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dure of Filip et al. (11). Proteins ..... 54:118-125. 11. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. ... enteritis in man and domestic animals. J. Am. Vet. Med.

Vol. 60, No. 9

INFECrION AND IMMUNrry, Sept. 1992, p. 3872-3877

0019-9567/92/093872-06$02.00/0 Copyright © 1992, American Society for Microbiology

Iron Acquisition and Hemolysin Production by Campylobacter jejuni CAROL L.

PICKETT,* TROY AUFFENBERG, EVERETT C. PESCI, VERA L. SHEEN, AND SRI S. D. JUSUF

Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0084 Received 26 March 1992/Accepted 19 June 1992

Campylobacterjejuni strains were tested for their ability to acquire iron from various iron sources present in humans. Hemin, hemoglobin, hemin-hemopexin, and hemoglobin-haptoglobin stimulated the growth of C. jejuni strains in low-iron medium. Transferrin, lactoferrin, and ferritin were unable to provide iron to the strains tested. Derivatives of the naturally transformable C. jejuni strain 81-176 were isolated on the basis of their inability to use hemin as an iron source. These mutants were also unable to use hemoglobin, heminhemopexin, or hemoglobin-haptoglobin as iron sources. Some mutants lacked a 71,000-Da iron-regulated outer membrane protein, while others appeared to retain all of their outer membrane proteins. Growth curves and a recombination experiment that exploited natural transformation were used to further characterize the mutants. A hemolytic activity was shown to be produced by several C. jejuni strains, but it did not appear to be iron regulated. Campylobacter jejuni is a gram-negative, microaerobic member of the family Spirillaceae (35). Although C. jejuni has been known for several years to be a cause of epizootic infectious abortion in sheep and to be associated with enteritis in calves and lambs, it is only in recent years that C. jejuni has become recognized as a frequent cause of diarrheal disease in humans (4, 26, 35). The severity of the disease and the observed symptoms in humans are variable, with stools ranging from mildly to very watery and often accompanied by blood (5, 42). The underlying reasons for this variability have not been conclusively demonstrated, but the immune status of the host is known to be important (42). Potential virulence factors include an enterotoxin, cytotoxins, and invasive capabilities, but the contribution of these to C jejuni pathogenesis has not been formally demonstrated (12, 15, 16, 22, 30, 42). The ability of pathogenic bacteria to acquire iron in the animal host has been shown to be of critical importance in establishing infection (2). Since there is essentially no free iron in humans, pathogenic bacteria must acquire it from one or more iron-containing compounds. Heme has been shown to provide iron to several different pathogens, including Neisseria gonorrhoeae, Haemophilus influenzae, Shigella flexneri, Yersinia pestis, and Vibrio cholerae (19, 23, 24, 39, 40). V. cholerae, Y pestis, H. influenzae, and some Neisseria isolates can also use hemoglobin as an iron source (23, 37, 39, 40). In addition, some heme-utilizing pathogens can also obtain heme (iron) from the serum heme-binding protein hemopexin (8, 13, 37). The mechanism by which iron (or heme) is acquired from these complexes is not understood, although it is known that the process does not require siderophores (21, 24, 39). The biosynthesis of siderophores by heme- or hemoglobin-utilizing bacteria apparently is associated with another iron acquisition system that requires siderophores. Transferrin, lactoferrin, and ferritin are other possible sources of iron in humans. Several species of

*

bacteria have been shown to use one or more of these to supply their required iron (19, 23, 27, 32, 36). It is not known what compounds in humans provide iron to C. jejuni. Because of this lack of knowledge, because C. jejuni is not closely related to any of the pathogens for which iron sources have been identified (18), and because it has been reported that iron enhances the disease-causing ability of C. jejuni in an animal model (38), we have undertaken a study of the iron acquisition systems of C. jejuni. MATERIALS AND METHODS Bacterial strains, media, and chemicals. C. jejuni 81-176, C31, and 1376 have been described (3, 10, 12, 17). Additional C. jejuni strains mentioned in this study were isolated from humans with diarrheal disease (1, 10). C. coli D115 and D126 were isolated from human stool samples (1). The C. jejuni mutant strains TA102, TA205, TA213, TA214, and TA218 were derived from strain 81-176 during the course of this study; they appear to be defective in their ability to use hemin as an iron source. C jejuni strains were stored at -70°C. C. jejuni routinely passaged once on brucella agar (Difco

cells were Laboratories, Detroit, Mich.) containing trimethoprim, vancomycin, and cephalothin at 5, 10, and 15 ,ug/ml, respectively, and subsequently either used immediately or passaged a minimum number of times on brucella agar without antibiotics (BA). Cultures were grown at 42°C in a microaerobic atmosphere of 5% 02, 10% CO2, and 85% N2. Campylobacter defined medium (CDM) was prepared as described by Tenover et al. (41), except that Fe(NO3)3 was ommitted. Additions to CDM were made for certain experiments and are described in the text. Ethylenediamine di-o-hydroxyphenylacetic acid (EDDA; Sigma Chemical Co., St. Louis, Mo.) was deferrated by the procedure of Rogers (28). Desferal (desferrioxamine B) was purchased from Ciba-Geigy. Preparation of iron reagents. All reagents were obtained from Sigma Chemical Co. except for hemopexin, which was kindly provided by Ursula Muller-Eberhard. Hemin and hemoglobin solutions were prepared fresh daily as described

Corresponding author. 3872

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by Dyer et al. (8). Complexes of hemin and hemoglobin with their respective serum-binding proteins, hemopexin and haptoglobin, were also prepared as described by Dyer et al. (8). Human transferrin and lactoferrin were deferrated by the method of Simonson et al. (34). Ferric citrate was then added to deferrated transferrin or lactoferrin to achieve iron saturation levels of 30 or 100% (7). Iron utilization assays. The ability of C. jejuni to use iron-containing compounds was tested by a method similar to that described by Field et al. (9) for testing the effect of siderophores on the growth of C. jejuni. Assay plates consisted of 20 ml of BA containing 55 FM EDDA and 5 x 107 CFU of the C jejuni strain being tested. Freshly made plates were incubated for 2 h, and then 10 ,ul of various ironcontaining compounds was spotted directly onto the plates. The plates were then incubated overnight, and the spot area was examined for growth. Transferrin and lactoferrin were tested in the plate assay at 30 ,uM each. Ferritin was used at 22 ,uM. Hemin was tested at concentrations ranging from 10 ,uM to 1 mM, and hemoglobin was tested at concentrations ranging from 4 to 600 ,uM. Complexes of hemin plus hemopexin and hemoglobin plus haptoglobin were prepared to 50% saturation. Hemopexin was tested at a concentration of 200 ,uM, and haptoglobin was tested at a concentration of 100 ,LM. Mutant isolation. Ethyl methanesulfonate mutagenesis and streptonigrin enrichment were performed in a manner similar to that described previously for the isolation of iron transport mutants (7, 44). A 1:1 solution of ethyl methanesulfonate in dimethyl sulfoxide was added to a final concentration of 0.5% (vol/vol) to early-exponential-phase brucella broth cultures of strain 81-176. After 60 min of incubation with gentle shaking, the ethyl methanesulfonate was removed by washing the cells three times in brucella broth. The cells were finally resuspended in an amount of brucella broth equivalent to the original culture and incubated overnight. Following overnight incubation, the cells were washed once, suspended in CDM without added iron, and used to inoculate fresh CDM medium in which hemin (5 ,uM) was the sole iron source. These CDM-hemin cultures were incubated for 6 h, and then streptonigrin was added to a final concentration of 0.2 ,ug/ml. After 60 min, the streptonigrin was removed by washing the cells three times with 100 mM Tris, pH 8.0, and the cells were immediately plated on complete CDM. All survivors were purified on complete CDM and subsequently screened for the inability to use hemin as an iron source on CDM containing 5 ,uM hemin and 55 ,uM EDDA. Growth curves. Strains were grown overnight on BA plates. The cells were suspended in CDM and used to inoculate Erlenmeyer flasks containing the same medium. After overnight growth in CDM, the bacteria were harvested and resuspended in CDM-D (CDM containing 10 FM Desferal, an iron chelator which C. jejuni cannot use [9]). These bacteria were then used to inoculate the growth curve flasks (20-ml cultures in 250-ml Erlenmeyer flasks) to an initialA560 of 0.4 to 0.5. Each strain was grown in CDM-D, CDM-D containing ferrous sulfate (20 ,uM), and CDM-D containing hemin (5 ,uM). All flasks were placed in a GasPak (BBL, Cockeysville, Md.) filled with 5% 02, 10% CO2, and 85% N2 and incubated with gentle shaking (100 rpm). Membrane isolation and SDS-PAGE. Membranes were isolated from cells grown on BA containing either 11 ,M EDDA (mutant strains, low iron) or 33 ,M EDDA (strain 81-176, low iron) or on BA (all strains, high iron). The cells were harvested and then sonicated, and membranes were pelleted by the method of Inouye and Guthrie (14). Outer

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TABLE 1. Stimulation of C. jejuni growth by different iron sources Growthb

Iron sourcea

Hemin Hemoglobin

Lactoferrin Transferrin Ferritin Hemin-hemopexin Hemoglobin-haptoglobin

1376

81-176

++

++

++ + +

++ + +

TA102C

NDd ND ND

a The lowest hemin concentration that consistently stimulated growth of all C. jejuni strains tested was 100 A.M. The lowest hemoglobin concentration that produced a similar result was 25 AM. The lowest ferrous sulfate concentration that stimulated growth was 1 mM. b Diameter of growth: -, no growth; +, 10 to 12 mm; ++, >15 mm. c TA205, TA213, TA214, and TA218 were also negative. d ND, not determined.

membrane proteins were obtained by the Sarkosyl procedure of Filip et al. (11). Proteins were solubilized and analyzed by sodium dodecyl sulfate-12% (wt/vol) polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (20). Hemolysin assay. The contact hemolysin assay described by Sansonetti et al. (31) was used with slight modification to test several C jejuni strains for hemolysin production. Briefly, C. jejuni cells were harvested, washed in phosphatebuffered saline (PBS), and resuspended to a concentration of 5 x 108 bacteria per ml. Aliquots of washed cells were mixed with an equal volume of sheep erythrocytes (washed and resuspended in PBS to 109 cells per ml) and centrifuged to produce a pellet of sheep erythrocytes covered with a layer of bacteria. The pellets were incubated microaerobically at 42°C for 4 h. After that time, they were resuspended and cold PBS was added, and after recentrifugation, the A54 of the supernatant fraction was determined. Control tubes were treated identically except that no bacteria were added. DNA isolation and natural transformation. Whole-cell Campylobacter DNA was isolated by the bacterial DNA isolation method described by Silhavy et al. (33). Natural transformation experiments were carried out by the procedure of Wang and Taylor (43) with only slight modification. Derivatives of strain 81-176 deficient in heme acquisition were grown overnight on BA and then suspended in brucella broth, and 108 cells were spread on the surface of a BA plate containing 55 ,uM EDDA and 20 ,uM hemin. The plates were dried briefly, and 0.25 ,ug of undigested C. jejuni DNA was spotted on the plates. Plates were incubated for 24 to 48 h and then examined for colonies within the spot. When colonies within the spot were picked and restreaked onto the same medium, they grew well, indicating stable acquisition of the wild-type genes. RESULTS Iron acquisition assay. The ability of C. jejuni strains to acquire iron from various iron- or heme-containing compounds was tested in a plate assay. C. jejuni cells were seeded into a low-iron solid medium in which they could not grow unless provided with an iron source. Hemin, hemoglobin, hemin-hemopexin, and hemoglobin-haptoglobin stimulated growth, whereas ferritin, transferrin, and lactoferrin did not (Table 1). Several additional C. jejuni strains (G13,

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B1B

A

B

C

D

68-

A560

0.1

43 -

0.1 r-

0

5

10

15

0

20

10

15

20

D

r

1

5

FIG. 2. SDS-PAGE of outer membrane proteins. (A) Strain 81-176 grown in high-iron medium; (B) 81-176 grown in low-iron medium; (C) TA205, high-iron; (D) TA205, low-iron. The arrow indicates the location of the missing 71,000-Da protein in strain TA205. Molecular sizes (in kilodaltons) of protein standards are indicated at the left.

A560

0.1

0.1

05 10 v1

0 5 10 15 20 25 10 15 20 25 Time (hours) Time (hours) FIG. 1. Growth curves of strain 81-176 and the heme utilization mutants. Each strain was grown in CDM-D supplemented with either ferrous sulfate (0) or hemin (V) or without an added iron source (0). (A) 81-176; (B) TA102; (C) TA213; (D) TA218. 0

5

G14, 289504, C31, 79-101, 79-193, 85-360, and 85-371) and two C. coli strains were tested for their abilities to utilize hemin and hemoglobin as iron sources, and all responded essentially as shown for strains 81-176 and 1376. Mutant isolation and characterization. Strain 81-176 was chosen for further study because its disease-causing potential for humans has been documented and because it is naturally transformable (3, 25). Derivatives of 81-176 that were unable to use hemin as an iron source were isolated by a chemical mutagenesis and enrichment procedure. The five mutants that were consistently unable to grow on BA-EDDA supplemented with hemin were studied further. These mutants, TA102, TA205, TA213, TA214, and TA218, were tested for the ability of heme-containing compounds to stimulate their growth in the plate assay. The mutants were not stimulated by any of the heme-containing compounds tested (Table 1). Higher concentrations of hemin (up to 1 mM) and hemoglobin (up to 300 ,uM) also failed to permit growth of the mutants. The ability of the mutants to be stimulated by hemin or ferrous sulfate was further examined by growing the strains in various CDM-D liquid media. Following growth on BA plates, all of the strains were grown overnight in CDM without added iron. These cells were then used to inoculate the growth curve flasks containing CDM-D without supplement or CDM-D supplemented with ferrous sulfate or hemin. Preliminary experiments indicated that overnight growth in CDM provided enough iron to ensure growth of the mutants in the subsequent CDM-D medium without providing so much iron that internal iron stores prevented differentiation of the mutants during growth in CDM-D. All of the mutant strains showed little or no stimulation by hemin (Fig. 1). However, two different growth patterns were exhibited by the five mutants. One pattern, illustrated by mutants TA102 (Fig. 1B) and TA205 (not shown), was only slightly different from that of the parent, 81-176 (Fig. 1A). These mutants were able to grow in the iron-restricted medium nearly as well as 81-176 but were not significantly

stimulated by hemin. However, TA213 and TA218 were able to reach a significant level of growth only when supplemented with ferrous sulfate, and they exhibited a lag phase that was longer than that of 81-176 or TA102 (Fig. 1C and D; TA214 was similar to TA213 and is not shown). The mutants were examined for the possible loss of iron-regulated outer membrane proteins. Strain 81-176 and all five mutants were grown on BA (iron replete) and BA supplemented with EDDA (low iron). Outer membrane proteins were obtained and analyzed by SDS-PAGE (Fig. 2). Strain 81-176 appeared to produce two iron-regulated outer membrane proteins in the 70- to 75-kDa range. One of these, with an apparent size of 71 kDa, was missing in profiles from strains TA102, TA205, and TA218 (Fig. 2, lane d, for TA205; TA102 and TA218 were identical to TA205 and are not shown here). The outer membrane proteins of TA213 and TA214 did not appear to be different from those of 81-176 (data not shown). Outer membrane proteins were also prepared from 81-176 grown in CDM-D or CDM-D supplemented with 25 ,uM hemin. The profiles obtained were essentially identical to those shown in lanes A and B of Fig. 2, indicating that the presence of hemin in the medium repressed the same proteins that ferrous iron did. The mutants were further classified in an experiment which took advantage of the fact that strain 81-176 is naturally transformable. Whole-cell DNA isolated from either 81-176 or the mutant derivatives was spotted onto low-iron plates containing hemin and seeded with the various mutant strains. Growth was never seen in spots where DNA from a particular mutant was spotted onto the same strain. DNA from 81-176 produced nearly confluent growth when spotted on any of the mutants but produced no growth when spotted onto a plate not spread with bacteria. When DNA from one mutant was spotted onto any other mutant, many colonies appeared within the spot except when TA102 and TA205 were tested together. DNA from TA102 did not lead to colony formation on a plate spread with TA205, and DNA from TA205 did not lead to growth of TA102 (Table 2). Hemolysin activity. Several bacterial species which can use hemin as an iron source produce a hemolysin that is regulated by iron (see, for example, reference 36). It has been reported that C. jejuni is not hemolytic on blood agar (35), and we have also found that when our C. jejuni strains are grown on BA medium supplemented with 10% sheep blood, zones of hemolysis are not apparent around colonies. However, when a contact hemolysin assay was used to test for hemolysin production, hemolytic activity was detected in several strains (Table 3). No evidence for iron regulation of this activity could be detected; the level of hemolysin production was not significantly different when cells were

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TABLE 2. Natural transformation: restoration of growth on heme Growtha promoted by DNA from strain:

Recipient strain

81-176

TA102

TA205

TA213

TA214

TA218

TA102 TA205 TA213 TA214 TA218

+ + + + +

+ + +

+ + +

+ + +

+ + + -

+

+

+ + + + -

a +, > 10 colonies within the spot of DNA; -, no colonies within spot. The frequency of transformation of 81-176 and derivatives was 2 x 10-' per cell, or 2 x 102 per ,ug of 81-176 DNA.

harvested from BA, from BA supplemented with 11 ,uM EDDA (Table 3), or from BA containing up to 100 ,uM hemin or 100 ,uM ferrous sulfate (data not shown). Also, the mutant strains were unaffected in hemolysin production. However, some C jejuni strain variation was apparent; some strains produced little or no hemolytic activity in this assay (Table 3). DISCUSSION We have shown that C. jejuni readily obtains iron from hemin and hemoglobin. All strains of C. jejuni that we tested had this capability, although some minor strain variation in the extent of hemin or hemoglobin stimulation was observed in the spot assay. Two strains of C. jejuni were tested for their ability to acquire iron from hemin-hemopexin and hemoglobin-haptoglobin, and both were capable of using these complexes as iron sources. Whether the intact hemin molecule or only the iron is taken up is not yet known. C. jejuni is capable of synthesizing its own heme (41), but it might be advantageous to assimilate heme directly. Initial characterization of the iron (heme) acquisition mutants suggested that at least four gene products are required for iron acquisition. The results of the natural transformation experiment should be interpreted with caution, but they indicate that the mutations in TA213, TA214, and TA218 are far enough away from the mutations in TA102 and TA205 and from each other that frequent recombination can occur between them. Therefore, the mutations in TA213, TA214, TA218, and TA102 (or TA205) may be TABLE 3. Hemolytic activity of C. jejuni strains grown on high- and low-iron media Hemolytic activity (A40) Low-iron medium

Strain

High-iron medium

79-193 85-360 85-371 1376 81-176 TA205 TA214 TA218

0 0

1.08 0.52 0.70 0.81 0.58 0.85

± ± ± ± t t

0 0

0.12 0.05 0.09 0.06 0.10 0.16

0.97 ± 0.09 0.44 + 0.04 0.54 t 0.06 0.77 ± 0.08 0.63 ± 0.06 0.93 t 0.04

a Hemolytic activity is expressed as the A540 of supernatant solutions. Complete hemolysis had a calculated A540 of 9.6. All values shown represent the mean and standard deviation of at least triplicate assays from a typical experiment. All experiments were repeated at least twice, and similar results were obtained each time. High-iron medium is BA, and low-iron medium is BA supplemented with 11 ,uM EDDA.

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located in different genes. Alternatively, these mutations may lie within the same gene, but their locations must be sufficiently separated to permit frequent recombination. TA218 grew like TA213 and TA214, but, in common with TA102 and TA205, it lacked the 71-kDa protein. DNA from TA218 readily restored the wild-type phenotype when used in the natural transformation experiment with all of the other mutants. Therefore, TA218 may not be a double mutant with a mutation in the 71-kDa protein gene and in one of the same gene(s) affected in TA213 and TA214. Perhaps the mutation in TA218 exerts a polar effect that eliminates more than one product but still easily allows recombination between the defect in TA218 and the mutations in the other mutant strains. Strains TA102 and TA205 were isolated in separate mutagenesis experiments, but the mutations in these strains are likely close to each other and may lie in the same gene, since the DNA from one strain did not restore the other to a wild-type phenotype in the transformation

experiment. Three of the mutants (TA102, TA205, and TA218) lack the iron-regulated 71-kDa outer membrane protein. It is therefore likely that this protein is involved in the acquisition of iron (heme) and that the gene that encodes this protein is defective in at least some of these mutants. The synthesis of this 71-kDa protein appeared to be repressed in cells grown on plates containing either iron or hemin. A likely interpretation of this result is that the protein is repressed when intracellular iron levels are above some critical level, regardless of the external source of the iron. Alternatively, both iron and heme may repress the synthesis of this protein. Iron acquisition spot assays of the mutants showed that in addition to being unable to utilize heme, they were all also incapable of using hemoglobin, hemin-hemopexin, or hemoglobin-haptoglobin as iron sources. This result implies that iron acquisition from these compounds occurs by a common pathway but does not eliminate the possibility that iron acquisition from compounds other than heme might also require additional functions that act at an earlier transport stage than the functions that are defective in these mutants. The growth studies reinforce our conclusion that these mutants are defective in hemin utilization. Neither 81-176 or the mutants attained a high level of growth in these experiments, but C. jejuni does not grow well in liquid medium unless high inocula, rich medium, and good microaerobic conditions are used (29). We were able to satisfy the last of these requirements, but owing to the nature of our experiment, we chose not to provide a rich medium or a higher inoculum. Three mutants (TA213, TA214, and TA218) were severely restricted in growth unless ferrous sulfate was added to the medium. TA102 and TA205 were not as deficient but nonetheless were apparently unable to efficiently use hemin. Why these mutants exhibited such different growth characteristics is unclear and requires further investigation into the exact nature of the mutations and the affected gene(s). The data presented here indicate that C. jejuni can make a hemolysin. This activity was detected in a manner similar to that used to detect hemolysins in other bacteria that do not appear to make hemolysins when plated on media supplemented with blood (29). The level of hemolysis by C. jejuni detected here is in the range of that reported for Shigella flexneri (6, 29). Hemolysin production was strain dependent, with some strains producing little or no hemolysin. Unlike the hemolysins produced by some heme-acquiring bacteria, the C. jejuni hemolysin activity appeared not to be

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regulated by iron, since neither low-iron- nor high-ironsupplemented growth affected hemolysin production. This suggests that the hemolytic activity may be constitutively expressed and may play a role in C. jejuni pathogenesis other than, or in addition to, iron acquisition. ACKNOWLEDGMENTS We thank A. O'Brien, M. Blaser, C. Patton, S. Payne, and R. Guerrant for providing bacterial strains and U. Muller-Eberhard for the gift of hemopexin. D. Cottle provided technical assistance. This work was supported in part by the University of Kentucky Medical Center Research Fund and by National Institutes of Health grant AI-27908.

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