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Response of Haemophilus somnus to iron limitation: expression and identification of a bovine-specific transferrin receptor
Microbial Pathogenesis 1990 ; 9 : 397-406
Response of Haemophilus somnus to iron limitation : expression and identification of a bovine-specific transferrin receptor Julius A . Ogunnariwo, Cindy Cheng, Jeffrey Ford and Anthony B . Schryvers* Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada (Received June 25, 1990 ; accepted in revised form September 5, 1990)
Cgunnariwo, J . A . (Dept of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada), C . Cheng, J . Ford and A . B . Schryvers . Response of Haemophilus somnus to iron limitation : expression and identification of a bovine-specific transferrin receptor . Microbial Pathogenesis 1990 ; 9 : 397-406 . Nine clinical isolates of Haemophilus somnus were screened for their ability to use different transferrins as a source of iron growth . All nine strains were capable of using bovine but not porcine, human or chicken transferrin . A screening assay for siderophore production did not show any evidence of siderophore production by these strains . When iron-deficient cells from these strains were screened for their ability to bind perixodase-conjugated transferrin, binding was detected with conjugated bovine, but not human or porcine transferrin . Competition binding studies demonstrated that the binding of peroxidase-conjugated bovine transferrin was competitively inhibited by unconjugated bovine transferrin but not transferrin from other species . The induction of receptor expression by low iron conditions was inhibited by chloramphenicol and rifampicin but not ampicillin indicating that new protein and mRNA synthesis was required for expression of receptor activity . Affinity isolation of receptor proteins with biotinylated bovine transferrin, but not human or porcine transferrin, yielded three proteins from H. somnus strain H74 . Two of the proteins were identified as 105 kDa and 73 kDa iron-regulated outer membrane proteins . A third protein of 85 kDa that was isolated did not co-migrate with any iron-regulated outer membrane protein . Affinity isolation of receptor proteins from other strains of H. somnus yielded a 73 kDa protein from all strains and a 105 kDa and 85 kDa protein in four of the six strains analysed . Key words : transferrin ; iron acquisition ; receptor ; Haemophilus somnus .
Introduction Haemophilus somnus is an important bacterial pathogen of cattle . It is the causative agent of infectious thromboembolic meningoencephalitis (TEME) and has been associated with other disease syndromes including pneumonia, abortion and infertility .' 3 TEM E tends to occur in herd outbreaks in feedlot cattle' yet the mechanism of disease transmission has not been firmly established . It has been suggested that respiratory tract disease may be a precursor to invasive H. somnus infection .' Although H. somnus can be isolated at low frequency from the reproductive and respiratory
*Author to whom correspondence should be addressed at : Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada . 0882-4010/90/120397+10 $03 .00/0
© 1990 Academic Press Limited
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tract of clinically normal cattle' it is not certain whether this is the only natural reservoir for infection . The pathogenesis of H. somnus infection in cattle is not fully understood . Histopathological studies have provided information on tissue distribution and on the pathological consequences of invasive H. somnus infection"' but the important factors involved in earlier phases of the infectious process are not known . Animal models of infection will be essential for investigation of the host and bacterial factors contributing to the infectious process but it has been difficult to reproduce consistently H. somnus infection experimentally, 12 particularly severe TEME . The observed differences in virulence amongst strains of H . somnus 2 obviously have to be taken into account in animal models of infection . It is well established that the ability of bacterial pathogens to acquire iron in the relatively hostile environment of the host is an important step in the pathogenesis of infection .` However, there is little or no reported information on iron acquisition by H. somnus. About half of the strains of H . somnus isolated from the reproductive tract of bulls were hemolytic and the hemolytic activity was stably maintained upon continued passage.' Whether hemolytic activity is uniformly present in clinical disease isolates and whether this hemolytic activity would be sufficient for accessing the intracellular iron pools in vivo is not known . In this study we report on the ability of clinical isolates of H . somnus to use the physiologically relevant iron source, transferrin, demonstrating a specificity for bovine transferrin that correlates with the identification and characterization of a bovine specific transferrin receptor .
Results Use of transferrin iron for growth In order to test the ability of different bacterial strains and species to use various protein sources of iron for growth we used a simple growth assay in which cell suspensions were spotted directly onto plates containing iron-deficient medium and the protein being tested . The media was rendered iron-deficient by the inclusion of ethylenediamine-di-(hydroxyphenylacetic acid) (EDDA) to 100 pm and initial experiments were performed to determine what concentration of cells in the suspension would result in no observable growth on iron-deficient plates yet evident growth on iron-sufficient plates . With cells resuspended directly from chocolate plates an A 600 of 0 .02 was adequate for H . somnus, Pasteurella haemolytica and Actinobacillus pleuropneumoniae but a more dilute suspension with an Asoo of 0 .005 was required for Neisseria meningitidis in order to avoid detectable growth on iron-deficient plates . As indicated in Table 1, all nine strains of H. somnus tested were able to grow on iron-deficient plates containing bovine transferrin as a source of iron for growth but were not able to use human, porcine or chicken transferrin . The inability to use human or porcine transferrin for growth could not be attributed to inadequacies of these preparations since the control bacteria N. meningitidis and A . p/europneumoniae were capable of using these transferrins as an exclusive source of iron for growth . Detection of siderophore production In an attempt to determine whether the strains of H. somnus were capable of producing siderophores, a simple screening assay originally described by Schwyn and Neilands 10 was used . In this assay, isolates are spotted onto medium containing iron in a bluecolored complex . The secretion of siderophores into the medium removes iron from the complex and results in a yellow-orange halo around siderophore-producing colonies . The inclusion of thiamine monophosphate in the medium" was sufficient
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Table 1 Growth of bacterial strains with different protein sources of irons Protein source of iron species Bacterium H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus N. meningitidis P. haemolytica A . pleuropneumoniae
e
6
pTf'
cTf b
-
-
+
-
-
-
+
-
-
+ -
+ + + +
-
Strain
hTf
bTf
H67 H68 H69 H70
-
+ +
-
H72
H73 H74 H76 B16B6 H95 H49
+
-
Growth was assessed after spotting 2 µl of a cell suspension (A 600 = 0 .02 for all strains except N. meningitidis which was 0 .005) onto plates containing 100 µnn EDDA and 10 pm of the indicated protein source of iron and incubating at 37'C for 24 h . + : obvious growth ; - : growth not detectable . ' hTf: human transferrin ; bTf : bovine transferrin ; pTf: porcine transferrin ; cTf : chicken transferrin .
to obtain growth of the H. somnus isolates . Pseudomonas cepacia strain 71 5j, a known siderophore-producing bacterium, was used as a positive control . After 72 h of incubation there was no evidence for siderophore production by any of the nine H. somnus strains tested although adequate growth of these isolates was observed (data not shown) . Expression of transferrin binding activity The specificity for bovine transferrin as a source of iron for growth that was observed in strains of H. somnus (Table 1) suggests the presence of a specific surface receptor involved in iron acquisition from this protein . To screen for the presence of bovine transferrin binding activity we used a solid-phase binding assay that we had developed previously for detecting receptors for human transferrin in N . meningitidis. 12 A conjugate of horseradish peroxidase and bovine transferrin (HRP-bTf) was prepared and then tested for binding to cells grown on iron-deficient plates to induce expression of iron-repressible proteins . As shown in Fig . 1, all nine isolates of H . somnus were capable of binding HRP-bTf but were incapable of binding HRP conjugates of human transferrin (HRP-hTf) or porcine transferrin (HRP-pTf) . The failure to bind HRP-hTf and HRP-pTf was not due to inadequacies in these preparations since the control bacteria N. meningitidis and A . pleuropneumoniae were capable of binding these conjugates . The binding of the HRP-bTf conjugate by the H. somnus strains illustrated in Fig . 1 was shown to be specifically due to the bTf component since the binding was eliminated by addition of excess unconjugated bTf (lane 2 ; Fig . 1) . To investigate the binding specificity further, a competitive binding assay was set up using irondeficient cells of H. somnus strain H74 as a target (Fig . 2) . In this competitive assay, immobilized, iron-deficient cells were exposed to mixtures of HRP-bTf and the indicated concentrations of unconjugated transferrins . It is evident from Fig . 2 that only bovine transferrin, and not transferrins from other species, was capable of competing with HRP-bTf for binding to the cells . The bTf binding activity shown in Figs 1 and 2 probably represents a surface receptor
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H. somnus H67 H. somnus H68 H. somnus H69 H. somnus H70 H. somnus H72 H. somnus H73 H. somnus H74 H. somnus H75
H. somnus H76
•
N meningitidis
P. haemolytica A . pleuropneumonioe
S
Fig . 1 . Detection of bovine transferrin binding activity in iron-deficient bacteria . Intact bacterial cells from the indicated species and strains grown on iron-deficient plates were spotted onto HA paper and exposed to binding mixtures containing the indicated reagents prior to washing and development with an HRP substrate mixture . Concentration of the unconjugated bTf in lane 2 was 20 hg/ml .
6 .4
1 .6
0.4
0 .1
bTf
pTf
hTf
rTf
cTf
Fig . 2 . Specificity of the transferrin in H. somnus strain H74. Intact, iron-deficient cells from H. somnus strain H74 were spotted onto HA paper and exposed to mixtures of HRP-bTf and the indicated concentration (mm) of unconjugated transferrins. After incubation with the binding mixtures, the paper was washed and exposed to HRP substrate mixture for color development . bTf, bovine transferrin ; pTf, porcine transferrin ; htF, human transferrin ; rTf, rabbit transferrin ; cTf, chicken transferrin .
Transferrin receptor in Haemophilus somnus
401 Time ( h )
Additions
0
I
2
3
4
None
EDDA
EDDA+CAP
EDDA+ Rif
EDDA+Amp
Fig . 3 . Effect of inhibitors on induction of transferrin receptor expression . H . somnus strain H74 grown on chocolate plates was used to inoculate broth cultures with the indicated additions to a starting A600 of 0 .05 . Samples taken at the indicated time periods were centrifuged and the cells were resuspended, spotted onto HA paper and assayed for HRP-bTf binding as described in Fig . 1 and the methods section . EDDA, 100 jinn EDDA ; EDDA+CAP, 100µM EDDA and 50 pg/ml chloramphenicol ; EDDA+Rif, 100 µm EDDA and 50 pg/ml rifampicin ; EDDA+Amp, 100 µM EDDA and 50 pg/ml ampicillin .
involved in iron acquisition from bTf . We had used iron-limiting conditions for growth in anticipation that the activity would be iron-regulated . To demonstrate the ironregulation of binding activity, strains of H. somnus were grown in broth alone, in broth containing a variety of iron chelators and in broth containing iron chelators plus excess iron . In these experiments it was observed in all strains that bTf binding activity was only detectable in cells grown in broth containing iron chelators (data not shown) . Since a variety of iron chelators were used, including chicken transferrin, it is unlikely that the iron chelators were inducing expression of receptor activity directly . Although the experiments described above demonstrate that expression of receptor activity was iron-regulated it was not known whether regulation was occurring at the transcriptional, translational or post-translational level . Thus an experiment was performed in which the effect of transcription and translation inhibitors on expression of bTf binding activity was evaluated . Figure 3 illustrates that cells inoculated into broth alone do not express detectable bTf binding activity even after 4 h of growth . In contrast, cells inoculated into broth containing 100 mm EDDA express detectable bTf binding activity after 1 h of incubation and increase the level of expression over the next 2-3 h period . Inclusion of the transcription inhibitor, rifampicin, or the translation inhibitor, chloramphenicol, essentially eliminated the induction of bTf receptor expression by EDDA . Ampicillin, an antibiotic that does not affect either of these processes, did not inhibit induction of receptor expression .
Identification of the bovine transferrin receptor proteins To identify the proteins involved in the binding of bTf to iron-deficient H . somnus cells we used an affinity isolation method, taking advantage of the highly specific binding of bTf . In this approach, biotinylated bTf or control proteins were prebound to iron-deficient membranes and, after solubilization, the biotinylated bTf-receptor complex was bound to streptavidin-agarose . After washing the resin, the receptor proteins were eluted from the streptavidin-agarose in sample buffer and analysed by
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B
C
D
E
F
116-
84-
58-
45-
Fig . 4 . Identification of the bovine transferrin receptor protein(s) in H . somnus strain H74 . Lanes A to E, membranes subjected to the affinity isolation procedure described in the methods section ; lane F, irondeficient outer membranes prepared as described in the Materials and methods section . The type of outer membranes used and the presence of biotinylated transferrin (bio-Tf) in the affinity isolation procedure were as follows : lane A, iron-sufficient outer membranes plus bio-bTf ; lane B, iron-deficient outer membranes without added bio-Tf ; lane C, iron-deficient outer membranes plus bio-pTf ; lane D, iron-deficient outer membranes plus bio-hTf; lane E, iron-deficient outer membranes plus bio-bTf .
SDS-PAGE . The results using a high salt wash buffer are shown in Fig . 4 . When the affinity isolation procedure was performed with iron-sufficient membranes prepared from strain H74 (lane A), no proteins were evident, which correlates with the lack of binding activity observed in iron-sufficient cells (Fig . 3) . When the affinity isolation procedure was performed with iron-deficient membranes and biotinylated bTf (lane E), three high molecular weight proteins (105 kDa, 85 kDa and 73 kDa) were isolated . The failure to isolate these bands when no biotinylated bTf was added (lane B) or when biotinylated porcine (lane C) or human (lane D) transferrin was used in place of biotinylated bTf confirms that these proteins were being isolated on the basis of specific binding to bovine transferrin . Comparison of the affinity-isolated bTf receptor proteins (lane E) with iron-deficient outer membranes (lane F) illustrates that the 105 kDa and 73 kDa affinity-isolated receptor proteins co-migrate with outer membrane proteins that are iron-regulated (data not shown) . In contrast, the 85 kDa protein does not co-migrate with any evident iron-regulated outer membrane protein . Although this band co-migrates with the upper band present in biotinylated bTf preparations, it does not react with HRPconjugated streptavidin after SDS-PAGE and electroblotting as does the original biotinylated stock (data not shown) . Attempts to detect binding of HRP-bTf by any of the putative receptor proteins in membrane or affinity-purified preparations after SDS-PAGE and electroblotting have not been successful . To determine whether similar bTf receptor proteins were present in other strains of H. somnus, the affinity isolation procedure was performed on iron-deficient membranes isolated from six representative strains . The results in Fig . 5 illustrate that a similar pattern of receptor proteins is observed in four of the six H. somnus strains tested (lanes A, D, E and F) . With two of the strains (lanes B and C), only the 73 kDa protein
Transferrin receptor in
Haemophilus somnus
A
B
403
C
D
E
F
45-
Fig . 5 . Comparison of the affinity-isolated bovine transferrin receptor proteins in different strains of H . Iron-deficient outer membranes were used in the affinity isolation procedure with biotinylated bovine transferrin as described in the Materials and methods section . The following strains were used : lane A, strain H67 ; lane B, strain H68; lane C, strain H70 ; lane D, strain 72 ; lane E, strain H74 ; lane F, strain H76 . somnus .
is evident in the affinity-isolated preparations . The additional protein bands migrating at approximately 50-55 kDa in these preparations are 'contaminating bands' not specifically associated with affinity isolation using biotinylated bTf . They are removed by more extensive washing conditions and are also observed when other transferrins (lane D, Fig . 4) are used in the isolation procedure . It is noteworthy that essentially the same pattern of proteins bands is observed when the affinity isolation is performed using a much cruder starting material, total membranes (data not shown), verifying the selectivity of the affinity isolation procedure . However, an additional band of 76 kDa is also present in the samples prepared from the total membranes . This band is also isolated when either iron-sufficient or irondeficient total membranes are solubilized and exposed to the streptavidin-agarose resin without addition of biotinylated transferrin indicating that it is a non-specific component unrelated to the observed transferrin receptor activity . Discussion The mammalian host provides an environment for potential bacterial pathogens that is restricted in the availability of iron .' , ' , ' Most of the iron is located intracellularly, .' complexed to the proteins ferritin, hemosiderin, myoglobin and especially hemoglobin In the extracellular compartment most of the iron is complexed to the high affinity iron-binding glycoproteins, transferrin and lactoferrin . These proteins effectively lower the concentration of unassociated iron to 10 -18 M which is insufficient to sustain growth of bacteria . Therefore, without access to intracellular iron pools, pathogenic bacteria must acquire iron from the transferrin and/or lactoferrin pools in order to sustain growth and cause infection . Two different mechanisms of acquiring iron from transferrin by pathogenic bacteria have been described . The first mechanism, which has been studied extensively, involves the production of small, soluble, high-affinity iron chelators called siderophores. 13 .14 After competing with transferrin for iron, the siderophore with complexed iron is bound to surface receptors and the iron is taken up into the cell ." A second type of mechanism involves direct binding of transferrin at the surface of the bacterium
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followed by removal and uptake of iron by an as yet unknown process .""" A characteristic of the 'receptor-mediated' iron uptake systems that have been studied in the human pathogens N. meningitidis, N. gonorrhoeae and Haemophilus influenzae is that the surface receptors are specific for transferrin from the host species, humans .","' 20 In this study we have shown that strains of H. somnus were capable of effectively obtaining iron for growth from bovine transferrin but not from transferrins from other mammalian species (Table 1 ) . In addition, whole cells or membranes from irondeficient H. somnus were capable of specifically binding bovine transferrin but not human, porcine, chicken or rabbit transferrin (Figs 1 and 2) . These results suggest that H. somnus acquires iron from bovine transferrin by a 'receptor-mediated' mechanism similar to that found in the human pathogens N. meningitidis, 1218 N. gonorrhoeae 19 and H. influenzae .20 Although there is no comprehensive evidence to exclude the presence of siderophore production and siderophore-mediated iron acquisition in H. somnus, our results are consistent with the absence of an effective siderophore-mediated mechanism . First, we have been unable to detect siderophore production by a screening assay that is based on ferric ion binding and is independent of the chemical structure of the siderophore ." Second, the strict specificity for bovine transferrin observed in our growth studies would not be expected if a siderophoremediated acquisition mechanism was operational . In the absence of a siderophore-mediated system, the ability of strains of H. somnus to acquire iron in vivo may be dependent upon the presence of the bovine transferrin receptor . The specificity of this receptor for bovine transferrin may be a partial explanation why H. somnus infection has not been described in other animal hosts . In addition, the requisite surface accessibility and the essential function of the bovine transferrin receptor indicates that this component would be an ideal candidate for vaccine development . The evidence discussed above, which suggested a 'receptor-mediated' iron uptake system, prompted us to attempt to identify the transferrin receptor protein(s) by adapting an affinity isolation method we had used previously for identification of the transferrin and lactoferrin receptor proteins in N. meningitidis ." As illustrated in Fig . 4, two iron-regulated outer membrane proteins of 105 and 73 kDa were isolated specifically by use of bovine transferrin in the affinity method . This pattern of two bands co-migrating with iron-repressible outer membrane proteins is reminiscent of the transferrin receptor proteins isolated from N. meningitidis .' $ However, a third protein of 85 kDa, which does not co-migrate with any iron-repressible outer membrane protein, is also observed in Fig . 4 . This band was shown not to be biotinylated bovine transferrin by its failure to bind with HRP-streptavidin and its variable molecular weight in different strains (Fig . 5) . This band was not observed in freshly prepared samples run on mini-gels but was evident after storage of the samples in SDS-PAGE sample buffer . It is possible that this band represents a specific derivative (perhaps a proteolytic cleavage product) of the 105 kDa band as both bands are apparently absent in samples from certain H . somnus isolates (lanes B and C, Fig . 5) . The apparent absence of the 105 kDa and 85 kDa proteins in affinity isolated preparations from two of the H . somnus strains (lanes B and C, Fig . 5) that were still capable of acquiring iron from bovin transferrin (Table 1) might lead one to propose that only the 73 kDa protein is essential for 'receptor-mediated' iron acquisition from bovine transferrin . However, careful observation reveals that these bands are present, albeit in considerably reduced quantities . It is clear that more definitive studies with specific mutations in the receptor protein gene(s) will be necessary to determine ultimately the precise role that these proteins play in iron acquisition from transferrin .
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Materials and methods Bacterial strains and growth conditions . Haemophilus somnus strains H67-H76 were disease isolates from cattle provided by Dr Jane Pritchard, Animal Health Laboratory, Alberta Agriculture, Airdrie, Alberta . Actinobacillus pleuropneumoniae strain H49 was a serotype 7 disease isolate from pig provided by Sharon Lundberg, Animal Health Laboratory, Alberta Agriculture, Airdrie, Alberta . Pasteurella haemolytica strain H95 was a serotype 1 disease isolate from cattle provided by Dr Andrew Potter, Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan . Neisseria meningitidis strain 1316136 is a standard serotyping strain originally provided by Dr Carl Frasch, Bethesda, Maryland . Pseudomonas cepacia strain 715j, a clinical isolate from a cystic fibrosis patient, was obtained from Dr Donald Woods . Bacterial stocks were maintained as frozen suspensions in 30% glycerol at -70°C . Isolates from frozen stocks were streaked onto chocolate-agar plates supplemented with CVA enrichment (Gibco Laboratories, Burlington, Ontario) and incubated at 37°C in an atmosphere containing 5% CO 2 for 16-20 h . Bacteria were routinely subcultured onto plates or into broth containing brain heart infusion (BHI) broth supplemented with 2 µM NAD, 5 µM thiamine monophosphate and 0 .5% yeast extract . Iron limited broth was prepared by the addition of EDDA to 100 µM . In the growth experiments, iron-saturated transferrin was added to 10 µM in plates composed of supplemented BHI agar containing 100 µM EDDA . In large scale cultures used for isolation of membranes the 0 .5% yeast extract supplement was omitted . The plates used to screen for siderophore production were composed of the media described by Schwyn and Neilands 10 containing thiamine monophosphate to 5 µM . In the expression experiments, cells resuspended from chocolate plates were used to iroculate prewarmed, supplemented BHI broth to an Asoo of 0 .05 and incubated with shaking at 37°C . Where indicated the broth contained EDDA to 100 µM and the indicated antibiotic to 50 µg/ml . Preparation of transferrins and derivatives . Human transferrin, bovin transferrin, rabbit transferrin and chicken transferrin were obtained from Sigma Chemical, St Louis, Missouri, and porcine transferrin was obtained from Binding Site, Birmingham, U .K . A conjugate of human transferrin and horseradish peroxidase was obtained from Jackson Immunoresearch Laboratories, Avondale, Pennsylvania . The commercial preparations of transferrin not fully iron-saturated were iron loaded as described previously ." Biotinylation of bovine transferrin with N-hydroxy succinimide biotin was performed essentially as described previously .' $ Horseradish peroxidase conjugates of bovine and porcine transferrin were prepared essentially by the method of Wilson and Nakane . 21 After chemical conjugation, the mixture of horseradish peroxidase and bovine or porcine transferrin was subjected to gel filtration on a Spherogel TSK 3000SW HPLC column (Beckman Industries, Fullerton, California) and the fractions from the peak corresponding to a 1 : 1 conjugate were pooled, dialysed, and aliquots frozen and stored at -70°C . Transferrin binding assay . The solid-phase binding assay was essentially a modification of the procedure previously described for detecting binding of hTf . 12 Intact cells grown on irondeficient plates were resuspended in normal saline (150 mm NaCI) to an Asoo of 5, and 2 111 of the cell suspensions were spotted onto nitrocellulose-cellulose acetate paper (HA paper, Millipore Corporation, Bedford, Massachusetts) and were allowed to dry . After drying, the HA paper was blocked with blocking solution 12 and the paper was exposed to blocking solution containing 450 ng/ml of either HRP-bTf, HRP-pTf or HRP-hTf conjugate . In competition binding experiments the indicated concentration of unconjugated protein was included in the initial binding mixture . The incubation times, washing steps and development with HRP substrate mixture was performed essentially as described previously . 12 Preparation of outer membranes and affinity-isolation of transferrin receptor proteins . Outer membranes were prepared from iron-sufficient or iron-deficient cells after french press lysis by selective detergent extraction essentially as described previously . 12 The bovine transferrin receptor proteins were isolated from iron-deficient outer membranes essentially as described previously 18 with the modification of using biotinylated bovine transferrin (bio-bTf) in place of biotinylated human transferrin (bio-hTf) . In the experiments illustrated in Figs 4 and 5, a high salt wash buffer (50 mm Tris-HCI, 1 M NaCl, 10 mm EDTA, 0 .5% Sarkosyl, pH 8 .0) was used . After the final wash step with 50 mm Tris-HCI, 100 mm NaCl, pH 8 .0 buffer, the gel pellets were suspended in 100-200 µl SDS-PAGE sample buffer without B-mercaptoethanol and incubated for 30 min at room temperature . The samples were centrifuged at 750xg for 5 min, the supernatants were transferred to a separate tube, and B-mercaptoethanol was added to a
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final concentration of 1 .4 M . A 25-50 id portion of this sample was applied to 10% polyacrylamide SDS gel . Polyacrylamide gel electrophoresis and silver staining of gels were performed as described previously ."
This work was supported by Medical Research Council of Canada grant MA10350 . We would like to thank Drs Pritchard and Potter and Ms Lundberg for the generous provision of strains .
References 1 . Stephens LR, Little PB, Wilkie BN, Barnum DA . Infectious thromboembolic meningoencephalitis in cattle: a review . J Am Vet Mad Assoc 1981 ; 178 : 378-84. 2 . Potgieter LND, Helman RG, Greene W, Breider MA, Thurber ET, Peetz RH . Experimental bovine respiratory tract disease with Haemophilus somnus . Vet Pathol 1988; 25 : 124-30 . 3 . Saunders JR, Thiessen WA, Janzen ED . Haemophilus somnus infections I . A ten year (1969-1978) retrospective study of losses in cattle herds in western Canada . Can Vet J 1980; 21 : 119-23 . 4 . Momotani E, Yabuki Y, Miho H, Ishikawa Y, Yoshino T . Histopathological evaluation of disseminated intravascular coagulation in Haemophilus somnus infection in cattle . J Comp Pathol 1985 ; 95 : 15-23 . 5 . Weinberg ED . Iron and infection . Microbiol Rev 1978 ; 42 : 45-66 . 6 . Bullen JJ, Rogers HJ, Griffiths E . Role of iron in bacterial infection . Curr Top Microbiol Immunol 1978 ; 80 : 1-35 . 7 . Payne SM, Finkelstein RA . The critical role of iron in host-bacterial interactions . J Clin Invest 1978 ; 610 :1428-40 . 8 . Griffiths E . Adaptation and multiplication of bacteria in host tissues . Phil Trans R Soc Lond 1983 ; 303 : 85-96 . 9 . Humphrey JD, Little PB, Stephens LR, Barnum DA, Doig PA, Thorsen J . Prevalence and distribution of Haemophi/us somnus in the male bovine reproductive tract . Am J Vet Res 1982 ; 43 : 791-5 . 10 . Schwyn B, Neilands JB . Universal chemical assay for the detection and determination of siderophores . Anal Biochem 1987 ; 160 : 47-56 . 11 . Asmussen MD, Baugh CL . Thiamine pyrophosphate (cocarboxylase) as a growth factor for Haemophilus somnus . J Clin Microbiol 1981 ; 14 :178-83 . 12 . Schryvers AB, Morris LJ . Identification and characterization of the transferrin receptor from Neisseria meningitidis . Mol Microbiol 1988; 2 : 281-8 . 13 . Crosa JH . The relationship of plasmid-mediated iron transport and bacterial virulence . Annu Rev Microbiol 1984; 38 : 69-89 . 14 . Neilands JB . Microbial iron compounds . Annu Rev Microbiol 1981 ; 50 : 715-31 . 15 . Neilands JB . Microbial envelope proteins related to iron . Annu Rev Microbiol 1982 ; 360 : 285-309 . 16 . Tsai J, Dyer DW, Sparling PF . Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source . Infect Immun 1988 ; 56 : 3132-8 . 17 . Archibald FS, DeVoe IW . Removal of iron from human transferrin by Neisseria meningitidis . Microbiol Lett 1979 ; 6 : 159-62 . 18 . Schryvers AB, Morris LJ . Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis . Infect Immun 1988; 56 : 1144-9 . 19 . Lee BC, Schryvers AB . Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae . Mol Microbiol 1988; 2 : 827-9. 20. Schryvers AB . Characterization of the human transferrin and lactoferrin receptors in Haemophi/us influenzae. Mol Microbiol 1988 ; 2 : 467-72 . 21 . Wilson MB, Nakane PK . Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies . In : Knapp W, Holubar K, Wick G, eds . Immunofluorescence and related staining techniques. Amsterdam : Elsevier/North Holland Biomedical Press, 1978 ; 215-24 .
Response of Haemophilus somnus to iron limitation : expression and identification of a bovine-specific transferrin receptor Julius A . Ogunnariwo, Cindy Cheng, Jeffrey Ford and Anthony B . Schryvers* Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada (Received June 25, 1990 ; accepted in revised form September 5, 1990)
Cgunnariwo, J . A . (Dept of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada), C . Cheng, J . Ford and A . B . Schryvers . Response of Haemophilus somnus to iron limitation : expression and identification of a bovine-specific transferrin receptor . Microbial Pathogenesis 1990 ; 9 : 397-406 . Nine clinical isolates of Haemophilus somnus were screened for their ability to use different transferrins as a source of iron growth . All nine strains were capable of using bovine but not porcine, human or chicken transferrin . A screening assay for siderophore production did not show any evidence of siderophore production by these strains . When iron-deficient cells from these strains were screened for their ability to bind perixodase-conjugated transferrin, binding was detected with conjugated bovine, but not human or porcine transferrin . Competition binding studies demonstrated that the binding of peroxidase-conjugated bovine transferrin was competitively inhibited by unconjugated bovine transferrin but not transferrin from other species . The induction of receptor expression by low iron conditions was inhibited by chloramphenicol and rifampicin but not ampicillin indicating that new protein and mRNA synthesis was required for expression of receptor activity . Affinity isolation of receptor proteins with biotinylated bovine transferrin, but not human or porcine transferrin, yielded three proteins from H. somnus strain H74 . Two of the proteins were identified as 105 kDa and 73 kDa iron-regulated outer membrane proteins . A third protein of 85 kDa that was isolated did not co-migrate with any iron-regulated outer membrane protein . Affinity isolation of receptor proteins from other strains of H. somnus yielded a 73 kDa protein from all strains and a 105 kDa and 85 kDa protein in four of the six strains analysed . Key words : transferrin ; iron acquisition ; receptor ; Haemophilus somnus .
Introduction Haemophilus somnus is an important bacterial pathogen of cattle . It is the causative agent of infectious thromboembolic meningoencephalitis (TEME) and has been associated with other disease syndromes including pneumonia, abortion and infertility .' 3 TEM E tends to occur in herd outbreaks in feedlot cattle' yet the mechanism of disease transmission has not been firmly established . It has been suggested that respiratory tract disease may be a precursor to invasive H. somnus infection .' Although H. somnus can be isolated at low frequency from the reproductive and respiratory
*Author to whom correspondence should be addressed at : Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada . 0882-4010/90/120397+10 $03 .00/0
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tract of clinically normal cattle' it is not certain whether this is the only natural reservoir for infection . The pathogenesis of H. somnus infection in cattle is not fully understood . Histopathological studies have provided information on tissue distribution and on the pathological consequences of invasive H. somnus infection"' but the important factors involved in earlier phases of the infectious process are not known . Animal models of infection will be essential for investigation of the host and bacterial factors contributing to the infectious process but it has been difficult to reproduce consistently H. somnus infection experimentally, 12 particularly severe TEME . The observed differences in virulence amongst strains of H . somnus 2 obviously have to be taken into account in animal models of infection . It is well established that the ability of bacterial pathogens to acquire iron in the relatively hostile environment of the host is an important step in the pathogenesis of infection .` However, there is little or no reported information on iron acquisition by H. somnus. About half of the strains of H . somnus isolated from the reproductive tract of bulls were hemolytic and the hemolytic activity was stably maintained upon continued passage.' Whether hemolytic activity is uniformly present in clinical disease isolates and whether this hemolytic activity would be sufficient for accessing the intracellular iron pools in vivo is not known . In this study we report on the ability of clinical isolates of H . somnus to use the physiologically relevant iron source, transferrin, demonstrating a specificity for bovine transferrin that correlates with the identification and characterization of a bovine specific transferrin receptor .
Results Use of transferrin iron for growth In order to test the ability of different bacterial strains and species to use various protein sources of iron for growth we used a simple growth assay in which cell suspensions were spotted directly onto plates containing iron-deficient medium and the protein being tested . The media was rendered iron-deficient by the inclusion of ethylenediamine-di-(hydroxyphenylacetic acid) (EDDA) to 100 pm and initial experiments were performed to determine what concentration of cells in the suspension would result in no observable growth on iron-deficient plates yet evident growth on iron-sufficient plates . With cells resuspended directly from chocolate plates an A 600 of 0 .02 was adequate for H . somnus, Pasteurella haemolytica and Actinobacillus pleuropneumoniae but a more dilute suspension with an Asoo of 0 .005 was required for Neisseria meningitidis in order to avoid detectable growth on iron-deficient plates . As indicated in Table 1, all nine strains of H. somnus tested were able to grow on iron-deficient plates containing bovine transferrin as a source of iron for growth but were not able to use human, porcine or chicken transferrin . The inability to use human or porcine transferrin for growth could not be attributed to inadequacies of these preparations since the control bacteria N. meningitidis and A . p/europneumoniae were capable of using these transferrins as an exclusive source of iron for growth . Detection of siderophore production In an attempt to determine whether the strains of H. somnus were capable of producing siderophores, a simple screening assay originally described by Schwyn and Neilands 10 was used . In this assay, isolates are spotted onto medium containing iron in a bluecolored complex . The secretion of siderophores into the medium removes iron from the complex and results in a yellow-orange halo around siderophore-producing colonies . The inclusion of thiamine monophosphate in the medium" was sufficient
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Table 1 Growth of bacterial strains with different protein sources of irons Protein source of iron species Bacterium H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus H. somnus N. meningitidis P. haemolytica A . pleuropneumoniae
e
6
pTf'
cTf b
-
-
+
-
-
-
+
-
-
+ -
+ + + +
-
Strain
hTf
bTf
H67 H68 H69 H70
-
+ +
-
H72
H73 H74 H76 B16B6 H95 H49
+
-
Growth was assessed after spotting 2 µl of a cell suspension (A 600 = 0 .02 for all strains except N. meningitidis which was 0 .005) onto plates containing 100 µnn EDDA and 10 pm of the indicated protein source of iron and incubating at 37'C for 24 h . + : obvious growth ; - : growth not detectable . ' hTf: human transferrin ; bTf : bovine transferrin ; pTf: porcine transferrin ; cTf : chicken transferrin .
to obtain growth of the H. somnus isolates . Pseudomonas cepacia strain 71 5j, a known siderophore-producing bacterium, was used as a positive control . After 72 h of incubation there was no evidence for siderophore production by any of the nine H. somnus strains tested although adequate growth of these isolates was observed (data not shown) . Expression of transferrin binding activity The specificity for bovine transferrin as a source of iron for growth that was observed in strains of H. somnus (Table 1) suggests the presence of a specific surface receptor involved in iron acquisition from this protein . To screen for the presence of bovine transferrin binding activity we used a solid-phase binding assay that we had developed previously for detecting receptors for human transferrin in N . meningitidis. 12 A conjugate of horseradish peroxidase and bovine transferrin (HRP-bTf) was prepared and then tested for binding to cells grown on iron-deficient plates to induce expression of iron-repressible proteins . As shown in Fig . 1, all nine isolates of H . somnus were capable of binding HRP-bTf but were incapable of binding HRP conjugates of human transferrin (HRP-hTf) or porcine transferrin (HRP-pTf) . The failure to bind HRP-hTf and HRP-pTf was not due to inadequacies in these preparations since the control bacteria N. meningitidis and A . pleuropneumoniae were capable of binding these conjugates . The binding of the HRP-bTf conjugate by the H. somnus strains illustrated in Fig . 1 was shown to be specifically due to the bTf component since the binding was eliminated by addition of excess unconjugated bTf (lane 2 ; Fig . 1) . To investigate the binding specificity further, a competitive binding assay was set up using irondeficient cells of H. somnus strain H74 as a target (Fig . 2) . In this competitive assay, immobilized, iron-deficient cells were exposed to mixtures of HRP-bTf and the indicated concentrations of unconjugated transferrins . It is evident from Fig . 2 that only bovine transferrin, and not transferrins from other species, was capable of competing with HRP-bTf for binding to the cells . The bTf binding activity shown in Figs 1 and 2 probably represents a surface receptor
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H. somnus H67 H. somnus H68 H. somnus H69 H. somnus H70 H. somnus H72 H. somnus H73 H. somnus H74 H. somnus H75
H. somnus H76
•
N meningitidis
P. haemolytica A . pleuropneumonioe
S
Fig . 1 . Detection of bovine transferrin binding activity in iron-deficient bacteria . Intact bacterial cells from the indicated species and strains grown on iron-deficient plates were spotted onto HA paper and exposed to binding mixtures containing the indicated reagents prior to washing and development with an HRP substrate mixture . Concentration of the unconjugated bTf in lane 2 was 20 hg/ml .
6 .4
1 .6
0.4
0 .1
bTf
pTf
hTf
rTf
cTf
Fig . 2 . Specificity of the transferrin in H. somnus strain H74. Intact, iron-deficient cells from H. somnus strain H74 were spotted onto HA paper and exposed to mixtures of HRP-bTf and the indicated concentration (mm) of unconjugated transferrins. After incubation with the binding mixtures, the paper was washed and exposed to HRP substrate mixture for color development . bTf, bovine transferrin ; pTf, porcine transferrin ; htF, human transferrin ; rTf, rabbit transferrin ; cTf, chicken transferrin .
Transferrin receptor in Haemophilus somnus
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Additions
0
I
2
3
4
None
EDDA
EDDA+CAP
EDDA+ Rif
EDDA+Amp
Fig . 3 . Effect of inhibitors on induction of transferrin receptor expression . H . somnus strain H74 grown on chocolate plates was used to inoculate broth cultures with the indicated additions to a starting A600 of 0 .05 . Samples taken at the indicated time periods were centrifuged and the cells were resuspended, spotted onto HA paper and assayed for HRP-bTf binding as described in Fig . 1 and the methods section . EDDA, 100 jinn EDDA ; EDDA+CAP, 100µM EDDA and 50 pg/ml chloramphenicol ; EDDA+Rif, 100 µm EDDA and 50 pg/ml rifampicin ; EDDA+Amp, 100 µM EDDA and 50 pg/ml ampicillin .
involved in iron acquisition from bTf . We had used iron-limiting conditions for growth in anticipation that the activity would be iron-regulated . To demonstrate the ironregulation of binding activity, strains of H. somnus were grown in broth alone, in broth containing a variety of iron chelators and in broth containing iron chelators plus excess iron . In these experiments it was observed in all strains that bTf binding activity was only detectable in cells grown in broth containing iron chelators (data not shown) . Since a variety of iron chelators were used, including chicken transferrin, it is unlikely that the iron chelators were inducing expression of receptor activity directly . Although the experiments described above demonstrate that expression of receptor activity was iron-regulated it was not known whether regulation was occurring at the transcriptional, translational or post-translational level . Thus an experiment was performed in which the effect of transcription and translation inhibitors on expression of bTf binding activity was evaluated . Figure 3 illustrates that cells inoculated into broth alone do not express detectable bTf binding activity even after 4 h of growth . In contrast, cells inoculated into broth containing 100 mm EDDA express detectable bTf binding activity after 1 h of incubation and increase the level of expression over the next 2-3 h period . Inclusion of the transcription inhibitor, rifampicin, or the translation inhibitor, chloramphenicol, essentially eliminated the induction of bTf receptor expression by EDDA . Ampicillin, an antibiotic that does not affect either of these processes, did not inhibit induction of receptor expression .
Identification of the bovine transferrin receptor proteins To identify the proteins involved in the binding of bTf to iron-deficient H . somnus cells we used an affinity isolation method, taking advantage of the highly specific binding of bTf . In this approach, biotinylated bTf or control proteins were prebound to iron-deficient membranes and, after solubilization, the biotinylated bTf-receptor complex was bound to streptavidin-agarose . After washing the resin, the receptor proteins were eluted from the streptavidin-agarose in sample buffer and analysed by
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B
C
D
E
F
116-
84-
58-
45-
Fig . 4 . Identification of the bovine transferrin receptor protein(s) in H . somnus strain H74 . Lanes A to E, membranes subjected to the affinity isolation procedure described in the methods section ; lane F, irondeficient outer membranes prepared as described in the Materials and methods section . The type of outer membranes used and the presence of biotinylated transferrin (bio-Tf) in the affinity isolation procedure were as follows : lane A, iron-sufficient outer membranes plus bio-bTf ; lane B, iron-deficient outer membranes without added bio-Tf ; lane C, iron-deficient outer membranes plus bio-pTf ; lane D, iron-deficient outer membranes plus bio-hTf; lane E, iron-deficient outer membranes plus bio-bTf .
SDS-PAGE . The results using a high salt wash buffer are shown in Fig . 4 . When the affinity isolation procedure was performed with iron-sufficient membranes prepared from strain H74 (lane A), no proteins were evident, which correlates with the lack of binding activity observed in iron-sufficient cells (Fig . 3) . When the affinity isolation procedure was performed with iron-deficient membranes and biotinylated bTf (lane E), three high molecular weight proteins (105 kDa, 85 kDa and 73 kDa) were isolated . The failure to isolate these bands when no biotinylated bTf was added (lane B) or when biotinylated porcine (lane C) or human (lane D) transferrin was used in place of biotinylated bTf confirms that these proteins were being isolated on the basis of specific binding to bovine transferrin . Comparison of the affinity-isolated bTf receptor proteins (lane E) with iron-deficient outer membranes (lane F) illustrates that the 105 kDa and 73 kDa affinity-isolated receptor proteins co-migrate with outer membrane proteins that are iron-regulated (data not shown) . In contrast, the 85 kDa protein does not co-migrate with any evident iron-regulated outer membrane protein . Although this band co-migrates with the upper band present in biotinylated bTf preparations, it does not react with HRPconjugated streptavidin after SDS-PAGE and electroblotting as does the original biotinylated stock (data not shown) . Attempts to detect binding of HRP-bTf by any of the putative receptor proteins in membrane or affinity-purified preparations after SDS-PAGE and electroblotting have not been successful . To determine whether similar bTf receptor proteins were present in other strains of H. somnus, the affinity isolation procedure was performed on iron-deficient membranes isolated from six representative strains . The results in Fig . 5 illustrate that a similar pattern of receptor proteins is observed in four of the six H. somnus strains tested (lanes A, D, E and F) . With two of the strains (lanes B and C), only the 73 kDa protein
Transferrin receptor in
Haemophilus somnus
A
B
403
C
D
E
F
45-
Fig . 5 . Comparison of the affinity-isolated bovine transferrin receptor proteins in different strains of H . Iron-deficient outer membranes were used in the affinity isolation procedure with biotinylated bovine transferrin as described in the Materials and methods section . The following strains were used : lane A, strain H67 ; lane B, strain H68; lane C, strain H70 ; lane D, strain 72 ; lane E, strain H74 ; lane F, strain H76 . somnus .
is evident in the affinity-isolated preparations . The additional protein bands migrating at approximately 50-55 kDa in these preparations are 'contaminating bands' not specifically associated with affinity isolation using biotinylated bTf . They are removed by more extensive washing conditions and are also observed when other transferrins (lane D, Fig . 4) are used in the isolation procedure . It is noteworthy that essentially the same pattern of proteins bands is observed when the affinity isolation is performed using a much cruder starting material, total membranes (data not shown), verifying the selectivity of the affinity isolation procedure . However, an additional band of 76 kDa is also present in the samples prepared from the total membranes . This band is also isolated when either iron-sufficient or irondeficient total membranes are solubilized and exposed to the streptavidin-agarose resin without addition of biotinylated transferrin indicating that it is a non-specific component unrelated to the observed transferrin receptor activity . Discussion The mammalian host provides an environment for potential bacterial pathogens that is restricted in the availability of iron .' , ' , ' Most of the iron is located intracellularly, .' complexed to the proteins ferritin, hemosiderin, myoglobin and especially hemoglobin In the extracellular compartment most of the iron is complexed to the high affinity iron-binding glycoproteins, transferrin and lactoferrin . These proteins effectively lower the concentration of unassociated iron to 10 -18 M which is insufficient to sustain growth of bacteria . Therefore, without access to intracellular iron pools, pathogenic bacteria must acquire iron from the transferrin and/or lactoferrin pools in order to sustain growth and cause infection . Two different mechanisms of acquiring iron from transferrin by pathogenic bacteria have been described . The first mechanism, which has been studied extensively, involves the production of small, soluble, high-affinity iron chelators called siderophores. 13 .14 After competing with transferrin for iron, the siderophore with complexed iron is bound to surface receptors and the iron is taken up into the cell ." A second type of mechanism involves direct binding of transferrin at the surface of the bacterium
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followed by removal and uptake of iron by an as yet unknown process .""" A characteristic of the 'receptor-mediated' iron uptake systems that have been studied in the human pathogens N. meningitidis, N. gonorrhoeae and Haemophilus influenzae is that the surface receptors are specific for transferrin from the host species, humans .","' 20 In this study we have shown that strains of H. somnus were capable of effectively obtaining iron for growth from bovine transferrin but not from transferrins from other mammalian species (Table 1 ) . In addition, whole cells or membranes from irondeficient H. somnus were capable of specifically binding bovine transferrin but not human, porcine, chicken or rabbit transferrin (Figs 1 and 2) . These results suggest that H. somnus acquires iron from bovine transferrin by a 'receptor-mediated' mechanism similar to that found in the human pathogens N. meningitidis, 1218 N. gonorrhoeae 19 and H. influenzae .20 Although there is no comprehensive evidence to exclude the presence of siderophore production and siderophore-mediated iron acquisition in H. somnus, our results are consistent with the absence of an effective siderophore-mediated mechanism . First, we have been unable to detect siderophore production by a screening assay that is based on ferric ion binding and is independent of the chemical structure of the siderophore ." Second, the strict specificity for bovine transferrin observed in our growth studies would not be expected if a siderophoremediated acquisition mechanism was operational . In the absence of a siderophore-mediated system, the ability of strains of H. somnus to acquire iron in vivo may be dependent upon the presence of the bovine transferrin receptor . The specificity of this receptor for bovine transferrin may be a partial explanation why H. somnus infection has not been described in other animal hosts . In addition, the requisite surface accessibility and the essential function of the bovine transferrin receptor indicates that this component would be an ideal candidate for vaccine development . The evidence discussed above, which suggested a 'receptor-mediated' iron uptake system, prompted us to attempt to identify the transferrin receptor protein(s) by adapting an affinity isolation method we had used previously for identification of the transferrin and lactoferrin receptor proteins in N. meningitidis ." As illustrated in Fig . 4, two iron-regulated outer membrane proteins of 105 and 73 kDa were isolated specifically by use of bovine transferrin in the affinity method . This pattern of two bands co-migrating with iron-repressible outer membrane proteins is reminiscent of the transferrin receptor proteins isolated from N. meningitidis .' $ However, a third protein of 85 kDa, which does not co-migrate with any iron-repressible outer membrane protein, is also observed in Fig . 4 . This band was shown not to be biotinylated bovine transferrin by its failure to bind with HRP-streptavidin and its variable molecular weight in different strains (Fig . 5) . This band was not observed in freshly prepared samples run on mini-gels but was evident after storage of the samples in SDS-PAGE sample buffer . It is possible that this band represents a specific derivative (perhaps a proteolytic cleavage product) of the 105 kDa band as both bands are apparently absent in samples from certain H . somnus isolates (lanes B and C, Fig . 5) . The apparent absence of the 105 kDa and 85 kDa proteins in affinity isolated preparations from two of the H . somnus strains (lanes B and C, Fig . 5) that were still capable of acquiring iron from bovin transferrin (Table 1) might lead one to propose that only the 73 kDa protein is essential for 'receptor-mediated' iron acquisition from bovine transferrin . However, careful observation reveals that these bands are present, albeit in considerably reduced quantities . It is clear that more definitive studies with specific mutations in the receptor protein gene(s) will be necessary to determine ultimately the precise role that these proteins play in iron acquisition from transferrin .
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Materials and methods Bacterial strains and growth conditions . Haemophilus somnus strains H67-H76 were disease isolates from cattle provided by Dr Jane Pritchard, Animal Health Laboratory, Alberta Agriculture, Airdrie, Alberta . Actinobacillus pleuropneumoniae strain H49 was a serotype 7 disease isolate from pig provided by Sharon Lundberg, Animal Health Laboratory, Alberta Agriculture, Airdrie, Alberta . Pasteurella haemolytica strain H95 was a serotype 1 disease isolate from cattle provided by Dr Andrew Potter, Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan . Neisseria meningitidis strain 1316136 is a standard serotyping strain originally provided by Dr Carl Frasch, Bethesda, Maryland . Pseudomonas cepacia strain 715j, a clinical isolate from a cystic fibrosis patient, was obtained from Dr Donald Woods . Bacterial stocks were maintained as frozen suspensions in 30% glycerol at -70°C . Isolates from frozen stocks were streaked onto chocolate-agar plates supplemented with CVA enrichment (Gibco Laboratories, Burlington, Ontario) and incubated at 37°C in an atmosphere containing 5% CO 2 for 16-20 h . Bacteria were routinely subcultured onto plates or into broth containing brain heart infusion (BHI) broth supplemented with 2 µM NAD, 5 µM thiamine monophosphate and 0 .5% yeast extract . Iron limited broth was prepared by the addition of EDDA to 100 µM . In the growth experiments, iron-saturated transferrin was added to 10 µM in plates composed of supplemented BHI agar containing 100 µM EDDA . In large scale cultures used for isolation of membranes the 0 .5% yeast extract supplement was omitted . The plates used to screen for siderophore production were composed of the media described by Schwyn and Neilands 10 containing thiamine monophosphate to 5 µM . In the expression experiments, cells resuspended from chocolate plates were used to iroculate prewarmed, supplemented BHI broth to an Asoo of 0 .05 and incubated with shaking at 37°C . Where indicated the broth contained EDDA to 100 µM and the indicated antibiotic to 50 µg/ml . Preparation of transferrins and derivatives . Human transferrin, bovin transferrin, rabbit transferrin and chicken transferrin were obtained from Sigma Chemical, St Louis, Missouri, and porcine transferrin was obtained from Binding Site, Birmingham, U .K . A conjugate of human transferrin and horseradish peroxidase was obtained from Jackson Immunoresearch Laboratories, Avondale, Pennsylvania . The commercial preparations of transferrin not fully iron-saturated were iron loaded as described previously ." Biotinylation of bovine transferrin with N-hydroxy succinimide biotin was performed essentially as described previously .' $ Horseradish peroxidase conjugates of bovine and porcine transferrin were prepared essentially by the method of Wilson and Nakane . 21 After chemical conjugation, the mixture of horseradish peroxidase and bovine or porcine transferrin was subjected to gel filtration on a Spherogel TSK 3000SW HPLC column (Beckman Industries, Fullerton, California) and the fractions from the peak corresponding to a 1 : 1 conjugate were pooled, dialysed, and aliquots frozen and stored at -70°C . Transferrin binding assay . The solid-phase binding assay was essentially a modification of the procedure previously described for detecting binding of hTf . 12 Intact cells grown on irondeficient plates were resuspended in normal saline (150 mm NaCI) to an Asoo of 5, and 2 111 of the cell suspensions were spotted onto nitrocellulose-cellulose acetate paper (HA paper, Millipore Corporation, Bedford, Massachusetts) and were allowed to dry . After drying, the HA paper was blocked with blocking solution 12 and the paper was exposed to blocking solution containing 450 ng/ml of either HRP-bTf, HRP-pTf or HRP-hTf conjugate . In competition binding experiments the indicated concentration of unconjugated protein was included in the initial binding mixture . The incubation times, washing steps and development with HRP substrate mixture was performed essentially as described previously . 12 Preparation of outer membranes and affinity-isolation of transferrin receptor proteins . Outer membranes were prepared from iron-sufficient or iron-deficient cells after french press lysis by selective detergent extraction essentially as described previously . 12 The bovine transferrin receptor proteins were isolated from iron-deficient outer membranes essentially as described previously 18 with the modification of using biotinylated bovine transferrin (bio-bTf) in place of biotinylated human transferrin (bio-hTf) . In the experiments illustrated in Figs 4 and 5, a high salt wash buffer (50 mm Tris-HCI, 1 M NaCl, 10 mm EDTA, 0 .5% Sarkosyl, pH 8 .0) was used . After the final wash step with 50 mm Tris-HCI, 100 mm NaCl, pH 8 .0 buffer, the gel pellets were suspended in 100-200 µl SDS-PAGE sample buffer without B-mercaptoethanol and incubated for 30 min at room temperature . The samples were centrifuged at 750xg for 5 min, the supernatants were transferred to a separate tube, and B-mercaptoethanol was added to a
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final concentration of 1 .4 M . A 25-50 id portion of this sample was applied to 10% polyacrylamide SDS gel . Polyacrylamide gel electrophoresis and silver staining of gels were performed as described previously ."
This work was supported by Medical Research Council of Canada grant MA10350 . We would like to thank Drs Pritchard and Potter and Ms Lundberg for the generous provision of strains .
References 1 . Stephens LR, Little PB, Wilkie BN, Barnum DA . Infectious thromboembolic meningoencephalitis in cattle: a review . J Am Vet Mad Assoc 1981 ; 178 : 378-84. 2 . Potgieter LND, Helman RG, Greene W, Breider MA, Thurber ET, Peetz RH . Experimental bovine respiratory tract disease with Haemophilus somnus . Vet Pathol 1988; 25 : 124-30 . 3 . Saunders JR, Thiessen WA, Janzen ED . Haemophilus somnus infections I . A ten year (1969-1978) retrospective study of losses in cattle herds in western Canada . Can Vet J 1980; 21 : 119-23 . 4 . Momotani E, Yabuki Y, Miho H, Ishikawa Y, Yoshino T . Histopathological evaluation of disseminated intravascular coagulation in Haemophilus somnus infection in cattle . J Comp Pathol 1985 ; 95 : 15-23 . 5 . Weinberg ED . Iron and infection . Microbiol Rev 1978 ; 42 : 45-66 . 6 . Bullen JJ, Rogers HJ, Griffiths E . Role of iron in bacterial infection . Curr Top Microbiol Immunol 1978 ; 80 : 1-35 . 7 . Payne SM, Finkelstein RA . The critical role of iron in host-bacterial interactions . J Clin Invest 1978 ; 610 :1428-40 . 8 . Griffiths E . Adaptation and multiplication of bacteria in host tissues . Phil Trans R Soc Lond 1983 ; 303 : 85-96 . 9 . Humphrey JD, Little PB, Stephens LR, Barnum DA, Doig PA, Thorsen J . Prevalence and distribution of Haemophi/us somnus in the male bovine reproductive tract . Am J Vet Res 1982 ; 43 : 791-5 . 10 . Schwyn B, Neilands JB . Universal chemical assay for the detection and determination of siderophores . Anal Biochem 1987 ; 160 : 47-56 . 11 . Asmussen MD, Baugh CL . Thiamine pyrophosphate (cocarboxylase) as a growth factor for Haemophilus somnus . J Clin Microbiol 1981 ; 14 :178-83 . 12 . Schryvers AB, Morris LJ . Identification and characterization of the transferrin receptor from Neisseria meningitidis . Mol Microbiol 1988; 2 : 281-8 . 13 . Crosa JH . The relationship of plasmid-mediated iron transport and bacterial virulence . Annu Rev Microbiol 1984; 38 : 69-89 . 14 . Neilands JB . Microbial iron compounds . Annu Rev Microbiol 1981 ; 50 : 715-31 . 15 . Neilands JB . Microbial envelope proteins related to iron . Annu Rev Microbiol 1982 ; 360 : 285-309 . 16 . Tsai J, Dyer DW, Sparling PF . Loss of transferrin receptor activity in Neisseria meningitidis correlates with inability to use transferrin as an iron source . Infect Immun 1988 ; 56 : 3132-8 . 17 . Archibald FS, DeVoe IW . Removal of iron from human transferrin by Neisseria meningitidis . Microbiol Lett 1979 ; 6 : 159-62 . 18 . Schryvers AB, Morris LJ . Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis . Infect Immun 1988; 56 : 1144-9 . 19 . Lee BC, Schryvers AB . Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae . Mol Microbiol 1988; 2 : 827-9. 20. Schryvers AB . Characterization of the human transferrin and lactoferrin receptors in Haemophi/us influenzae. Mol Microbiol 1988 ; 2 : 467-72 . 21 . Wilson MB, Nakane PK . Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies . In : Knapp W, Holubar K, Wick G, eds . Immunofluorescence and related staining techniques. Amsterdam : Elsevier/North Holland Biomedical Press, 1978 ; 215-24 .