DETECTION AND CHARACTERIZATION BY DIFFERENTIAL PCR OF HOST EUKARYOTICCELL GENES DIFFERENTIALLY TRANSCRIBED FOLLOWING UPTAKE OF INTRACELLULAR BACTERIA

Host eukaryotic cell genes that are differentially transcribed after p hagocytosis of various pathogenic and nonpathogenic bacterial cells we re identified by a differential PCR (DPCR) system, This DPCR procedure favors detection and isolation of host genes affected at the transcri ptional 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 d ownregulated following uptake of Listeria monocytogenes into J774 mous e macrophage cells were initially defined by this DPCR procedure, Beca use of the sensitivity of the DPCR technique, all of the genes exhibit ed less than a 10-fold difference in transcription compared with nonin fected cells as measured by limiting-dilution PCR, One of the gene fra gments has a very high level of homology with a mitogen-activated prot ein kinase phosphatase (MKP-1), whereas the other affected fragments s howed no homologies to known gene sequences, In addition, one of the g ene fragments (WS30-B2/1) was specifically downregulated after L. mono cytogenes uptake and another gene was repressed by uptake of either Sh igella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activ ated following general phagocytosis (i.e., following uptake of any spe cies of bacterium tested). Further characterization of the affected ge nes was conducted by using mutants of L. monocytogenes. A hemolysin-ne gative mutant of L. monocytogenes failed to elicit transcriptional reg ulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosom e may be required to initiate these responses, Furthermore, mutants wi th mutations in mpl and actA, two genes whose gene products are involv ed in actin polymerization and intrahost spread, also did not induce r egulation of WS10-B4/14. These results demonstrate that (i) DPCR can i dentify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these gen es may be new or may never before have been linked to interactions bet ween hosts and pathogens.