Serratia marcescens has long been recognized as an important opportunistic pathogen, but the underlying pathogenesis mechanism is not completely clear. Here, we report a key pathogenesis pathway in S. marcescens comprising the RssAB two-component system and its downstream elements, FlhDC and the dominant virulence factor hemolysin ShlBA. Expression of shlBA is under the positive control of FlhDC, which is repressed by RssAB signaling. At 37°C, functional RssAB inhibits swarming, represses hemolysin production, and promotes S. marcescens biofilm formation. In comparison, when rssBA is deleted, S. marcescens displays aberrant multicellularity favoring motile swarming with unbridled hemolysin production. Cellular and animal infection models further demonstrate that loss of rssBA transforms this opportunistic pathogen into hypervirulent phenotypes, leading to extensive inflammatory responses coupled with destructive and systemic infection. Hemolysin production is essential in this context. Collectively, a major virulence regulatory pathway is identified in S. marcescens.The Gram-negative bacterium Serratia marcescens is an important opportunistic pathogen that causes a wide range of diseases and clinical presentations with high morbidity (25). S. marcescens frequently causes outbreaks in intensive and neonatal care units, and the occurrence of multiple-antibioticresistant strains has further exacerbated clinical treatment difficulties (3, 39). Despite years of study, the mechanism of pathogenesis of S. marcescens and why it behaves as an opportunistic pathogen remain poorly understood. Unraveling the underlying mechanism of pathogenesis is thus very important for developing strategies to prevent and treat S. marcescens infection.The hemolysin ShlA was shown to be a dominant virulence factor in S. marcescens pathogenesis using a murine lung infection model (35). ShlA is responsible for the hemolytic and cytotoxic effects on erythrocytes and cultured cells, with the aid of an outer membrane protein, ShlB (28,29,47,53,54). ShlA also contributes to the release of inflammatory mediators, increases uropathogenicity, and triggers microtubule-dependent invasion of S. marcescens into epithelial cells (27,30,34,40). However, the mechanism by which the expression of shlA is regulated, especially in response to any bacterial signaling system control, remains uncharacterized. Only one reported study has indicated that iron is involved in the regulation of shlBA expression in S. marcescens (46). S. marcescens exhibits swarming, which is recognized as a highly coordinated multicellular surface migration behavior (24,51,62) that is correlated with virulence capability, antibiotic resistance, and hemolysin production in other bacteria (1,17,44). S. marcescens swarms on Luria-Bertani (LB) agar surfaces at 30°C, but not at 37°C (36). Our previous studies showed that activation of a bacterial two-component system, RssAB, comprising a sensor kinase, RssA, and a response regulator, RssB, inhibited swarming and reduced hemolysin productio...

Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising a two-component system (TCS) and the TCS-regulated iron chelator 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) that directly senses and modulates environmental ferric iron (Fe3+) availability to determine swarming initiation and biofilm formation. We demonstrate that the two-component system RssA-RssB (RssAB) directly senses environmental ferric iron (Fe3+) and transcriptionally modulates biosynthesis of flagella and the iron chelator ICDH-Coumarin whose production requires the pvc cluster. Addition of Fe3+, or loss of ICDH-Coumarin due to pvc deletion results in prolonged RssAB signaling activation, leading to delayed swarming initiation and increased biofilm formation. We further show that ICDH-Coumarin is able to chelate Fe3+ to switch off RssAB signaling, triggering swarming initiation and biofilm reduction. Our findings reveal a novel cellular system that senses iron levels to regulate bacterial surface lifestyle.

Activation of the epidermal growth factor receptor (EGFR) during tumor development can trigger the MEK signaling pathway. In the present study, we investigated the MEK signaling pathway in non-small cell lung cancer (NSCLC) cells with respect to the effect of epidermal growth factor (EGF) on expression of Ret finger protein like 3 (RFPL3) and human telomerase reverse transcriptase (hTERT), and the effect of RFPL3 overexpression on other MEK signaling proteins. In vitro, A549 and H1299 cells were treated with different concentrations of EGF for 24 h or 48 h. Expression of RFPL3 and hTERT at the mRNA and protein levels was determined by real-time quantitative PCR (RT-qPCR) and western blot analysis; cell viability was detected by MTT assay, and apoptosis was assayed via flow cytometry. We also pretreated A549 and H1299 cells with EGFR tyrosine kinase inhibitors, AG1478 and erlotinib, and MEK-specific inhibitor (PD98059) in the presence of EGF. We used western blot analysis to assess the expression levels of RFPL3, hTERT and related MEK-pathway proteins in A549 and H1299 cells transfected with RFPL3-overexpression plasmids. EGF significantly upregulated RFPL3 and hTERT protein levels in the NSCLC cells. RFPL3 and hTERT proteins upregulation by EGF were attenuated by pretreatment with AG1478 and erlotinib. EGF promoted proliferation and inhibited apoptosis; PD98059 decreased RFPL3 and hTERT protein expression; and RFPL3 overexpression increased the expression of hTERT and related MEK-pathway proteins. EGF upregulated RFPL3 and hTERT protein expression in NSCLC cells via the MEK pathway, promoted cell proliferation and inhibited apoptosis. RFPL3 overexpression increased expression of hTERT and related MEK signaling proteins (Ras, Raf, ERK and p-ERK), which implies that RFPL3 is a potential therapeutic target for NSCLC.

2,3-BTD activates NK cells cytotoxicity by NKG2D/NCRs pathways. The natural compound 2,3-BTD has diverse physiological effects in a range of organisms, including acting as a detoxifying product of liver alcohol metabolism in humans and ameliorating endotoxin-induced acute lung injury in rats. In this study, we reveal that 2,3-BTD enhances NK cell cytotoxic activity in human pNK cells and NK92 cells. Treatment of NK cells with 2,3-BTD increased perforin expression in a dose-dependent manner. This was accompanied by elevated JNK and ERK1/2 MAPK activities and enhanced expression of NKG2D/NCRs, upstream signaling molecules of the MAPK pathways. The 2,3-BTD effect was inhibited by pretreatment with inhibitors of JNK (SP) or ERK1/2 (PD) or by depleting NKG2D/NCRs or JNK1 or ERK2 with siRNA. These results indicate that 2,3-BTD activates NK cell cytotoxicity by NKG2D/NCR pathways and represent the first report of the 2,3-BTD effect on activation of innate immunity cells.

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