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

摘要

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.