Vol. 32, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1994, p. 32-390095-1137/94/$04.00+0Copyright C) 1994, American Society for Microbiology

Comparison of Siderophore Production and Utilization inPathogenic and Environmental Isolates of

Yersinia enterocoliticaCATHERINE E. CHAMBERS AND PAMELA A. SOKOL*

Department ofMicrobiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary,Alberta, Canada T2N 4N1

Received 12 May 1993/Returned for modification 13 July 1993/Accepted 11 October 1993

Yersinia enterocolitica strains of serotypes lethal to mice have been reported previously to produce anendogenous siderophore. In this study, an ethyl acetate-extractable siderophore was characterized and giventhe name yersiniophore. Yersiniophore was produced by 16 of 16 human isolates of serotypes 0:4, 0:4,32, 0:8,0:21, and one nonhuman isolate of serotype 0:21. It was not produced by isolates of serotype 0:3, 0:5, or 0:9.One strain of Yersinia pseudotuberculosis produced yersiniophore, but strains of Yersinia kristensenii, Yersiniafrederiksenii, and Yersinia intermedia did not produce or utilize yersiniophore. Food and water isolates of Y.enterocolitica produced a water-soluble siderophore but not yersiniophore. Sixty-two strains of Y. enterocoliticaincluding 42 isolates from human infections, 2 animal isolates, and 18 water and food isolates were examinedfor utilization of yersiniophore, the water-soluble siderophore, and ferrioxamine. Yersiniophore promotedgrowth rate, iron binding, and uptake in 17 of 62 strains, all of which produced yersiniophore. Ten of 17 foodand water isolates and one human isolate were capable of utilizing the water-soluble siderophore. Utilizationstudies suggest that at least one additional water-soluble siderophore may be produced. Ferrioxaminepromoted the growth of 60 of 62 strains examined; however, only the 17 strains which produced yersiniophoreactively accumulated [59FeJferrioxamine. Yersiniophore production and utilization may be important inclinical infections since all human strains belonging to serotype 0:8 produced yersinophore. The water-solublesiderophore was not detected in human isolates.

Yersinia enterocolitica is an invasive bacterial pathogenrecognized as an important cause of enteritis in small chil-dren. Septicemia has also been reported (5, 16); however,this complication is rare and is generally associated with anunderlying disorder. In a review of 100 cases of Y entero-colitica septicemia, Larigakis et al. (16) reported that 30% ofsepticemic patients suffered from disorders of iron metabo-lism such as siderosis, thalassemia, and hemochromatosis,while 80% had underlying disorders such as cirrhosis, dia-betes mellitus, and renal failure.Y enterocolitica strains are classified into more than 50

serotypes on the basis of 0 antigenic factors (4). The mostcommon serotype worldwide is 0:3 of biogroup 4. Serotypes0:9 and 0:5,27 of biogroup 2 are frequently isolated inEurope, Japan, southern Africa, and China, whereas sero-type 0:8 is the predominant type in the United States.Serotypes 0:8, 0:4, 0:13, 0:20, and 0:40 have been re-ported to be lethal to mice (14, 24).To establish an infection, pathogenic bacteria must com-

pete with the host to acquire iron essential for their growth(28). Most bacteria acquire iron from the host by secretinghigh-affinity iron-binding factors known as siderophores(20). Siderophore production has been reported for someYersinia species. In 1975, Wake et al. reported indirectevidence of siderophore production in Yersinia pestis bydemonstrating that certain strains which grew on deferratedbrain heart infusion agar could promote the growth of other

* Corresponding author. Mailing address: Dept. of Microbiologyand Infectious Diseases, University of Calgary Health SciencesCentre, Calgary, Alberta, Canada T2N 4N1. Phone: (403) 220-6037.Fax: (403) 283-8814. Electronic mail address: PSokol@acs.ucalgary.ca.

Y pestis strains on this medium (27). However, Perry andBrubaker (21) were unable to detect siderophore productionin Y pestis by using the standard chemical assays. Y pestiswas also unable to complement an enterochelin-deficientmutant of S. typhimurium LT2 enb-7, which is capable ofutilizing numerous exogenous siderophores (21). Heese-mann reported siderophore production in Y enterocoliticadetectable with the Chrome Azurol S (CAS) assay (13).Siderophore production was detected in strains of serotypes0:8, 0:13, 0:20, and 0:40 but not in serotypes which areless virulent in the septic mouse model (13). The siderophoreproduced by these serotypes was not characterized further.Environmental isolates of Yersinia frederiksenii, Yersiniaintennedia, and Yersinia kristensenii have been reported toproduce the siderophore aerobactin (26).

Patients with iron overload are susceptible to infectionbecause of the increased concentration of iron in the blood(28). Treatment with a therapeutic chelator can removeexcess iron (17) and render it unavailable to most potentialpathogens. Desferrioxamine B is a trihydroxymate sidero-phore produced by Actinomyces species (20). The methanesulfonate salt of this compound is licensed and commerciallyavailable as Desferal, which is used therapeutically as aniron chelator for patients with iron overload (17). Y entero-colitica septicemia has been reported in patients treated withDesferal (5). Y enterocolitica has been shown to utilizeferrioxamine to acquire iron in vitro (7) and in vivo (23).Intraperitoneally administered Desferal has been shown toreduce the median lethal dose of Y enterocolitica strains ofserotypes 0:3 and 0:9 by 105 CFU/ml in a mouse septicmodel (23). The median lethal dose of a serotype 0:8 strain,which is normally more virulent than serotypes 0:3 and 0:9,was also reduced but only by 10- to 100-fold.

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SIDEROPHORES IN YERSINLA ENTEROCOLITICA 33

The utilization of siderophores requires the presence ofiron-regulated membrane receptor proteins (19). Recently,the gene for the ferrioxamine B receptor (foxA4) was clonedand sequenced from Y enterocolitica (2). Carniel et al. havedescribed several iron-regulated membrane proteins includ-ing two high-molecular-mass proteins (HMWP) of 190 and240 kDa (9). The two HMWP were expressed by virulentserotypes of Y pestis, Yersinia pseudotuberculosis, and Yenterocolitica serotypes, which are lethal in mice (10), butnot by serotypes 0:3 and 0:9, which are not virulent in micein the absence of desferrioxamine.

In this study, we have characterized and compared sidero-phore production and utilization in 62 clinical and environ-mental isolates of Y enterocolitica. We have identified twodifferent siderophores produced by Y enterocolitica isolatesand have characterized ferrioxamine utilization in thesestrains. These studies indicate that there are at least threecommon mechanisms of iron acquisition in Y enterocolitica.

MATERIALS AND METHODS

Strains. Sixty-two Y enterocolitica isolates from food,water, and patients with yersiniosis were generously pro-vided by C. H. Pai, Department of Clinical Pathology, AsianMedical Centre, Seoul, Korea. The majority of these strainswere obtained from S. Toma, The National Reference Cen-tre for Yersinia, Laboratory Services Branch, Toronto,Ontario, Canada. These strains have been examined previ-ously for heat-stable enterotoxin production and the abilityto penetrate HeLa cells and evoke keratoconjunctivitis inguinea pigs (Sereny test) (18). Y pseudotuberculosis YPT01,a chinchilla isolate, was provided by A. Potter of theVeterinary Infectious Disease Organization, Saskatoon,Saskatchewan, Canada. Y pseudotuberculosis ATCC29833, Y intermedia ATCC 29909, Y frederiksendi ATCC33642, and Y kristensenii ATCC 33638 were obtained fromthe American Type Culture Collection, Rockville, Md. Sal-monella typhimurium enb mutant strain was obtained fromA. Schryvers, University of Calgary.

Culture conditions. Cultures were maintained on L agar(Difco Laboratories, Detroit, Mich.). For the production ofsiderophore and iron-regulated proteins, cultures weregrown in Yersinia minimal medium (YMM) as described byCamiel et al. (9) but with the addition of 0.4 mM MgSO4.Cultures were made iron rich with 150 ,uM FeCl3 and ironpoor with 50 FM 2,2'-dipyridyl (Sigma Chemical Co.).Cultures were grown initially in peptone broth (Bacto Pep-tone; Difco) for 24 h at 28°C and washed once in 0.5 volumeof sterile water (4°C). These cultures were used to inoculateYMM to anA6. of 0.1, and the medium was incubated for 36to 48 h at 28°C. Growth promotion was examined onagarose-solidified minimal medium supplemented with ferricsiderophore. YMM agarose plates were prepared with 200FM 2,2'-dipyridyl. Plates were supplemented with 100 nmolof ferric siderophore spread on the surface. Ten-microliteraliquots of a 0-4 dilution of the peptone broth cultures wereapplied to plates supplemented with ferric siderophore andincubated for 48 h at 28°C. Plates without siderophoresserved as negative controls. All glassware was acid washed,and all reagents were prepared with water purified by theMilli-Q System (Millipore Corp., Bedford, Mass.). Cultureswere screened for the virulence plasmid by using an alkalinelysis procedure (3).

Detection of siderophores. The CAS assay (25) with thefollowing modifications was used to detect siderophoreproduction. YMM was substituted for the minimal salts. Ten

milliliters of 20% Casamino Acids and 10 ml of 10% fructosewere added per liter of agar after autoclaving. The dyecomplex of CAS (Sigma) and hexadecyl-trimethylammo-nium bromide (Fisher Scientific) was prepared as describedoriginally. CAS assay solution (25) was used to detectsiderophores by mixing 0.5 ml of culture supernatant fluidwith 0.5 ml of CAS solution. The reaction was allowed toreach equilibrium for 6 to 18 h before theA630 was measured.

Sixty-two strains were screened for yersiniophore produc-tion by thin-layer chromatography. Ethyl acetate or dichlo-romethane extracts (20 ml) of 50-ml cultures were dried byrotary evaporation, and the residue was resuspended in 500,ul of methanol (MeOH). Fifty microliters of the MeOHsolution was chromatographed on a thin-layer Silica Gel Gplate (Analtech, Inc., Newark, Del.) with CHCl3-ethyl alco-hol-acetic acid-ether (90:5:2.5:2.5) as the solvent. A fluores-cent yellow-green band (Rf = 0.26) visible under shortwaveUV light was characteristic of yersiniophore. Water-soluble(WS) siderophore was detected by CAS assay of the aqueousphase after ethyl acetate extraction of culture supernatants.

Siderophore isolation. Yersiniophore was isolated by ex-tracting 1 to 2 liters of culture supematant fluid with dichlo-romethane (2:5 [vol/vol]). The extract was dried by rotaryevaporation, resuspended in

34 CHAMBERS AND SOKOL

itself was negligible. By using this estimate, all ferric sidero-phore solutions were made 20% saturated. It was assumedthat the concentration of free iron under these conditionswould be insignificant.

Iron uptake assays. Cultures were grown in iron-poorYMM to a density of 108 CFU/ml. The cells were harvested,washed once with sterile water, and resuspended in 10 ml ofmedium to a final A6. of 0.2. Cell suspensions were incu-bated for 5 min at 28°C with shaking prior to the addition ofiron. Ferric siderophore solutions were prepared with59FeC13 and allowed to equilibrate at room temperature forseveral hours. The assay was initiated by the addition of 600pmol of 59Fe-siderophore per 10-ml culture. Samples (1 ml)were removed at 0, 2, 5, and 10 min, filtered through0.2-,um-pore-size cellulose acetate filters (Sartorius GmbH,Goettingen, Germany), and washed with 5 ml of Tris-saline(10 mM Tris [pH 7.5], 0.9% NaCl). The amount of 59Feaccumulated on the filters was measured in an LKB Com-pugamma counter. The experiments were repeated threetimes with comparable results.

Solid-phase binding assays. A solid-phase dot assay wasused to examine binding of ferric siderophores to wholecells. Bacteria were grown in iron-rich and iron-poor condi-tions for 48 to 72 h at 28°C. Cells were pelleted andresuspended in 50 mM Tris (pH 7.5). Protein concentrationswere determined by the method of Bradford (6) with acommercially prepared protein assay (Bio-Rad Laborato-ries, Richmond, Calif.). Whole cells were filtered onto anitrocellulose membrane (Bio-Rad) in quantities equivalentto 1 ,ug of protein. The membrane was blocked for 2 h at 37°Cwith 5% bovine serum albumin BSA (Sigma) and reactedwith 50 pmol of 59Fe-siderophore per ml of 10 mM Tris (pH7.5) for 1 h at room temperature. After being washed threetimes with Tris-saline, the membranes were exposed toX-ray film (X-Omat; Kodak, Rochester, N.Y.). Experimentswere performed in triplicate.

Analysis of membrane proteins. Total membranes wereisolated by the method of Carniel et al. (9). Briefly, strainswere grown overnight in peptone broth at 28°C, washed oncein sterile water, and resuspended in either iron-rich oriron-poor YMM to anA6. of

SIDEROPHORES IN YERSINlA ENTEROCOLITICA 35

TABLE 1. Comparison of yersiniophore production, siderophore utilization, and expression of HMWP in Yersinia species'

No. ofisolates No. of isolates Siderophore utilizatione

Strain and serotype Source No. of expressing producing(biotype) isolates HMWP/total Ysp/total no.

no. of of isolates' Ysp Dfx WSisolates'

Y. enterocolitica0:3 (4) Human, extraintestinal 7 0/7 0/7 0/7 7/7 0/70:4 (ND)e Human stool 1 1/1 1/1 1/1 1/1 0/10:4 (ND) Human, extraintestinal 3 3/3 3/3 3/3 3/3 0/30:4,32 (ND) Human, extraintestinal 2 2/2 2/2 2/2 2/2 0/20:4,33 (1) Raw milk 2 0/2 0/2 0/2 2/2 2/20:5 (1) Stool 1 0/1 0/1 0/1 1/1 0/10:5 (1 or 2) Human, extraintestinal 4 0/4 0/4 0/4 4/4 0/40:5 (1) Raw milk 2 0/2 0/2 0/2 2/2 0/20:6,30 (1) Human, extraintestinal 4 0/4 0/4 0/4 4/4 0/40:6,31 (1) Water 1 0/1 0/1 0/1 1/1 0/10:8 (1) Human stool 5 5/5 5/5 5/5 5/5 0/50:8 (1) Human, extraintestinal 4 4/4 4/4 4/4 4/4 0/40:8 (1) Nonhuman 2 0/2 0/2 0/2 2/2 0/20:9 (2) Human stool 1 0/1 0/1 0/1 0/1 0/10:9 (2) Monkey 1 0/1 0/1 0/1 0/1 0/10:14 (1) Food and water 2 0/2 0/2 0/2 2/2 2/20:16 (1) Human stool 4 0/4 0/4 0/4 4/4 0/40:16 (1) Human eye 1 0/1 0/1 0/1 1/1 0/10:16 (1) Water 1 0/1 0/1 0/1 1/1 1/10:17 (1) Human, extraintestinal 2 0/2 0/2 0/2 2/2 1/20:17 (1) Water 1 0/1 0/1 0/1 1/1 1/10:18 (1) Water 1 0/1 0/1 0/1 1/1 1/10:21 (1) Human stool 1 1/1 1/1 1/1 1/1 0/10:21 (1) Human, infected thigh 1 1/1 1/1 1/1 1/1 0/10:21 Nonhuman 4 1/4 1/4 1/4 4/4 1/40:34 (1) Human stool 1 0/1 0/1 0/1 1/1 0/10:34 (2) Water 2 0/2 0/2 0/2 2/2 1/2Summary Human 42 16/42 16/42 16/42 40/42 0/42Summary Nonhuman 20 1/20 1/20 1/20 20/20 9/20

Y pseudotuberculosis YPTO1 1 1/1 1/1 1/1 0/1 1/1Y pseudotuberculosis ATCC 29833 1 0/1 0/1 0/1 0/1 1/1Y kristensii ATCC 33638 1 0/1 0/1 0/1 1/1 0/1Y frederiksenii ATCC 33642 1 0/1 0/1 0/1 1/1 0/1Y intermedia ATCC 29909 1 0/1 0/1 0/1 1/1 1/1

aAbbreviations: Ysp, yersiniophore; Dfx, desferrioxamine; WS, WS siderophore.bExpression of the 190- and 240-kDa HMWP was determined by SDS-polyacrylamide gel electrophoresis of total membrane preparations.Yersiniophore production was determined by thin-layer chromatography of ethyl acetate extract of culture supernatants.

d Siderophore utilization refers to growth promotion on dipyridyl medium and whole-cell binding of '9Fe-labeiled siderophore assays as described in Materialsand Methods.

' ND, not determined.

these siderophores. The ability of clinical and environmentalisolates of different serotypes to use endogenous sidero-phores and desferrioxamine has not been examined previ-ously. In this study, we examined siderophore utilization bydetermining the effects of yersiniophore, ferrioxamine, and aWS siderophore on growth promotion, iron binding, and ironuptake in isolates from a variety of sources. The ability ofyersiniophore, YE1111 WS siderophore, and ferrioxamine topromote growth of 62 Y enterocolitica isolates in iron-restricted medium was determined on YMM agarose supple-mented with 200 ,M 2,2'-dipyridyl and 100 nmol of ferricsiderophore (Table 1). Sixty of 62 isolates grew in thepresence of ferrioxamine. Yersiniophore only promoted thegrowth of those strains which produce yersiniophore (17 of62). YE1111 WS siderophore promoted the growth of 9 of 62strains. Only one of these strains, a urine isolate, was ofhuman origin; the other eight were isolates from water ormilk. Two strains which produced a WS siderophore detect-able by CAS assay did not show growth promotion in the

presence of YE1111 WS siderophore, suggesting that adifferent WS siderophore may be produced by these strains.The growth promotion studies suggested that there may be

a difference in siderophore receptor expression between Yenterocolitica strains of different serotypes and differentsources of isolation. To address this hypothesis, the abilityof each strain to bind 59Fe-labelled siderophores was exam-ined in a whole-cell dot blot binding assay (Table 1). Arepresentative blot of YE1111 WS siderophore binding towhole cells is shown in Fig. 1. Cultures grown in iron-richYMM were used as negative controls. When a dot blot of thesame strains was reacted with 59Fe-yersiniophore, only thefour strains in the top row, UC310, YE7260, YE748, andYE1373, bound yersiniophore (data not shown). When anidentical blot was reacted with [ 9Fe]ferrioxamine, all of thestrains shown with the exceptions of YE1472 and Y pseudo-tuberculosis ATCC 29833 bound ferrioxamine (data notshown). The relative binding of 59Fe-yersiniophore andYE1111 WS siderophore was consistently greater than that

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36 CHAMBERS AND SOKOL

UC310

+

YE 1472

YE1111

YE1 260

YE235

0* *YEl140

YE 148

YE1096

YE.1060

YE1 373

#9YE275

YE1.181'B

w * i .K pI

FIG. 1. Solid-phase binding assay of whole cells reacted with59Fe-WS siderophore. Bacteria were grown in iron-rich and iron-poor conditions for 36 to 48 h. Cells were centrifuged and resus-pended in Tris (pH 7.5). Cells were then filtered in triplicate ontonitrocellulose in concentrations equivalent to 1 p,g protein per dotand blocked with BSA. The membrane was reacted with 50 pmol of59Fe-siderophore per ml for 1 h, washed, and subjected to autora-diography. +, cells grown with iron; -, cells grown without iron; F,Y frederiksenii; K, Y kristensii; P, Y pseudotuberculosis ATCC29833; I, Y internedia. Strains are described in Table 2.

of ferrioxamine (data not shown). The ability of these threesiderophores to promote5growth correlated 100% with theability of strains to bind 5'Fe-siderophore (Table 1).

Siderophore-mediated iron uptake. To determine whetherthere was a correlation between siderophore binding towhole cells and uptake of ferric siderophore, representativeisolates were examined for their ability to accumulate ironcomplexed to the three siderophores (Table 2). Eleven

TABLE 2. Comparison of the ability of siderophores to promotegrowth and iron uptake in representative strains of

Yersinia speciesa

Growth Uptake'Strain (serotype) Source promotion U

Ysp Dfx WS Ysp Dfx WS

Y. enterocoliticaYE235 (0:3) Human - + - - - NDYE619 (0:3) Human - + - - - NDYE1260 (0:4) Human + + - + + NDYE1500 (0:4) Human + + - + + NDYE1171 (0:4,33) Raw milk - + + - - +UC310 (0:8) Human + + - + + -YE420 (0:8) Swine - + - - + NDYE1481 (0:8) Human + + - + + NDYE1472 (0:9) Human . . . . . NDYE748 (0:21) Human + + - + + NDYE1111(0:21) Rawmilk - + + - - +

Y pseudotuberculosis YPT01 + - + + - +ATCC 29833 - - + - - +

Y intermedia ATCC 29909 - + + - - +Y. frederiksenii ATCC 33642 - + - - - -Y k,istensenii ATCC 33638 - + - - + -

a Abbreviations: Ysp, yersiniophore; Dfx, desferrioxamine; WS, WS si-derophore.

b Symbols: +, growth promotion in the presence of siderophore on dipy-ridyl medium;-, no growth.C Symbols: +, iron accumulation in the presence of siderophore; -, no ironaccumulation. ND, not determined.

0 20

I0E

10

o 2 4 6 a 10 12

time (min)

FIG. 2. Promotion of iron uptake in Y enterocolitica by theaddition of desferrioxamine (Dfx), yersiniophore (Ysp), and YE1111WS siderophore (WS). Reaction mixtures contained 108 bacteria perml. Uptake was initiated by the addition of 60 pmol of 59Fe-siderophore per ml. One-milliliter aliquots were removed at theindicated intervals, and the amount of -'Fe accumulated was deter-mined. Symbols: - - -0- - , YE619, with Dfx; - -C---, YE619, withYsp; -*-, YE1111, with Dfx; -A-, YE1111, with WS; -0-,UC310, with Dfx; -l-, UC310, with Ysp; -A-, UC310, withWS. Strains are described in Table 2.

isolates of Y enterocolitica and five strains of the otherYersinia species were examined for yersiniophore- andferrioxamine-mediated iron uptake. Representative Y en-terocolitica isolates are shown in Fig. 2, and the Yersinia

40

30-

iL 20

toS0

E

10

-=- - -- - - - - ---0 2 4 6 6 10 12

time (min)

FIG. 3. Promotion of iron uptake in Y kristensii, Y pseudotu-berculosis, and Y intermedia by the addition of desferrioxamine(Dfx), yersiniophore (Ysp), and YE1111 WS siderophore (WS).Symbols for Y. k,istensii culture additions: -0-, Dfx; -0-,Ysp; -A-, WS. Symbols for Y pseudotuberculosis ATCC 29833culture additions: -*-, Dfx; -U-, Ysp; -A-, WS. Symbolsfor Y internedia culture additions: --O--, Dfx; --0--, Ysp;- -A--, WS. Reactions were performed as described in the legendto Fig. 2. Strains are described in Table 2.

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SIDEROPHORES IN YERSINL ENTEROCOLITICA 37

t 2 3 4 5 6 7 8 9 10 11 12 13 14 15

*

*

*

180

1 006

84

66

48-5

FIG. 4. Expression of iron-regulated outer membrane proteins in Y enterocolitica. Cultures were grown in iron-poor and iron-rich YMMfor 36 to 48 h. Total membrane proteins were isolated and electrophoresed on SDS-7.5% polyacrylamide gels. The 190- and 240-kDa HMWPdescribed by Carniel et al. are indicated by an asterisk; these proteins were coexpressed with three other iron-regulated proteins of 67, 71,and 80 kDa and are indicated by a period in yersiniophore-positive strains. Cultures for lanes 1, 3, 5, 8, 10, 12, and 14 were grown with 50pIM dipyridyl. Cultures for lanes 2, 4, 6, 9, 11, 13, and 15 were grown in medium with 150 p,M FeC13. Lanes: 1 and 2, YE591, 0:5,27, humanappendix; 3 and 4: YE1472, 0:9, human stool; 5 and 6, YE619, 0:3, human abscess; 7, molecular weight markers (sizes are noted on the rightof the figure in thousands); 8 and 9: YE1251, 0:21, water isolate; 10 and 11, YE748, 0:21, water isolate; 12 and 13: YE314, 0:8, nonhumanisolate; 14 and 15, UC310, 0:8, human appendix.

species are shown in Fig. 3. Accumulation of 59Fe-yersinio-phore was seen only in those Y enterocolitica strains whichproduce the yersiniophore (Fig. 2 and Table 2). The strain ofY pseudotuberculosis (YPT01) previously shown to produceyersiniophore also demonstrated yersiniophore-mediatediron uptake (data not shown); however, Y. pseudotubercu-losis ATCC 29833 was unable to utilize yersiniophore (Fig.3). YE1111 WS siderophore-mediated uptake was examinedin Y enterocolitica UC310, YE111, and YE1171 and fivestrains of other Yersinia species (Fig. 2 and 3 and Table 2).Y enterocolitica YE1171 and YE1111, Y intermedia, andboth strains of Y pseudotuberculosis (Table 2) were able totake up iron with this siderophore. The iron accumulationassay results for both endogenous siderophores correlatewith the growth promotion and binding assays in the strainsexamined.The exogenous siderophore ferrioxamine showed a strik-

ing discrepancy between active uptake and growth promo-tion. Two phenotypes for ferrioxamine utilization were seen.One phenotype actively bound and accumulated [59FeJferri-oxamine and used it to promote growth. A second phenotypedid not accumulate ferrioxamine after 30 min, althoughferrioxamine binding and growth promotion were demon-strated. Ferrioxamine uptake was not induced when strainsof the second phenotype were grown in the presence ofpartially iron-saturated desferrioxamine (data not shown). Itshould be noted that although both phenotypes demon-strated growth promotion in the presence of ferrioxamine,the appearance of colonies of clinical isolates of the latterphenotype lagged by 1 or 2 days. All clinical strains exam-ined which belong to serotypes that produce yersiniophorealso accumulated ferrioxamine as demonstrated by UC310 inFig. 2. Yersiniophore-negative serotypes or strains whichproduced a WS siderophore did not accumulate ferrioxamineas demonstrated by YE619 and YE1111.

Correlation of siderophore production with expression ofiron-regulated membrane proteins. Total membrane prepara-tions of 62 clinical, food, and water isolates of Y entero-colitica were examined for the expression of the 190- and240-kDa HMWP (9) on SDS-7.5% polyacrylamide gels. Thetwo HMWP were detectable in all human isolates of sero-types 0:8, 0:4, 0:21, and 0:4,32 and one nonhuman 0:21isolate which was shown to produce yersiniophore. Thirty-four isolates were further examined for iron-regulated pro-teins. An additional three iron-regulated proteins of 80, 71,and 67 kDa were coexpressed with the HMWP (Fig. 4, lanes10 and 14). There was 100% correlation between this patternof iron-regulated proteins (240, 190, 80, 71, and 67 kDa) andthe production of yersiniophore in Y. enterocolitica. One Y.pseudotuberculosis isolate (YPT01) which produces yersin-iophore also expressed the iron-regulated HMWP (data notshown). Non-yersiniophore-producing human isolates ex-pressed iron-regulated proteins of 80 and 71 kDa but did notexpress the 67 kDa protein (Fig. 4, lanes 1, 3, and 5). Totalmembranes of food and water isolates which produce a WSsiderophore were more heterogeneous, although there weretypically three iron-regulated proteins in the 65- to 80-kDarange (Fig. 4, lane 8). Isolates which did not fall into theabove categories had slight differences in iron-regulatedprotein patterns. These strains often expressed one or twoiron-regulated proteins with sizes similar to those alreadydescribed; however, no consistent pattern emerged.

DISCUSSION

It has been known for several years that Y enterocoliticais able to assimilate iron complexed to several exogenoussiderophores, including desferrioxamine B (21). It has alsobeen shown by CAS plate assays that mouse-lethal sero-types of Y enterocolitica secrete an iron-binding compound

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38 CHAMBERS AND SOKOL

(13). In the present study, two distinct endogenous sidero-phores were detected and partially purified. Although thestructure remains to be determined, evidence suggests thatyersiniophore is a siderophore. It binds iron and reacts in theCAS assay. Yersiniophore is iron regulated and stimulatesgrowth of cells under conditions of low iron. It does notstimulate the growth of a Salmonella enb mutant, suggestingthat it requires a receptor not present in Salmonella spp.Further studies are in progress to elucidate the structure ofyersiniophore.

Yersiniophore was produced predominantly by isolatesfrom human infections, whereas WS siderophore(s) wasproduced by nonhuman isolates. Yersiniophore, which isextractable with dichloromethane, was negative for theArnow (1), Rioux et al. (22), and periodate and Csaky (12)assays, suggesting that it may have a unique structure. TheYE1111 WS siderophore was also negative for the chemicalassays described above. This siderophore was not soluble indichloromethane and therefore was easily differentiatedfrom yersiniophore. Since YE1111 WS siderophore wasnegative in the hydroxamate and Csaky assays, it cannot beaerobactin, which is produced by some strains of Y fred-eriksenii, Y internedia, and Y kristensenii (26). Two strainswhich produce a WS siderophore could not use the WSsiderophore from YE1111, suggesting that these strainsproduced a different WS siderophore than YE1111. It ispossible the WS siderophore produced by these strains isaerobactin, although Stuart et al. (26) were unable to detecthydroxymate production in any of 50 Y enterocoliticastrains examined. The siderophore from these two strainswas not purified or further characterized.

Yersiniophore appears to be more clinically significantthan the WS siderophore. Production was found in all humanisolates of serotypes 0:4, 0:4,32, 0:8, and 0:21 (Table 1).No other serotypes were found to produce yersiniophore,although not all serotypes were available for testing. Produc-tion and utilization of the WS siderophore was associatedwith nonhuman isolates. Interestingly, Y enterocoliticastrains produced and utilized either yersiniophore or the WSsiderophore but not both. One strain of Y pseudotubercu-losis was the only strain examined which was capable ofusing both of these siderophores. Many gram-negative bac-teria possess receptors for siderophores which they do notproduce. Y enterocolitica appears to be unique, however,since only strains which produce the siderophores charac-terized in this study appear to have receptors for them.

Ferrioxamine promoted the growth of many strains whichdid not appear to have an active ferrioxamine uptake system.These strains may have an alternative mechanism of acquir-ing iron from ferrioxamine. The assimilation of desferriox-amine-bound iron was reported previously in a strain of Yenterocolitica of unknown serotype by using a 2-h assay (7).In this assay, 5.0 x 109 bacteria were incubated with 1.8nmol of [59Fe]desferrioxamine. After 2 h at 37°C, approxi-mately 2.6 pmol of 59Fe per 108 cells was assimilated, a rateof accumulation which would not be detected by the assayused in our study. Strains of the second phenotype may havea low affinity and/or slower rate of uptake for ferrioxamine.This would also account for the lower growth rate in strainsof this type.

Strains grown in iron-rich medium did not bind any of the59Fe-siderophores, demonstrating that the expression of thereceptor proteins was iron regulated. At least three patternsof iron-regulated proteins have been identified in a compar-ison of 34 total membrane preparations from Y enterocolit-ica isolates resolved on SDS-7.5% polyacrylamide gels (Fig.

4). Two common patterns were observed in human isolates.The first pattern correlated with yersiniophore-producingstrains and strains which accumulated ferrioxamine. Thesestrains expressed iron-regulated proteins of 240, 190, 80, 71,and 67 kDa. The second pattern correlated with clinicalisolates which did not accumulate ferrioxamine. Thesestrains expressed iron-regulated proteins of 80 and 71 kDa.Comparison of these data with those of Baumler and Hantke(2) suggests that the 71-kDa protein, present in all clinicalstrains examined, may be the ferrioxamine receptor. Strainsproducing a WS siderophore were more heterogenous butusually expressed three iron-regulated proteins with a mo-lecular mass between 65 and 80 kDa. The difference inproteins in these strains may correlate with different WSsiderophores.The serotype 0:3 strains examined in this study did not

actively take up ferrioxamine or yersiniophore. Serotype0:3 is the most prevalent serotype worldwide, althoughserotypes 0:8 and 0:9 are also common (4). Human isolatesof serotype 0:3 must have an alternative mechanism of ironacquisition not involving siderophores, or they may usesiderophores provided by other organisms. Yersiniophore-producing serotypes are more virulent in the mouse septicmodel (13), suggesting that this mechanism of iron acquisi-tion may be required for optimal iron acquisition from mice.It appears that several mechanisms may be involved inacquiring iron from human hosts, including yersiniophoreand ferrioxamine. WS siderophores were detected in onlyone human isolate examined, suggesting that these sidero-phores may not be effective in acquiring iron from mamma-lian hosts.

ACKNOWLEDGMENTS

This study was supported by the Canadian Bacterial DiseasesNetwork of Centres of Excellence. P.A.S. is an Alberta HeritageFoundation for Medical Research Scholar.The authors acknowledge J. Dennis and C. Kooi for critical

review of the manuscript.

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