Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Infection and Immunity logoLink to Infection and Immunity
. 1997 Dec;65(12):5057–5066. doi: 10.1128/iai.65.12.5057-5066.1997

Characterization of a new region required for macrophage killing by Legionella pneumophila.

G Segal 1, H A Shuman 1
PMCID: PMC175729  PMID: 9393796

Abstract

In a previous study, a collection of 55 Legionella pneumophila mutants defective for macrophage killing was isolated by transposon mutagenesis. In this study, nine of these mutants that belong to the same DNA hybridization group (group 3) were characterized. A wild-type DNA fragment that covers this DNA hybridization group was cloned and sequenced. This region was found to contain six new genes (designated icmT, icmS, icmR, icmQ, icmP, and icmO), five of which contain at least one transposon insertion. No transposon insertion was found in icmS. However, this gene was found to be required for macrophage killing, since a kanamycin resistance cassette introduced into icmS by gene replacement resulted in a mutant that was attenuated for macrophage killing. A plasmid containing the DNA fragment that covers this region complements all the mutants for macrophage killing, although various levels of complementation were observed for mutants in different genes. Complementation tests were also performed with plasmids containing one or two of these genes, as well as with plasmids containing nonpolar in-frame deletions. The results from these complementation tests indicated that all six genes located in this region are needed for macrophage killing and that they are probably arranged as two transcriptional units (icmTS and icmPO) and two genes (icmR and icmQ). A region upstream of the coding sequence of several icm genes may contain a potential promoter and/or regulatory site. Homology searches show that icmP and icmO bear significant homology to the trbA and trbC genes from the Salmonella R64 plasmid, respectively. The sequences of the other four genes do not show significant homology with any entries in sequence databases.

Full Text

The Full Text of this article is available as a PDF (281.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abu Kwaik Y., Pederson L. L. The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol Microbiol. 1996 Aug;21(3):543–556. doi: 10.1111/j.1365-2958.1996.tb02563.x. [DOI] [PubMed] [Google Scholar]
  2. Armstrong J. A., Hart P. D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med. 1971 Sep 1;134(3 Pt 1):713–740. doi: 10.1084/jem.134.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1992 May 11;20 (Suppl):2013–2018. doi: 10.1093/nar/20.suppl.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bellinger-Kawahara C., Horwitz M. A. Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes. J Exp Med. 1990 Oct 1;172(4):1201–1210. doi: 10.1084/jem.172.4.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berger K. H., Isberg R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol. 1993 Jan;7(1):7–19. doi: 10.1111/j.1365-2958.1993.tb01092.x. [DOI] [PubMed] [Google Scholar]
  6. Berger K. H., Merriam J. J., Isberg R. R. Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol Microbiol. 1994 Nov;14(4):809–822. doi: 10.1111/j.1365-2958.1994.tb01317.x. [DOI] [PubMed] [Google Scholar]
  7. Bjellqvist B., Hughes G. J., Pasquali C., Paquet N., Ravier F., Sanchez J. C., Frutiger S., Hochstrasser D. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis. 1993 Oct;14(10):1023–1031. doi: 10.1002/elps.11501401163. [DOI] [PubMed] [Google Scholar]
  8. Brand B. C., Sadosky A. B., Shuman H. A. The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol Microbiol. 1994 Nov;14(4):797–808. doi: 10.1111/j.1365-2958.1994.tb01316.x. [DOI] [PubMed] [Google Scholar]
  9. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  10. Cianciotto N. P., Eisenstein B. I., Mody C. H., Engleberg N. C. A mutation in the mip gene results in an attenuation of Legionella pneumophila virulence. J Infect Dis. 1990 Jul;162(1):121–126. doi: 10.1093/infdis/162.1.121. [DOI] [PubMed] [Google Scholar]
  11. Cianciotto N. P., Eisenstein B. I., Mody C. H., Toews G. B., Engleberg N. C. A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun. 1989 Apr;57(4):1255–1262. doi: 10.1128/iai.57.4.1255-1262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Collins S. J., Ruscetti F. W., Gallagher R. E., Gallo R. C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl Acad Sci U S A. 1978 May;75(5):2458–2462. doi: 10.1073/pnas.75.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feeley J. C., Gibson R. J., Gorman G. W., Langford N. C., Rasheed J. K., Mackel D. C., Baine W. B. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol. 1979 Oct;10(4):437–441. doi: 10.1128/jcm.10.4.437-441.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fields B. S. The molecular ecology of legionellae. Trends Microbiol. 1996 Jul;4(7):286–290. doi: 10.1016/0966-842x(96)10041-x. [DOI] [PubMed] [Google Scholar]
  15. Friis R. R. Interaction of L cells and Chlamydia psittaci: entry of the parasite and host responses to its development. J Bacteriol. 1972 May;110(2):706–721. doi: 10.1128/jb.110.2.706-721.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Furuya N., Komano T. Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region. J Bacteriol. 1996 Mar;178(6):1491–1497. doi: 10.1128/jb.178.6.1491-1497.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gabay J. E., Blake M., Niles W. D., Horwitz M. A. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. J Bacteriol. 1985 Apr;162(1):85–91. doi: 10.1128/jb.162.1.85-91.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hoffman P. S., Ripley M., Weeratna R. Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila. J Bacteriol. 1992 Feb;174(3):914–920. doi: 10.1128/jb.174.3.914-920.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horwitz M. A. Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes. J Exp Med. 1987 Nov 1;166(5):1310–1328. doi: 10.1084/jem.166.5.1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horwitz M. A. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med. 1983 Oct 1;158(4):1319–1331. doi: 10.1084/jem.158.4.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horwitz M. A. Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil. Cell. 1984 Jan;36(1):27–33. doi: 10.1016/0092-8674(84)90070-9. [DOI] [PubMed] [Google Scholar]
  22. Horwitz M. A., Silverstein S. C. Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin. J Clin Invest. 1983 Jan;71(1):15–26. doi: 10.1172/JCI110744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horwitz M. A., Silverstein S. C. Legionnaires' disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J Clin Invest. 1980 Sep;66(3):441–450. doi: 10.1172/JCI109874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Horwitz M. A. The Legionnaires' disease bacterium (Legionella pneumophila) inhibits phagosome-lysosome fusion in human monocytes. J Exp Med. 1983 Dec 1;158(6):2108–2126. doi: 10.1084/jem.158.6.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Imai Y., Matsushima Y., Sugimura T., Terada M. A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res. 1991 May 25;19(10):2785–2785. doi: 10.1093/nar/19.10.2785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jones T. C., Hirsch J. G. The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J Exp Med. 1972 Nov 1;136(5):1173–1194. doi: 10.1084/jem.136.5.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marra A., Horwitz M. A., Shuman H. A. The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium. J Immunol. 1990 Apr 1;144(7):2738–2744. [PubMed] [Google Scholar]
  28. Morales V. M., Bäckman A., Bagdasarian M. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene. 1991 Jan 2;97(1):39–47. doi: 10.1016/0378-1119(91)90007-x. [DOI] [PubMed] [Google Scholar]
  29. Nakai K., Kanehisa M. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins. 1991;11(2):95–110. doi: 10.1002/prot.340110203. [DOI] [PubMed] [Google Scholar]
  30. Payne N. R., Horwitz M. A. Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med. 1987 Nov 1;166(5):1377–1389. doi: 10.1084/jem.166.5.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roy C. R., Isberg R. R. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages. Infect Immun. 1997 Feb;65(2):571–578. doi: 10.1128/iai.65.2.571-578.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sadosky A. B., Wiater L. A., Shuman H. A. Identification of Legionella pneumophila genes required for growth within and killing of human macrophages. Infect Immun. 1993 Dec;61(12):5361–5373. doi: 10.1128/iai.61.12.5361-5373.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Segal G., Ron E. Z. Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure. J Bacteriol. 1993 May;175(10):3083–3088. doi: 10.1128/jb.175.10.3083-3088.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stewart G. S., Lubinsky-Mink S., Jackson C. G., Cassel A., Kuhn J. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid. 1986 May;15(3):172–181. doi: 10.1016/0147-619x(86)90035-1. [DOI] [PubMed] [Google Scholar]
  35. Swanson M. S., Isberg R. R. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun. 1995 Sep;63(9):3609–3620. doi: 10.1128/iai.63.9.3609-3620.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Szeto L., Shuman H. A. The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect Immun. 1990 Aug;58(8):2585–2592. doi: 10.1128/iai.58.8.2585-2592.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wiater L. A., Sadosky A. B., Shuman H. A. Mutagenesis of Legionella pneumophila using Tn903 dlllacZ: identification of a growth-phase-regulated pigmentation gene. Mol Microbiol. 1994 Feb;11(4):641–653. doi: 10.1111/j.1365-2958.1994.tb00343.x. [DOI] [PubMed] [Google Scholar]
  38. Wintermeyer E., Ludwig B., Steinert M., Schmidt B., Fischer G., Hacker J. Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells. Infect Immun. 1995 Dec;63(12):4576–4583. doi: 10.1128/iai.63.12.4576-4583.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES