Abstract

Author summary Salmonella is a bacterial genus that causes foodborne illnesses worldwide. The ability of Salmonella to cause disease is related to the presence of Pathogenicity Islands (PAIs), which are clusters of genes within the bacterial chromosome that are involved in virulence. Interestingly, some PAIs excise and re-integrate into the bacterial chromosome, which is a process probably involved in the capacity of Salmonella to cause infection in their hosts. Here we show that the excision of Region of Difference 21 (ROD21), one of the excisable PAIs within the genome of Salmonella enterica serovar Enteritidis, occurs with high frequency in the mesenteric lymph node at early stages of infection, suggesting that excision is required by S. Enteritidis to reach this organ from the intestinal tract. Accordingly, S. Enteritidis strains unable to excise ROD21 are unable to invade intestinal host cells, delaying the infective cycle and showing attenuated virulence. We propose that ROD21 excision in vivo is required by S. Enteritidis to cross the intestinal barrier, a fundamental step to further colonize deep organs, due to modulation of virulence genes transcription. Thus, ROD21 excision may play an important role in the capacity of the bacteria to cause a successful systemic infection in the mouse. Our data suggest that the excision of PAIs is a mechanism used by Salmonella and probably other Gram-negative enterobacteria to modulate the expression of virulence genes and may provide insights to design novel therapies to control the infection caused by these pathogens. Pathogenicity island excision is a phenomenon that occurs in several Salmonella enterica serovars and other members of the family Enterobacteriaceae. ROD21 is an excisable pathogenicity island found in the chromosome of S. Enteritidis, S. Dublin and S. Typhi among others, which contain several genes encoding virulence-associated proteins. Excision of ROD21 may play a role in the ability of S. Enteritidis to cause a systemic infection in mice. Our previous studies have shown that Salmonella strains unable to excise ROD21 display a reduced ability to colonize the liver and spleen. In this work, we determined the kinetics of ROD21 excision in vivo in C57BL/6 mice and its effect on virulence. We quantified bacterial burden and excision frequency in different portions of the digestive tract and internal organs throughout the infection. We observed that the frequency of ROD21 excision was significantly increased in the bacterial population colonizing mesenteric lymph nodes at early stages of the infective cycle, before 48 hours post-infection. In contrast, excision frequency remained very low in the liver and spleen at these stages. Interestingly, excision increased drastically after 48 h post infection, when intestinal re-infection and mortality begun. Moreover, we observed that the inability to excise ROD21 had a negative effect on S. Enteritidis capacity to translocate from the intestine to deeper organs, which correlates with an abnormal transcription of invA in the S. Enteritidis strain unable to excise ROD21. These results suggest that excision of ROD21 is a genetic mechanism required by S. Enteritidis to produce a successful invasion of the intestinal epithelium, a step required to generate systemic infection in mice.