Salmonella Enteritidis activates inflammatory storm via SPI-1 and SPI-2 to promote intracellular proliferation and bacterial virulence

Abstract

<i>Salmonella</i> Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. <i>S.</i> Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of <i>Salmonella</i> pathogenicity island-1 (SPI-1) and SPI-2 to <i>S.</i> Enteritidis virulence <i>in vitro</i> and <i>in vivo</i>, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that <i>S.</i> Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. <i>S.</i> Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. <i>S.</i> Enteritidis <i>ΔSPI-1</i>-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in <i>ΔSPI-2-</i> and <i>ΔSPI-1/SPI-2</i>-infected mice. A survival assay showed that <i>ΔSPI-1</i> mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to <i>S.</i> Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways.