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Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

ABSTRACT: A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive.

SUBMITTER: Schwan WR

PROVIDER: S-EPMC175682 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

Schwan W R WR Kopecko D J DJ

Infection and immunity 19971101 11

A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mu ...[more]

PMID: 9353061

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Project description: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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Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

Publications

Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

Similar Datasets

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