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

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.

Lactobacillus gasseriK7 modulates the blood cell transcriptome of conventional mice infected withEscherichia coliO157:H7

“…RT-PCR also confirmed the slight up-regulation of two genes of the mitogen-activated protein (MAP) kinase family, Map4k2 and Mapk11. MAP kinase cascades are crucial in signal transduction pathways in the regulation of the host inflammatory response to infection (Schwan et al 1996;Dahan et al 2002;Zhong and Kyriakis 2007). Also, E. coli O157:H7 activates MAP kinases in cultured intestinal epithelial cells (Ledesma et al 2010).…”

Rickettsia rickettsii infection of cultured human endothelial cells induces NF-kappaB activation

“…1). This oligonucleotide binds to several NF-B species in EC, which are composed predominantly of homo-and heterodimers of p50 and p65 subunits, including a homodimer of a 50-kDa subunit (p50) and a heterodimer of p50 and a 65-kDa subunit (p65 or relA) (24). The nuclear complexes activated by R. rickettsii infection reacted in gel supershift assays with polyclonal antibodies to both p50 and p65 ( Fig.…”

“…2), implicating R. rickettsii entry. L. monocytogenes infection of macrophage-like cells results in activation of p50-p65 prior to invasion and escape from the phagolysosome (7,24). The late phase of activation during R. rickettsii infection could perhaps be explained by production of a rickettsial metabolite or replication of the organisms and the early phase could be induced by cell surface interaction, entry, or simple contact of organisms with the host cell cytoplasm.…”

Expression of listeriolysin O by intracellular Listeria monocytogenes following infection of lipopolysaccharide-treated or untreated J774.1 macrophage-like cells

“…When L. monocytogenes infected J774.1 cells, several host proteins were altered. Expression of host heat shock proteins [21] and mitogen-activated protein kinase phosphatase [22] by 5774.1 cells was upregulated upon infection by L. monocytogenes of 5774.1 cells. In addition, the infection by L. monocytogenes of J774.1 cells decreased unknown host proteins [22].…”

“…Expression of host heat shock proteins [21] and mitogen-activated protein kinase phosphatase [22] by 5774.1 cells was upregulated upon infection by L. monocytogenes of 5774.1 cells. In addition, the infection by L. monocytogenes of J774.1 cells decreased unknown host proteins [22]. A hemolysin-negative mutant failed to elicit regulation or elicited it only minimally, suggesting that escape into the host cytoplasm may be required for initiation of these responses.…”