Deciphering the role of spermidine in Salmonella Typhimurium pathogenesis

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative anaerobic bacterium. It infects a broad range of hosts, causing symptoms such as gastroenteritis, abdominal pain, inflammatory diarrhoea and systemic typhoid fever (in mice). Upon entering through the faeco-oral route, it breaches the intestinal epithelium to encounter macrophages in the underlying lamina propria. They are then taken up by the macrophages and spread systemically through the reticuloendothelial system (RES). Inside the host macrophages, they are exposed to different types of stress, such as oxidative and nitrosative conditions, low nutrient availability, and acidic pH. Salmonella utilises multiple stress response regulators to overcome such stress conditions and promote its survival within the hostile host niches. One of these stress response regulators is a polycationic molecule known as polyamines. Many studies have linked polyamines to bacterial virulence, such as Streptococcus pneumonaie and Yersinia pestis and stress response in Shigella spp. Putrescine and spermidine are the two significant polyamines in Salmonella that it can synthesise and transport from the extracellular milieu. Salmonella has specialised transporters, namely PotABCD, and PotFGHI, for importing spermidine and putrescine, respectively. Studies show that polyamines are crucial in the virulence of pathogenic bacteria, including Salmonella. However, the mechanism behind it remains unknown. Thus, we were interested in studying polyamines' role in Salmonella Typhimurium pathogenesis and delineating the mechanism behind the same. In our study we show that spermidine mediates the initial attachment and adhesion of Salmonella Typhimurium to Caco-2 cells, facilitating its invasion. Polyamines have previously been shown to regulate the transcription of multiple genes in both eukaryotes and prokaryotes. We show that spermidine controls the RNA expression of the two-component system, BarA/SirA, that further regulates multiple fimbrial and non-fimbrial adhesins in Salmonella. Flagella is also a vital surface structure aiding in motility and attachment to surfaces of host cells and gall stones. Spermidine regulated the expression of flagellin genes by enhancing the translation of sigma28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, we show that spermidine tunes the expression of the Salmonella pathogenicity island-1 encoded genes, which are essential for invasion into non-phagocytic intestinal epithelial cells. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by host macrophages, and the immune cells help disseminate the bacteria to secondary sites of infection through the RES. Inside the macrophages, it faces multiple stresses such as oxidative and nitrosative stresses, acidic pH, etc. However, Salmonella employs multiple countermeasures to cope with the hostile environment of macrophages. We show spermidine activates a stress response in Salmonella by regulating multiple antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels and less survival in RAW264.7 macrophages. We further show that it regulates a novel enzyme, Glutathonylspermidine synthase(Gsp), in Salmonella, producing a conjugate of spermidine and glutathione. This conjugate in E.coli has been previously shown to bind to protein thiols, thereby preventing oxidation of proteins in the bacteria. Here, we show that mutant of this enzyme shows significantly lesser survival in the presence of hydrogen peroxide in-vitro and also in RAW264.7 macrophages like the spermidine mutants. A rescue in fold proliferation was observed upon infection in macrophages isolated from gp91phox-/- mice. Our results were further validated in a mice model of Salmonella infection. Thus, elucidating the role of spermidine in stress response regulation in STM. Spermidine also plays multiple essential functions in eukaryotes, from parasites to plants to animals. Likewise, spermidine transport and synthesis are well-defined in eukaryotes. We further show that infection of Salmonella Typhimurium into RAW264.7 cells upregulates the polyamine biosynthesis pathway in the host cell. Upon knockdown of odc1 that synthesises the first member of polyamine in the host, Salmonella Typhimurium showed attenuated survival in RAW264.7 macrophages. Since polyamines are highly upregulated in tumour cells owing to their critical role in cell division, polyamine-targeted therapies have been developed to treat cancer. In our study, we used the anti-cancer drug to inhibit polyamine synthesis in host cells and show that, interestingly, it reduces Salmonella Typhimurium survival in RAW264.7 macrophages. Our study was further validated in mice, a model of Salmonella infection. Thus, our study solves the enigma of how polyamine spermidine assists in the pathogenesis of Salmonella. We show that spermidine acts as a novel regulatory molecule in Salmonella Typhimurium, regulating the expression of multiple genes and assisting in virulence and stress response.