Expression levels of human transferrin receptors in Neisseria species

Journal of Microbiological Methods 15 (1992) 321 - 326 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167- 7012/92/$ 5.00

321

MIMET 00503

Expression levels of human transferrin receptors in Neisseria species Mar Pintor, Carlos Ferreir6s, Maria Teresa Criado and Lucia Ferr6n Departamento de Micro~iologia y Parasitologia, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago cl2 Corapostela, Spain (Received 4 November 1991; revision received and accepted l0 February 1992)

Summary All pathogenic Neisseria strains synthesize transferrin receptors. To determine if transferrin receptor expression is quantitatively different in Neisseria meningitidis strains from two separate origins (carrier and invasive) and in commensal Neisseria, we applied an enzyme-linked receptor assay (ELRA) and a radiolabelled receptor assay (RLRA) to evaluate the receptor expression levels after iron-limited growth. There were significant differences in receptor expression in commensal Neisseria and Neisseria meningitidis as determined by both methods and between the two origins (invasive and carrier) when the ELRA method was used, but in the latter case, the biological significance is questionable ard the amount of transferrin receptor expressed may not be a useful indicator of the potential pathogenicity of strains. The RLRA method was more suitable for measuring transferrin receptor expression since it provided a lower background in strains lacking transferrin receptor and, consequently, made interpretation of the results easier.

Key words: Neisserie: Receptor; Transferrin

Introduction

There is considerable evidence linking pathogenicity of many human pathogens with the presence of high affinity iron uptake systems [1 ]. In spite of :he abundance and ubiquity of iron in nature, it tends to form highly insoluble complexes and is sequestered by specific macromolecules (lactoferrin and transferrin) in humans, the only ecological niche for Neisseria meningitidis, and consequently iron is not readily available for the growth of invading microorganisms. To circumvent this problem and to ensure their survival, many bacteria produce siderophores, low molecular weight soluble Fe3+-complexing substances. Nevertheless, N. meningitidis acquires Correspondence to: M.T. Criado, Departamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

322 iron via a siderophore-independent mechanism which involves a direct interaction between transferrin and the bacterial cell surface [2, 3] mediated by receptors for this proLein. Transferrin is the main source of iron during the invasive processes [1] and the mechanisms used by meningococci for iron uptake from this glycoprotein are not completely understood. The complete expression of this energy-dependent iron uptake system requires induction by growth of the bacteria under conditions of iron limitation [2, 4]. The molecular identity of the transferrin receptor is now known [5 - 9] from studie~ employing binding assays between transferrin and high numbers of N. meningitidis strains. Transferrin receptors seem to be essential since mutants blocked in their synthesis are unable to scavenge iron from ferric-transferrin complexes [ 10]. This suggests that existence of transferrin receptors is a prerequisite to the removal of iron from this glycoprotein, and as such, expression of transferrin receptors plays an important role in meningococcal pathogenesis. In this work we estimate expression levels of transferrin receptors and correlate expression with virulence in long numbers of N. meningitidis strains from different origins: 29 isolated from individuals with meningococcal illness and 20 isolated from the oropharynx of healthy carriers and, for comparative purposes, 20 commensai Neisseria (N. sicca and N. lactamica). The estimation methods used included an ELISA-derived assay (by coupling horse radish peroxidase to transferrin, ELRA) and an RIA-based assay (using radioiodinated transferrin, RLRA). Materials and Methods

Bacterial strains and growth conditions Sixty N. meningitidis strains, 20 each of invasive (obtained from infected individuals), carrier (obtained from the nasopharynx of healthy people) and commensal (strains obtained from the nasopharynx of healthy carriers and classified in species other than N. meningitidis and N. gonorrhoeae) origin taken from our laboratory collection were used in this study. The s:rains were cultured a n C h a - l ~ a .g.,- .t~,,~ for 24 h at 37 °C in a 5% CO 2 atmosphere and then one isolated colony was subcultured for 8 h under the same conditions before inoculation of 100 ml of MuellerHinton broth (MH; normal iron) or Mueller-Hinton broth with 39 #M ethylenediamine di(O-hydroxyphenyl acetic acid (MH-EDDA; iron limitation; 39 #M is the maximum concentration of EDDA which allows growth in our culture conditions). _

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Chemicals Horseradish peroxidase-coupled transferrin (HRP-Tf) was obtained from Jackson Immunoresearch Laboratories (West Grove, PA, U.S.A.). Human transferrin (Tf) was purchased from Sigma Chemical Co., St. Louis, MO, and it was at least 98% pure and significantly iron-free. 125I-Tf was obtained from Amersham International (U.K). Enzyme linked receptor assay (ELRA) For this assay, 5 ml aliquots of culture were centrifuged at 2000 x g for 5 min and the pellet was washed twice in Tris-buffered saline (TBS). The cells obtained were suspended in 2 ml of blocking solution (TBS with 1 mg of bovine serum albumin per

323 ml), incubated for 2 h at ambient temperature, and again centrifuged at 2000 × g for 10 min. The pellet was washed twice with TBS and resuspended to 108 CFU/mi. One hundred microliters of this suspension were added to wells of previously blocked Millipore MultiScreen-HV Filtration plates (with 0.2 um filters attached to the bottom of each well) followed by 100 #1 of HRP-Tf (500/~g/ml in blocking solution) and incubation for 2 h at ambient temperature in a humid chamber. After incubation, the suspensions were filtered and washed 5 times with 200 #1 of TBS followed by the addition of 100 #1 of 5-aminosalicylic acid (1 g/l in 10 mM sodium phosphate, 100 mM EDDA, pH 6) [11]. After incubation at ambient temperature for 1 h, 100 #1 of 0.3 M NaOH were added to stop the reaction. Absorbances of the filtrates were read at 450 nm in a microtiter plate reader.

Radiolabelled receptor assay (RLRA) Five milliliters aliquots of bacterial suspension as described above were centrifuged at 10000 × g for 10 min and the pellet obtained was washed twice in 5 ml of 50 mM MOPS buffer, pH 7.5. Bacteria were recovered by centrifugation and then suspended tO 5 × 10 7 CFU/ml in 5 ml of blocking solution (MOPS buffer with 1 mg of BSA per ml). One hundred microliters of :25I-Tf (corresponding to 40000 cpm) and 100 #1 of the bacterial cell suspension, previously treated with 5 mM KCN for 5 min, were added to each well of filtration plates previously blocked for 1 h with blocking solution. The plates were incubated for 20 min at ambient temperature and then reaction mixtures were filtered and washed three times with 200 #l of MOPS buffer. The filters were dried and counted in a gamma counter. Results and Discussion

Figs. 1 and 2 show the transferrin receptor levels in all strains used in this work as estimated by ELRA and RLRA, respectively. As can be seen from Fig. 1 the levels 1.5 o

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Fig. 2. Expression of the transferrin receptor in Neisseria strains evaluated by a radiolabelled receptor assay. Bars indicate mean + S.E., and points show individual measures. Open bars show results from controls grown under iron sufficience. Dashed bars indicate results obtained with cells grown in the presence of EDDA.

of expression are only slightly different in carrier and inva~ive strains when analyzed by ELRA, and they are not significantly different by RLRA (Fig. 2). This similar ability of N. meningitidis strains of different sources to bind transferrin suggests that the potential virulence of the strains may be similar and that the reason for their different behaviour could be related to the functional characteristics of the receptors, possibly by having different affinity constants, and so different capacities for iron uptake. Alternatively, other components of the iron uptake system may function at significantly different levels. As expected, there were very significant differences between levels of receptor expression under normal and iron-limited culture conditions, regardless of the assay method employed (Figs. i, 2) for evaluation. As seen in Fig. 3, the increase in expression of the receptors when induced by growth under iron-limiting conditions varies considerably between strains and also with the method used for estimation. Significant differences (p< 0.05) in the level of transferrin receptor expression were found between both sources of N. meningitidis and between these and the commensal Neisseria, either under normal growth conditions or when cells were grown in ironrestricted media (Figs. 1 and 2). Consequently, since the transferrin-binding activity is displayed by a great number of strains it cannot be considered a determinant of virulence. Also, if transferrin-binding were correlated with iron-uptake ability (which remains to be demonstrated) although it is essential for invasion, the last still could not be considered a determinant of the pathogenic potential of the strains. Whereas the statistical analyses indicate (when the ELRA method is used) significant differences between both sources of N. meningitidis (carrier and invasive isolates, Fig. 1), in this case the level of significance is low, suggesting that their biological significance may not be relevant. Lee and Bryan [15] were also unable to demonstrate differences in the level of transferrin-binding activity between gonococcal strains causing localized and invasive diseases. Figs. 1 and 2 also show the diversity of receptor expression in strains from the same

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Per cent variations in the expression of the transferrin receptor of individual Neisseria strains when grown under iron deprivation.

source. Due to the above variation, the level of expression of transferrin receptor may not be a useful indicator to determine the origin of Neisseria strains or their potential virulence. Colonization of mucosal surfaces by Neisseriaceae is a frequent phenomenon, especially colonization of the oropharynx by N. meningitidis and commensal Neisseria species (N. sicca, N. lactamica and others). In this environment iron is unavailable for growth of bacteria because it is bound to lactoferrin. In the bloodstream meningococcal infections are prevented because iron is sequestered by transferrin. Thus, if bacterial cells are to grow in these two environments they must possess mechanisms to remove ferric iron from lactoferrin and transferrino Consequently, the ability to use transferrin as an iron source would be expected to be an important virulence factor. Our results indicate that all N. meningitidis strains tested express transferrin :eceptors, independent of their origin or the assay method employed. However, only 35°70 and 40070 of the commensal Neisseria strains possessed receptors as determined by ELRA and RLRA, respectively. These results disagree with those of Mickelsen and Sparling [12] who indicated that 22070 of their strains were able to grow with iron scavenged from transferrin, and with the results of Schryvers and Lee [13] who found that 80070 of their commensal strains were able to bind transferrin in a dot-blot assay with HRP-Tf. Perhaps in the latter case, transferrin binding could be an overestimate of the actual ability to obtain sequestered iron from transferrin. The enzyme-linked receptor assay used in this work is similar to a whole-cell ELISA, but uses filters to retain bacteria instead of allowing their adsorption to the plate surfaces in order to prevent cell numbers from changing during washing procedures. Abdillahi and Poolman [14] consider whole-cell ELISA for the screening of N. meningitidis antigens in their native state since it is a rapid, easy, sensitive and reproducible technique. Nevertheless, we determined that the levels of reaction of some of the commensal strains used, which lack transferrin receptors (as determined by both a whole-cell dot-blott assay and SDS-PAGE-electrotransfer of membrane

326 protein, revealed with HRP-Tf) are relatively high (A540 =0.1 -0.2). In contrast, the radiolabelled receptor assay results in essentially no detectable binding of transferrin to the strains lacking receptors, results similar to those obtained in controls without bacteria.

Acknowledgements This work was supported by Grants PM88-0209 of the Direcci6n General de la Investigaci6n Cientifica y T6cnica (DGICYT; Spanish Government) and XUGA81503988 of the Direcci6n Xeral de Universidades de la Xunta de Galica (Autonomic Government).

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