A Macrophage Invasion Mechanism of Pathogenic Mycobacteria
10.1126/science.277.5329.1091
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Tuberculosis is the leading cause of death due to an infectious organism, killing an estimated 3 million people annually. Mycobacterium tuberculosis , the causative agent of tuberculosis, and other pathogenic mycobacteria require entry into host macrophages to initiate infection. An invasion mechanism was defined that was shared among pathogenic mycobacteria including M. tuberculosis , M. leprae , and M. avium but not by nonpathogenic mycobacteria or nonmycobacterial intramacrophage pathogens. This pathway required the association of the complement cleavage product C2a with mycobacteria resulting in the formation of a C3 convertase. The mycobacteria-associated C2a cleaved C3, resulting in C3b opsonization of the mycobacteria and recognition by macrophages.

Bibliography

Schorey, J. S., Carroll, M. C., & Brown, E. J. (1997). A Macrophage Invasion Mechanism of Pathogenic Mycobacteria. Science, 277(5329), 1091–1093.

Authors 3
  1. Jeffrey S. Schorey (first)
  2. Michael C. Carroll (additional)
  3. Eric J. Brown (additional)
References 36 Referenced 145
  1. Fenton M. J., Vermeulen M. W., Infect. Immun. 64, 683 (1996). (10.1128/iai.64.3.683-690.1996) / Infect. Immun. by Fenton M. J. (1996)
  2. Weissler J. C., Am. J. Med. Sci. 305, 52 (1993). (10.1097/00000441-199301000-00009) / Am. J. Med. Sci. by Weissler J. C. (1993)
  3. Ellner J. J., Goldberger M. J., Parenti D. M., J. Infect. Dis. 163, 1326 (1991). (10.1093/infdis/163.6.1326) / J. Infect. Dis. by Ellner J. J. (1991)
  4. Schlesinger L. S., Horwitz M. A., J. Immunol. 147, 1983 (1991); (10.4049/jimmunol.147.6.1983) / J. Immunol. by Schlesinger L. S. (1991)
  5. Schlesinger L. S., Bellinger-Kawahara C. G., Payne N. R., Horwitz M. A., ibid. 144, 277 (1990). / ibid. by Schlesinger L. S. (1990)
  6. Launois P., et al., Int. J. Lepr. Other Mycobact. Dis. 60, 225 (1992). / Int. J. Lepr. Other Mycobact. Dis. by Launois P. (1992)
  7. 10.1126/science.8303277
  8. Equine serum (250 ml) was heat treated by incubation at 56°C for 30 min. The serum was diluted to 1 liter with Hanks' buffered saline 1 mM CaCl 2 MnCl 2 and MgCl 2 (HBSS 3+ ) and run through a column containing 4 ml of packed concanavalin A (Con A)–agarose equilibrated in HBSS 3+ at a flow rate of 0.35 ml/min. The column was washed with HBSS 3+ and the bound proteins eluted with 14 ml of HBSS 0.1 M α-methyl mannoside. The sample (13.5 ml) was diluted to 140 ml with 20 mM tris (pH 7.6) and loaded onto a heparin-Sepharose column containing 8 ml of heparin-Sepharose resin equilibrated in 20 mM tris (pH 7.6). The bound protein was eluted with 14 ml of 20 mM tris 1.2 M NaCl (pH 7.6). The heparin-Sepharose elution (13.5 ml) was diluted to 140 ml with 20 mM tris (pH 7.6) (final NaCl concentration = 116 mM). This material was loaded onto a Mono Q fast protein liquid chromatography (FPLC) HR 5/5 anion-exchange column equilibrated in 20 mM tris 100 mM NaCl (pH 7.6) with a Waters FPLC at a flow rate of 1.0 ml/min. Flow-through was collected and used in the subsequent purification step. The column flow-through was concentrated and the buffer exchanged into 25 mM triethylamine 100 mM NaCl (pH 11.0). Final volumes of sample equaled 3.5 ml. A portion of this sample (3.4 ml) was loaded onto a Mono P FPLC 5/5 chromatofocusing column equilibrated in 25 mM triethylamine 100 mM NaCl (pH 11.0) buffer. Bound proteins were eluted with Pharmalyte (pH 8.0 to 10.5) 100 mM NaCl (pH 8.0). Fractions (1 ml) were collected and the pH adjusted to neutrality. Fractions were analyzed for protein purity by SDS–polyacrylamide gel electrophoresis (PAGE) silver stained and examined for activity in the mycobacteria-ingestion assay. Fraction 22 which showed the highest activity gave only a single 70-kD band on SDS-PAGE indicating that the protein was purified to apparent homogeneity. A sample (0.5 ml) of this fraction was sent to Midwest Analytical (St. Louis MO) for analysis of NH 2 -terminal sequence. Partially purified C2a used in some experiments was obtained by Con A fractionation of heat-treated (56°C) human serum (mixed lymphocyte reaction–C + serum North American Biologicals Miami FL) or equine serum as described above or by batch mode. Protein eluted with HBSS 0.1 M α-methyl mannoside was exchanged into phosphate-buffered saline (PBS) and used in the experiments at ∼0.15 mg/ml.
  9. J. S. Schorey et al. data not shown.
  10. Seventy-kilodalton protein (NH 2 -terminus) KIQIQRSGHLNLYLLLD A SQ; human C2a (NH 2 -terminus) KIQIQRSGHLNLYLLLD C SQ. The single–amino acid difference between the purified equine protein and the NH 2 -terminus of human C2a is shown in bold. Abbreviations for the amino acid residues: A Ala; C Cys; D Asp; G Gly; H His; I Ile; K Lys; L Leu; N Asn; Q Gln; R Arg; S Ser; and Y Tyr.
  11. Sitomer G., Stroud R. M., Mayer M. M., Immunochemistry 3, 57 (1966); (10.1016/0019-2791(66)90282-5) / Immunochemistry by Sitomer G. (1966)
  12. Muller-Eberhard H. J., Polley M. J., Calcott M. A., J. Exp. Med. 125, 359 (1967). (10.1084/jem.125.2.359) / J. Exp. Med. by Muller-Eberhard H. J. (1967)
  13. Stroud R. M., Mayer M. M., Miller J. A., McKenzie A. T., Immunochemistry 3, 163 (1966); (10.1016/0019-2791(66)90182-0) / Immunochemistry by Stroud R. M. (1966)
  14. Polley M. J., Muller-Eberhard H. J., J. Exp. Med. 128, 533 (1968) . (10.1084/jem.128.3.533) / J. Exp. Med. by Polley M. J. (1968)
  15. To make the protein A–anti-C2 we incubated 0.05 ml of the rabbit polyclonal serum against human C2 which cross-reacts with equine C2 with 0.05 ml of packed protein A–Sepharose (Sigma) in 0.9 ml of PBS for 2 hours at 4°C. The protein A–Sepharose with coupled antibody was washed twice with PBS and resuspended in 0.1 ml of PBS. Approximately 0.2 mg of Con A–fractionated proteins from heat-treated equine serum or human serum (obtained from a mixed lymphocyte reaction) in PBS was incubated with protein A–Sepharose or protein A–Sepharose with bound anti-C2 for 3 hours at 4°C. The protein A was subsequently removed and the treated samples tested for the ability to support M. avium invasion of human monocyte-derived macrophages. No detectable C2 or C2 fragments were observed in the anti-C2–treated samples by protein immunoblot analysis with goat anti-C2 (human).
  16. Wessels M. R., et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11490 (1995). (10.1073/pnas.92.25.11490) / Proc. Natl. Acad. Sci. U.S.A. by Wessels M. R. (1995)
  17. Stokes R. W., Haidl J. D., Jefferies W. A., Speert D. P., J. Immunol. 151, 7067 (1993); (10.4049/jimmunol.151.12.7067) / J. Immunol. by Stokes R. W. (1993)
  18. Schlesinger L. S., ibid. 150, 2920 (1993) . / ibid. by Schlesinger L. S. (1993)
  19. F. S. Cole W. J. Matthews Jr. J. T. Marino D. J. Gash H. R. Colten ibid. 125 1120 (1980); (10.4049/jimmunol.125.3.1120)
  20. Schorlemmer H. U., Bitter-Suermann D., Allison A. C., Immunology 32, 929 (1977); / Immunology by Schorlemmer H. U. (1977)
  21. Schorlemmer H. U., Allison A. C., ibid. 31, 781 (1976). / ibid. by Schorlemmer H. U. (1976)
  22. Brostoff J., Lenzini L., Rottoli P., Rottoli L., Tubercle 62, 169 (1981); (10.1016/0041-3879(81)90002-7) / Tubercle by Brostoff J. (1981)
  23. Carr R. I., et al., Clin. Exp. Immunol. 39, 562 (1980). / Clin. Exp. Immunol. by Carr R. I. (1980)
  24. K. S. S. Sai-Baba K. D. Moudgil R. C. Jain L. M. Srivastava Tubercule 71 103 (1990). (10.1016/0041-3879(90)90004-R)
  25. Lepow I. H., Naff G. B., Todd E. W., Pensky J., Hinz C. F., J. Exp. Med. 117, 983 (1963). (10.1084/jem.117.6.983) / J. Exp. Med. by Lepow I. H. (1963)
  26. Zheleznyak A., Brown E. J., J. Biol. Chem. 267, 12042 (1992). (10.1016/S0021-9258(19)49803-7) / J. Biol. Chem. by Zheleznyak A. (1992)
  27. The human or mouse macrophages were plated onto eight-well LabTek chamber Permanox slides 2 to 14 hours before the infection experiments at 5 × 10 4 cells per well. A mycobacteria to macrophage ratio of 10:1 was used in all experiments. This ratio was also used when other organisms were tested. The macrophages mycobacteria and C2a-containing material were incubated for 2 hours at 37°C 5% CO 2 in 0.2 ml of RPMI 0.5% human serum albumin (infection media). The cells were washed with PBS fixed with acetone/methanol (1:1) and stained for pathogenic mycobacteria with a TB Fluorescent Stain Kit O (Becton Dickinson Cockney MD). Macrophages infected with M. smegmatis M. vaccae and M. phlei were fixed in 2.5% glutaraldehyde and stained with acridine orange. The number of macrophages containing attached and internalized mycobacteria was scored visually with a Nikon Microphot-FX fluorescent microscope. The number of macrophages with associated mycobacteria per 100 cells is given as percent infected. For some experiments the data are presented as the mean ± SD for 300 macrophages counted and represent variability within a given experimental chamber well. Defining the number of macrophages infected with organisms other then mycobacteria was done by visual inspection with a Zeiss Axioskop microscope.
  28. A C1-containing fraction was isolated from human serum as described (18). Antibody-coated erythrocytes (EA) (Sigma) were incubated in 0.9 ml of Isogevers Veronal-buffered saline 0.15 mM CaCl 2 1 mM MgCl 2 (GVBS 2+ ) sequentially with 0.1 ml of C1 fraction purified commercial C4 (50 000 U) and C2 (25 000 U) (Advanced Research Technologies San Diego CA). Each incubation was for 30 min at 30°C except for the last incubation after addition of C2 which was for 2 hours. EA were washed with GVBS 2+ after incubation of GVBS 2+ with C1 and C4. After the final 2-hour incubation EA were removed by centrifugation and 0.1 ml of the supernatant was tested for the ability to support M. avium invasion of human monocyte-derived macrophages.
  29. Roach T. I., Barton C. H., Chatterjee D., Blackwell J. M., J. Immunol. 150, 1886 (1993). (10.4049/jimmunol.150.5.1886) / J. Immunol. by Roach T. I. (1993)
  30. Blood from C3 −/− mice were obtained through the eye orbital. The blood was allowed to clot for 30 min at 25°C and then 30 min at 4°C. The serum was cleared of clotted blood by centrifugation and immediately treated with a bovine serum albumin (BSA)–anti-BSA immune complex for 1 hour at 37°C. The immune complex was removed by centrifugation and the serum incubated at 56°C for 30 min to inactivate the complement pathways and stored at −70°C until needed.
  31. Wright S. D., et al., Proc. Natl. Acad. Sci. U.S.A. 80, 5699 (1983). (10.1073/pnas.80.18.5699) / Proc. Natl. Acad. Sci. U.S.A. by Wright S. D. (1983)
  32. Gresham H. D., Graham I. L., Anderson D. C., Brown E. J., J. Clin. Invest. 88, 588 (1991). (10.1172/JCI115343) / J. Clin. Invest. by Gresham H. D. (1991)
  33. Hoffman R. A., Kung P. C., Hansen W. P., Goldstein G., Proc. Natl. Acad. Sci. U.S.A. 77, 4914 (1980). (10.1073/pnas.77.8.4914) / Proc. Natl. Acad. Sci. U.S.A. by Hoffman R. A. (1980)
  34. Brown E., Hooper L., Ho T., Gresham H., J. Cell Biol. 111, 2785 (1990). (10.1083/jcb.111.6.2785) / J. Cell Biol. by Brown E. (1990)
  35. Gresham H. D., Goodwin J. L., Allen P. M., Anderson D. C., Brown E. J., ibid. 108, 1935 (1989). / ibid. by Gresham H. D. (1989)
  36. We thank C. Hammer (NIH) for polyclonal anti-C2 and D. Russell (Washington University) for the L. mexicana and L. monocytogenes. Supported in part by NIH grant AI33348. J.S.S. is an American Foundation for Urologic Disease–Dornier Medical System Research Scholar.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:50 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:20 p.m.)
Indexed 3 months, 2 weeks ago (May 20, 2025, 5:16 a.m.)
Issued 28 years ago (Aug. 22, 1997)
Published 28 years ago (Aug. 22, 1997)
Published Print 28 years ago (Aug. 22, 1997)
Funders 0

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@article{Schorey_1997, title={A Macrophage Invasion Mechanism of Pathogenic Mycobacteria}, volume={277}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.277.5329.1091}, DOI={10.1126/science.277.5329.1091}, number={5329}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Schorey, Jeffrey S. and Carroll, Michael C. and Brown, Eric J.}, year={1997}, month=aug, pages={1091–1093} }