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. 1996 Jan;64(1):91–99. doi: 10.1128/iai.64.1.91-99.1996

Detection and characterization by differential PCR of host eukaryotic cell genes differentially transcribed following uptake of intracellular bacteria.

W R Schwan 1, S Kügler 1, S Schüller 1, D J Kopecko 1, W Goebel 1
PMCID: PMC173732  PMID: 8557379

Abstract

Host eukaryotic cell genes that are differentially transcribed after phagocytosis of various pathogenic and nonpathogenic bacterial cells were identified by a differential PCR (DPCR) system. This DPCR procedure favors detection and isolation of host genes affected at the transcriptional level by selecting for poly(A) tails but differs substantially from reverse transcription-PCR. Several unidentified macrophage gene fragments from genes that were either transcriptionally activated or downregulated following uptake of Listeria monocytogenes into J774 mouse macrophage cells were initially defined by this DPCR procedure. Because of the sensitivity of the DPCR technique, all of the genes exhibited less than a 10-fold difference in transcription compared with noninfected cells as measured by limiting-dilution PCR. One of the gene fragments has a very high level of homology with a mitogen-activated protein kinase phosphatase (MKP-1), whereas the other affected fragments showed no homologies to known gene sequences. In addition, one of the gene fragments (WS30-B2/1) was specifically downregulated after L. monocytogenes uptake and another gene was repressed by uptake of either Shigella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activated following general phagocytosis (i.e., following uptake of any species of bacterium tested). Further characterization of the affected genes was conducted by using mutants of L. monocytogenes. A hemolysin-negative mutant of L. monocytogenes failed to elicit transcriptional regulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosome may be required to initiate these responses. Furthermore, mutants with mutations in mpl and actA, two genes whose gene products are involved in actin polymerization and intrahost spread, also did not induce regulation of WS10-B4/14. These results demonstrate that (i) DPCR can identify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these genes may be new or may never before have been linked to interactions between hosts and pathogens.

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Selected References

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  1. Adam T., Arpin M., Prévost M. C., Gounon P., Sansonetti P. J. Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells. J Cell Biol. 1995 Apr;129(2):367–381. doi: 10.1083/jcb.129.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bliska J. B., Galán J. E., Falkow S. Signal transduction in the mammalian cell during bacterial attachment and entry. Cell. 1993 Jun 4;73(5):903–920. doi: 10.1016/0092-8674(93)90270-z. [DOI] [PubMed] [Google Scholar]
  4. Brundage R. A., Smith G. A., Camilli A., Theriot J. A., Portnoy D. A. Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11890–11894. doi: 10.1073/pnas.90.24.11890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Charles C. H., Abler A. S., Lau L. F. cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein. Oncogene. 1992 Jan;7(1):187–190. [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dabiri G. A., Sanger J. M., Portnoy D. A., Southwick F. S. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6068–6072. doi: 10.1073/pnas.87.16.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Domann E., Wehland J., Rohde M., Pistor S., Hartl M., Goebel W., Leimeister-Wächter M., Wuenscher M., Chakraborty T. A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. EMBO J. 1992 May;11(5):1981–1990. doi: 10.1002/j.1460-2075.1992.tb05252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flamm R. K., Hinrichs D. J., Thomashow M. F. Introduction of pAM beta 1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect Immun. 1984 Apr;44(1):157–161. doi: 10.1128/iai.44.1.157-161.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Francis C. L., Ryan T. A., Jones B. D., Smith S. J., Falkow S. Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria. Nature. 1993 Aug 12;364(6438):639–642. doi: 10.1038/364639a0. [DOI] [PubMed] [Google Scholar]
  14. Galán J. E., Pace J., Hayman M. J. Involvement of the epidermal growth factor receptor in the invasion of cultured mammalian cells by Salmonella typhimurium. Nature. 1992 Jun 18;357(6379):588–589. doi: 10.1038/357588a0. [DOI] [PubMed] [Google Scholar]
  15. HENLE G., DEINHARDT F. The establishment of strains of human cells in tissue culture. J Immunol. 1957 Jul;79(1):54–59. [PubMed] [Google Scholar]
  16. Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
  17. Karunasagar I., Senghaas B., Krohne G., Goebel W. Ultrastructural study of Listeria monocytogenes entry into cultured human colonic epithelial cells. Infect Immun. 1994 Aug;62(8):3554–3558. doi: 10.1128/iai.62.8.3554-3558.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kocks C., Gouin E., Tabouret M., Berche P., Ohayon H., Cossart P. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell. 1992 Feb 7;68(3):521–531. doi: 10.1016/0092-8674(92)90188-i. [DOI] [PubMed] [Google Scholar]
  19. Kuhn M., Goebel W. Induction of cytokines in phagocytic mammalian cells infected with virulent and avirulent Listeria strains. Infect Immun. 1994 Feb;62(2):348–356. doi: 10.1128/iai.62.2.348-356.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leimeister-Wächter M., Haffner C., Domann E., Goebel W., Chakraborty T. Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8336–8340. doi: 10.1073/pnas.87.21.8336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liang P., Averboukh L., Pardee A. B. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 1993 Jul 11;21(14):3269–3275. doi: 10.1093/nar/21.14.3269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  23. Mou L., Miller H., Li J., Wang E., Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun. 1994 Mar 15;199(2):564–569. doi: 10.1006/bbrc.1994.1265. [DOI] [PubMed] [Google Scholar]
  24. Pace J., Hayman M. J., Galán J. E. Signal transduction and invasion of epithelial cells by S. typhimurium. Cell. 1993 Feb 26;72(4):505–514. doi: 10.1016/0092-8674(93)90070-7. [DOI] [PubMed] [Google Scholar]
  25. Patanjali S. R., Parimoo S., Weissman S. M. Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1943–1947. doi: 10.1073/pnas.88.5.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pistor S., Chakraborty T., Niebuhr K., Domann E., Wehland J. The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton. EMBO J. 1994 Feb 15;13(4):758–763. doi: 10.1002/j.1460-2075.1994.tb06318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Platzer C., Richter G., Uberla K., Müller W., Blöcker H., Diamantstein T., Blankenstein T. Analysis of cytokine mRNA levels in interleukin-4-transgenic mice by quantitative polymerase chain reaction. Eur J Immunol. 1992 May;22(5):1179–1184. doi: 10.1002/eji.1830220511. [DOI] [PubMed] [Google Scholar]
  28. Plum G., Clark-Curtiss J. E. Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages. Infect Immun. 1994 Feb;62(2):476–483. doi: 10.1128/iai.62.2.476-483.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ralph P., Prichard J., Cohn M. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. J Immunol. 1975 Feb;114(2 Pt 2):898–905. [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schwan W. R., Goebel W. Host cell responses to Listeria monocytogenes infection include differential transcription of host stress genes involved in signal transduction. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6428–6432. doi: 10.1073/pnas.91.14.6428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schwan W. R., Seifert H. S., Duncan J. L. Growth conditions mediate differential transcription of fim genes involved in phase variation of type 1 pili. J Bacteriol. 1992 Apr;174(7):2367–2375. doi: 10.1128/jb.174.7.2367-2375.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sokolovic Z., Riedel J., Wuenscher M., Goebel W. Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions. Mol Microbiol. 1993 Apr;8(2):219–227. doi: 10.1111/j.1365-2958.1993.tb01566.x. [DOI] [PubMed] [Google Scholar]
  34. Southwick F. S., Purich D. L. Arrest of Listeria movement in host cells by a bacterial ActA analogue: implications for actin-based motility. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5168–5172. doi: 10.1073/pnas.91.11.5168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tang P., Rosenshine I., Finlay B. B. Listeria monocytogenes, an invasive bacterium, stimulates MAP kinase upon attachment to epithelial cells. Mol Biol Cell. 1994 Apr;5(4):455–464. doi: 10.1091/mbc.5.4.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Theriot J. A., Rosenblatt J., Portnoy D. A., Goldschmidt-Clermont P. J., Mitchison T. J. Involvement of profilin in the actin-based motility of L. monocytogenes in cells and in cell-free extracts. Cell. 1994 Feb 11;76(3):505–517. doi: 10.1016/0092-8674(94)90114-7. [DOI] [PubMed] [Google Scholar]
  37. Welsh J., Chada K., Dalal S. S., Cheng R., Ralph D., McClelland M. Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 1992 Oct 11;20(19):4965–4970. doi: 10.1093/nar/20.19.4965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wong K. K., McClelland M. Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):639–643. doi: 10.1073/pnas.91.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhao S., Ooi S. L., Pardee A. B. New primer strategy improves precision of differential display. Biotechniques. 1995 May;18(5):842-6, 848, 850. [PubMed] [Google Scholar]
  40. Zheng C. F., Guan K. L. Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen-induced Thr/Tyr protein phosphatase. J Biol Chem. 1993 Aug 5;268(22):16116–16119. [PubMed] [Google Scholar]
  41. Zychlinsky A., Prevost M. C., Sansonetti P. J. Shigella flexneri induces apoptosis in infected macrophages. Nature. 1992 Jul 9;358(6382):167–169. doi: 10.1038/358167a0. [DOI] [PubMed] [Google Scholar]

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