Intracellular replication of Edwardsiella tarda in murine macrophage is dependent on the type III secretion system and induces an up-regulation of anti-apoptotic NF-κB target genes protecting the macrophage from staurosporine-induced apoptosis (original) (raw)
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2005
Certain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-B, which depends on IB kinase  (IKK). To better understand how p38 and NF-B maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival.
Immunity, 2005
Certain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-B, which depends on IB kinase  (IKK). To better understand how p38 and NF-B maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival.
Inflammation Research, 2006
Objective and Background: Macrophages are known to be one of the initial responders to bacterial infection. While infection of macrophages with bacteria induces apoptosis, a pro-inflammatory response is also elicited. Thus, the aim of this study is to further elucidate the differential effect of infections with bacteria on the survival and function of macrophages. Methods and results: THP-1 monocytic cells induced to differentiate into macrophages were infected with non-pathogenic Escherichia coli (E. coli) and analyzed for apoptosis and inflammatory response over time. Following infection with E. coli macrophages underwent apoptosis which was reduced by the general caspase inhibitor, zVAD.fmk. Inhibition of caspase activity resulted in increased DNA binding activity of NF-κB and enhanced production of NF-κB-dependent reporter gene expression following infection. Increased activity of NF-κB was independent of IκBα since IκBα degradation was unaffected by zVAD.fmk. Further, suppression of caspase activity reduced the proteolytic cleavage of NF-κB. The increased activity of NF-κB in the zVAD.fmk-treated macrophages was associated with a markedly enhanced production of pro-inflammatory cytokines and elimination of E. coli. Conclusion: These data indicate that infection of macrophages with E. coli induces a caspase-dependent inhibition of NF-κB that results in a reduced production of pro-inflammatory cytokines and impaired clearance of bacteria.
Infection and Immunity, 2004
Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA ؊ E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA ؉ E. coli induces the expression of Bcl XL , an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA ؊ E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA ؉ E. coli-infected macrophages. In addition, OmpA ؉ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA ؊ E. coli with a plasmid containing the ompA gene restored the ability of OmpA ؊ E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.
FEMS microbiology letters, 2004
Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever. In human infections, the primary target of R. rickettsii infection is vascular endothelium. Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R. rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3. To understand upstream signaling mechanisms, we have determined the effect of NF-kappaB blockade on the status of different Bcl-2 (B-cell lymphoma 2) proteins in this study. Quantitative analysis following TUNEL and Hoechst staining confirmed that infection of endothelial cells with R. rickettsii for 6 h in the presence of a specific NF-kappaB inhibitor, MG132, resulted in induction of apoptosis. Infection-induced apoptosis of EC was associated with decreased level of Bid and accumulation of Ba...
Diseases of Aquatic Organisms, 2009
The Type III secretion system is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. We identified the secreted proteins of the Type III secretion system by comparing the wild-type strain and the Type III mutant mET1229. The wild-type strain secreted 55, 25, and 22 kDa proteins into the culture supernatant, whereas the Type III mutant did not. These proteins were identified as EseB, EseC, and EseD and are similar in sequence to Salmonella SseB, SseC, and SseD that function as a translocon. The EseB, EseC, and EseD knockout mutants did not replicate in murine macrophages, suggesting that these proteins are essential for intracellular replication of E. tarda. Highest secretion of EseBCD proteins was observed when bacterial cells were cultured in neutral and alkaline pHs but not in acidic pH. When the pH of the phagosomes was examined using an acidotropic probe, the phagosomes containing the wild-type strain showed neutral pH, whereas those containing the Type III mutant exhibited acidic pH. These results suggest that the Type III-dependent interference with formation of the acidic environment in phagosomes is essential for intracellular replication of bacteria in murine macrophages.
African journal of microbiology research
The activation and nuclear translocation of NF-B and the expression of the pro-inflammatory cytokine genes by macrophages infected with the attenuated Brucella abortus RB51 and virulent 2308 strains were evaluated. pIBα and NF-B were determined by immunoblot, and cytokines IFN- and IL12 mRNA were determined by reverse transcriptase polymerase chain reaction (qPCR) and translocation of NF-B protein to the nucleus was determined by electrophoretic mobility shift assay (EMSA). We demonstrate that the attenuated B. abortus RB51 strain stimulates cells resulting in NF-B activation and nuclear translocation, during experimental infection in macrophages J774A.1 which induced a pro-inflammatory response producing IL-6, 12 TNF-s INF-g and iNOS. The virulent strain B. abortus 2308 also stimulated the cells but induced a p50 homodimer of NF-B which is inactive. The p50 homodimer of NF-B binds to DNA, and thus blocked the activation of pro-inflammatory cytokines genes. Therefore, an evasion mechanism of the strain 2308 is to produce an inactive homodimer of NF-B which does not give rise to pro-inflammatory response to eliminate the bacteria.
Journal of Leukocyte Biology, 2005
Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor ␣ (TNF-␣), interleukin (IL)-1, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-␣, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1, IL-1, and