Abstract
Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl3 suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.
Highlights
Abbreviations SubAB Subtilase cytotoxin Locus for Enterocyte Effacement (LEE) Locus of enterocyte effacement STEC Shiga-toxigenic Escherichia coli LPS Lipopolysaccharide C/EBP CCAAT enhancer binding protein CHOP C/EBP homology protein PERK RNA-dependent protein kinase-like endoplasmic reticulum (ER) kinase TM Tunicamycin eukaryotic translation initiation factor 2α (eIF2α) Eukaryotic translation initiation factor 2α LCN2 Lipocalin 2 PARP Poly (ADP ribose) polymerase cleaved PARP (cPARP) Cleaved PARP NC Non-targeting control TM Tunicamycin RT-qPCR Real-time quantitative reverse transcription polymerase chain reaction
SubAB on defense factors generated by host cells, we focused on LCN2, a protein that was induced by ER stress[38] and acts as an anti-microbial defense factor by binding to a subset of bacterial siderophores[25]
Since overexpression of LCN2 was protective from inflammation-associated cell death by LPS50, we examined the effect of LCN2 overexpression on SubAB-induced apoptosis (Fig. 5C)
Summary
Abbreviations SubAB Subtilase cytotoxin LEE Locus of enterocyte effacement STEC Shiga-toxigenic Escherichia coli LPS Lipopolysaccharide C/EBP CCAAT enhancer binding protein CHOP C/EBP homology protein PERK RNA-dependent protein kinase-like ER kinase TM Tunicamycin eIF2α Eukaryotic translation initiation factor 2α LCN2 Lipocalin 2 PARP Poly (ADP ribose) polymerase cPARP Cleaved PARP NC Non-targeting control TM Tunicamycin RT-qPCR Real-time quantitative reverse transcription polymerase chain reaction. STEC O113:H21 98KN2 strain was associated with an outbreak of HUS in Australia This LEE-negative STEC strain produced two cytotoxins, Stx[2] and subtilase cytotoxin (SubAB)[7]. SubAB-induced apoptosis in HeLa cells was suppressed by steroids or diacylglycerol a nalogues[22]. Cathelicidins and defensins bind directly to bacterial membranes, inducing membrane damage and death[24] Besides these AMPs, mammalian cells inhibit bacterial growth by producing Lipocalin-2 (LCN2), a secretary glycoprotein that binds siderophores and prevents delivery of iron to the bacteria[25]. Our findings demonstrate that SubAB induced a novel non-secreted form of LCN2, which would promote survival of LEE-negative STEC
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