the caseof L. acidophilus, which requires a low redox potential todeconjugate bile salts (Gilliland and Speck, 1977). In con-trast, the BSHs from strict anaerobes such as Clostridiumand Bacteroides are oxygen insensitive (Masuda, 1981).Once ingested, L. monocytogenes encounters oxygentensions that are much lower than atmospheric air (159 mmHg). Interestingly, microenvironments in whichbacteria can encounter bile salts, i.e. the intestine and theliver (two tissues that are L. monocytogenes targetsduring invasive listeriosis), constitute microaerophilicenvironments. Measurement of intraluminal oxygenationin the gastrointestinal tract of mice shows an oxygen gra-dient ranging from 58 mmHg in the stomach to 32 mmHgin the duodenum and 3 mmHg in the sigmoid colon (Heet al., 1999). When the invasion process starts, L. mono-cytogenes faces low oxygen tensions inside cells, i.e. 5–40 mmHg (Jiang et al., 1996). Like temperature, reducedoxygen tension in the host could be a signal sensed byListeria to switch on the expression of its virulencemachinery where, when and to the extent needed. Theactivator PrfA and its interaction with target genes havebeen shown to respond to various environmental signalssuch as temperature (Renzoni et al., 1997) and iron concentration (Bockmann et al., 1996). However, asdescribed in this work, the level of production of the PrfAprotein itself does not seem to be influenced by oxygentension, and transcription of the bsh gene was not alteredby hypoxia. These data suggest that the oxygen-dependent regulation of BSH activity is independent ofPrfA. The post-transcriptional mechanism by which theBSH is activated and/or stabilized by hypoxia is notknown. The enzyme itself might be sensitive to oxygenand/or regulated post-transcriptionally by a redox sensor.
Little is known about the molecular mechanismsevolved by enteric bacteria to resist bile toxicity. Specificinducible bile tolerance responses might co-exist withnon-specific constitutive mechanisms of resistance tobile, such as the outer membrane, which is an importantbarrier to bile (Gunn, 2000). Some enteric pathogens notonly produce proteins that confer resistance to bile, butalso sense bile and use this signal to know where and
when to express virulence factors (Gunn, 2000; Proutyand Gunn, 2000). Bile has been shown to alter the patternof expression of several virulence factors such as Vibrioparahaemolyticus haemolysin (Osawa and Yamai, 1996),the toxin co-regulated pilus and the cholera toxin from Vibrio cholerae (Gupta and Chowdhury, 1997;Schuhmacher and Klose, 1999). The influence of bile onvirulence factors and the role of bile tolerance mecha-nisms in establishing intestinal commensalism led us tostudy the effect of BSH deficiency in vivo. Faecal carriage,a reflection of intestinal persistence, was determined afteroral infection of guinea pigs. L. monocytogenes BSH wasshown to be critical for bacterial faecal carriage in stoolsas the multiplication of the BSH-deficient mutant was dra-matically reduced compared with the wild-type strain. IfBSH contributes to survival in the gastrointestinal tract byconferring resistance to bile toxicity in the intestine, onecould hypothesize that it could also promote colonizationby providing an advantage to L. monocytogenes whencompeting with other intestinal pathogens or the normalenteric microflora. This could be tested in a gnotobioticmodel of infection. In addition, the BSH-deficient mutantwas less virulent than its parental strain in the BALB/cmouse model of infection after intravenous injection, andwas impaired in its ability to infect liver, spleen and, at alater stage, brain. The presence of bile salts in hepato-cytes most probably accounts for this defect in the liver.The reduced bacterial counts in the spleen and braincould be secondary to, and possibly a consequence of,the impaired multiplication in the liver.
BSH could thus have a role in the intestinal and hepaticphases of invasive human listeriosis and/or during humanfebrile gastroenteritis associated with L. monocytogenes(Dalton et al., 1997; Aureli et al., 2000). More importantly,our data argue for a role for BSH in L. monocytogenesoral–faecal transmissibility (Wing and Gregory, 2002).
Experimental procedures
Bacterial strains, media and growth conditions
Listeria monocytogenes EGDe and L. monocytogenes EGDeDprfA was obtained from Dr M. Kuhn. All other Listeria strainswere obtained from the Centre National de Référence desListeria, Institut Pasteur, Paris. Listeria were grown routinelyin BHI medium (Difco) at 37∞C. When appropriate, Listeriawere grown under microaerophilic conditions in jars usingCampygen sachets (Oxoid). When required, antibiotics wereadded at the following concentrations: chloramphenicol, 7 mg ml-1; kanamycin, 20 mg ml-1. E. coli strains were grown inLB medium (Difco) at 37∞C. When required, antibiotics wereincluded at the following concentrations: ampicillin, 100 mgml-1; kanamycin, 20 mg ml-1. For detection of BSH activity, theplate assay developed by Dashkevicz and Feighner (1989)was used with minor modifications. Bacteria were grown ontoMan Rogosa and Sharpe (MRS) agar medium (Difco) sup-
plemented with 0.5% (w/v) glycodeoxycholic acid (Sigma),and plates were incubated at various oxygen tensions.Colonies of bacteria producing BSH were surrounded with white halos of free bile acids, which precipitated at acidic pH.
DNA techniques
DNA manipulations were performed by standard proceduresas described previously (Sambrook et al., 1989). DNA frag-ments used in the cloning procedures and PCR productswere isolated from agarose gels with the Qiaquick gel extrac-tion kit (Qiagen) according to the manufacturer’s instructions.Plasmid DNA from E. coli was isolated and purified with aQiaprep spin miniprep kit (Qiagen) or a Qiagen plasmid midikit. Isolation of genomic DNA from Listeria was performedusing the DNeasy tissue kit (Qiagen), starting with digestionof the bacterial cell wall in 4 mg ml-1 lysozyme-containing TEbuffer for 1 h at 37∞C. For PCR on Listeria colonies, bacteriawere microwaved for 4 min at 620 W before adding the PCRreagents.
Mutagenesis
The aminoglycoside 3¢-phosphotransferase gene frompUC4K (Amersham) was amplified by PCR using oligonu-cleotides Kan-KpnI (5¢-GCTCTAGAGCGATTAGAAAAA-3¢)and Kan-XbaI (5¢-TACCCCAAAGCCACGTT-3¢). The KpnI–XbaI PCR product was cloned into the thermosensitiveplasmid pKSV7 (Smith and Youngman, 1992), creatingpOD23. A DNA fragment containing 0.5 kb of the bshupstream sequence was generated by PCR using oligonu-cleotides U50 and L50 (5¢-GGGGTACCCCAAATTTTTCACCTTACAT-3¢). The fragment was cloned into EcoRI–KpnI-digested pOD23, constructing pOD30. A DNA fragmentcontaining 0.5 kb of the bsh downstream sequence was gen-erated by PCR using oligonucleotides U51 (5¢-GCTCTAGAGCCCTTGTCATAGTTTTTTT-3¢) and L51 (5¢-ACCTGCAGGTTAAAACCAACTATTATAA-3¢). The fragment was clonedinto XbaI–PstI-digested pOD30, constructing pOD38. Toachieve allelic exchange, pOD38 was electroporated into L. monocytogenes EGDe at 2500 V, 200 W and 25 mF. Trans-formants were selected at 30∞C on BHI medium containingchloramphenicol (BHI-Cm). One colony was grown in BHI-Cm broth at 43.5∞C, and the culture was plated onto BHI-Cmagar at 43.5∞C. One colony was resuspended in 100 ml ofBHI, and 10 ml of BHI broth was inoculated with 1ml of thissuspension and incubated at 30∞C. BHI broth (10 ml) wasinoculated with 1 ml of a 1:10 dilution of the previous cultureand incubated at 43.5∞C. Tenfold serial dilutions of thisculture were plated onto BHI agar and incubated at 43.5∞C.Colonies were screened onto BHI agar, BHI-Cm agar and BHI agar containing kanamycin. Kanamycin-resistant chloramphenicol-sensitive colonies were analysed by PCRusing oligonucleotides P11 (5¢-TAACTTATACAACGAAGG-3¢) and P12 (5¢-GACGAGTGGATAAATAGC-3¢), which werecomplementary to the sequence between nucleotides 14–31and 928–945, respectively, relative to the BSH start codon.The gene replacement of bsh was verified by Southern blot-ting of genomic DNA from clones that did not lead to ampli-fication of the bsh gene by PCR. Southern blot analysis was
performed using Hybond N+ nylon membrane (Amersham)according to standard protocols (Sambrook et al., 1989). Thebsh gene, used as a probe, was 5¢ end labelled with T4polynucleotide kinase and [g-32P]-ATP (Amersham). Detec-tion was carried out by autoradiography.
Complementation and transformation
A DNA fragment containing the bsh gene and its promoterwas amplified by PCR using oligonucleotides U50 (5¢-AGGAATTCCTTATTGTGGATCCAACTAA-3¢) and L68(5¢-ACAACTGCAGGTTTTGGTTTTTCCTCAC-3¢). The PCRproduct was cut with EcoRI and PstI and cloned into thereplicative plasmid pMK4 (Sullivan et al., 1984), creatingpOD48. Plasmid pOD48 was electroporated into L. monocy-togenes EGDe, L. monocytogenes EGDe Dbsh and L. innocua.
RNA techniques
RNA from Listeria was isolated and purified with the RNeasykit (Qiagen), starting with digestion of the bacterial cell wallin 4 mg ml-1 lysozyme-containing TE buffer for 1 h at 37∞C.Purified RNA was treated with DNase I (Ambion) accordingto the manufacturer’s procedure. The absence of any signif-icant DNA contamination of the RNA was checked by PCR.RT-PCR was performed using Superscript II reverse tran-scriptase and Taq DNA polymerase in a single tube accord-ing to the protocol of the Superscript one-step RT-PCRsystem (Life Technologies). Oligonucleotides P11 (5¢-TAACTTATACAACGAAGG-3¢) and P12 (5¢-GACGAGTGGATAAATAGC-3¢), and oligonucleotides U141 (5¢-TTGCTCTTCCAATGTTAG-3¢) and L142 (5¢-GAGTGCTTAATGCGTTAG-3¢),were used for the cDNA synthesis and PCR amplification ofa 930 bp bsh DNA fragment and a 806 bp rrnA DNA fragmentrespectively.
Primer extension experiments
The bsh transcriptional start point was determined by primerextension analysis using the primer extension system fromPromega. Briefly, oligonucleotide L140 (5¢-TACCCTCGGAGTTTGGAG-3¢), complementary to the sequence betweennucleotides -43 and -60 relative to the BSH start codon, was5¢ end labelled with T4 polynucleotide kinase and [g-32P]-ATP(Amersham). The labelled oligonucleotide was annealed to20 mg of RNA and extended with AMV reverse transcriptasefor 30 min at 42∞C. To determine the size of the extendedproduct, a PCR was first performed on L. monocytogeneschromosomal DNA using oligonucleotides U50 (5¢-AGGAATTCCTTATTGTGGATCCAACTAA-3¢) and L133 (5¢-AAAATAGTGATCCTTCGTTG-3¢). Then, the PCR product corre-sponding to the region upstream from the bsh gene andoligonucleotide L140 were used to generate a sequencingladder by the dideoxy-chain termination method with theSequenase DNA polymerase (Sequenase PCR productsequencing kit; Amersham). The primer extension productand the sequencing ladder were loaded onto a 8% polyacry-lamide sequencing gel and run for 3 h at 1200 V.
Northern blot analyses
Quantitative detection of RNA was carried out using theNorthernmax-Gly glyoxal-based system (Ambion). Briefly, 1 mg of RNA was denatured in glyoxal, loaded onto a 1%agarose gel and run at 5 V cm-1. The RNA was transferred toa positively charged nylon membrane by downward transferfor 2 h and cross-linked for 3 min at 312 nm. The blot was prehybridized in Ultrahyb buffer (Ambion) for 30 min at 42∞C.Approximately 500 ng of DNA corresponding to the bsh geneobtained by PCR was labelled with psoralen-biotin for 45 minat 365 nm according to the Brightstar psoralen-biotin proto-col (Ambion). Labelled DNA probes were denatured by heattreatment and added to the blot at a final concentration of ª 1 ng ml-1 in Ultrahyb buffer. The blot was hybridizedovernight at 42∞C, washed, and the biotinylated probes were detected by chemiluminescence using the Brightstarbiodetection kit according to the manufacturer’s procedures(Ambion).
Sensitivity to bile salts and bile
Listeria were grown to log phase in BHI broth at 37∞C. Cultures were diluted in BHI, and ª 5 ¥ 103 bacteria ml-1 werechallenged with increasing concentrations of bile salts andbile in microtitre plates. Plates were incubated at 37∞C inaerobic or microaerophilic conditions. MIC was determinedby assessing bacterial growth at 600 nm using a spec-trophotometer (Multiskan RC; Labsystems).
Animal studies
Oral infections were performed as described previously(Lecuit et al., 2001). Briefly, 300 g male Hartley guinea pigs(Charles River) were starved for 2 days and anaesthetized(15 mg ml-1 ketamine injected intramuscularly). Five millilitresof 25 mg ml-1 CaCO3 in PBS was injected intragastrically fol-lowed by 1 ml of a sublethal bacterial inoculum, ª 1010 cfu.Stool specimens were recovered at 24, 48 and 72 h afterinfection. Stool pellets were weighed, homogenized in 8 ml ofsterile BHI broth, serially diluted in BHI broth and plated onOxford agar (Oxoid) for quantification of Listeria.
Fifty per cent lethal dose (LD50) experiments were per-formed by injecting 4- to 6-week-old specific pathogen-freefemale BALB/c mice (Charles River) intravenously with 0.3 ml of serial dilutions of bacteria.
Bacterial growth in vivo was studied by injecting mice intra-venously with ª 104 cfu. At 24, 48 and 72 h after infection, thenumber of cfu was determined by plating serial dilutions oforgan homogenates on BHI agar medium.
Acknowledgements
We thank Michael Kuhn for providing L. monocytogenesEGDe DprfA, Michel Doumith, Paul Martin and ChristineJacquet for sharing unpublished results and providing Liste-ria type strains. This research was supported by InstitutPasteur, the Ministère de l’Education Nationale, de laRecherche et de la Technologie (Programme de RechercheFondamentale en Microbiologie et Maladies Infectieuses
et Parasitaires) and by the European Commission (contractQLG2-CT 1999-00932). P.C. is an international scholar fromthe Howard Hughes Medical Institute.
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