The Virulence of Salmonella enterica Serovar Typhimurium in the Insect Model Galleria mellonella Is Impaired by Mutations in RNase E and RNase III

Abstract

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a Galleria mellonela model in S. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in hfq, a wellestablished Salmonella virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in Salmonella virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions. Salmonella infections are a serious medical and veterinary problem worldwide. This pathogenic bacterium is able to invade and replicate within eukaryotic host cells. For infection, Salmonella relies upon a range of laterally acquired virulence regions, the so-called Salmonella pathogenicity islands (SPIs). Of these, SPI-1 and SPI-2 contain genes that encode type III secretion systems (TTSS), which deliver effector proteins into host cells to facilitate either cellular invasion or intracellular survival, respectively (for a review, see reference 1). Hundreds of genes are upregulated during infection and play important roles in adaptation, survival, and proliferation within mammalian cells (2). Transcriptome analysis of Salmonella enterica serovar Typhimurium within epithelial cells and macrophages revealed distinct patterns of expression linked to the different stages of infection (3, 4).Both the evolutionarily close relationship with Escherichia coli and the pathogen-specific aspects make Salmonella a very good model for studying the influence of RNA determinants in bacterial pathogenicity. In addition to transcriptional control, regulation of RNA decay has emerged as a major pathway in the fast adaptive process of bacteria to changes in the environment. RNAs may also act as regulatory molecules that can directly sense environmental clues and modulate the expression of target RNAs (for a review, see reference 5). The fate of RNA transcripts can be also controlled by proteins, including ribonucleases (RNases) and RNA chaperones.RNases are enzymes that govern the maturation and degradation of RNA molecules. RNA decay in Gram-negative bacteria usually begins with an endonucleolytic cleavage at one or more internal sites on the RNA molecule. This cleavage is normally performed by RNase E and/or RNase III (6, 7). The single-stranded specific endori...

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

“…Altogether, we detected a total of 2,557 mRNAs cleaved by RNase E, with a different number of cleavage sites per transcript (Figure 2B); these represent 78% of 3,286 Salmonella mRNAs that are well expressed (RPKM > 10, Table S2) in the early stationary phase. Notably, the assay captured many essential genes and virulence genes required for intracellular growth (Table S3), which provide insights into the processing of transcripts from indispensable genes and the roles of RNase E in Salmonella pathogenesis (Viegas et al., 2013), respectively. Longer transcripts generally tend to contain a higher number of cleavage sites (Figures S1D and S1E).…”

New Perspectives on Galleria mellonella Larvae as a Host Model Using Riemerella anatipestifer as a Proof of Concept

“…Evidence accumulated in recent years has demonstrated that G. mellonella larvae can be used as an attractive alternative model to identify the virulence of various pathogens (42,(47)(48)(49)(50). In this study, we investigated the extent to which the virulence factors required for pathogenesis in both ducklings and G. mellonella larvae overlap.…”

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis

“…Larvae of the wax moth Galleria mellonella are an abundantly used in vivo infection model, including those with enteric pathogens such as enteropathogenic E. coli (EPEC) and Salmonella (Leuko and Raivio, 2012;Viegas et al, 2013). It is particularly suited to study interactions between pathogens and phagocytic cells in a living organism (Harding et al, 2012).…”

“…Therefore, other animal models such as the murine lung and rabbit ligated ileal loop models have been developed for defining some of the immune and inflammatory components of the disease (Philpott et al, 2000). Recently, the great moth, G. mellonella, has gained popularity as a model for assessing virulence and a number of pathogens have been studied using this model, including enteropathogenic E. coli (EPEC), Salmonella typhimurium and Listeria monocytogenes (Leuko and Raivio, 2012;Harding et al, 2013;Viegas et al, 2013). To our knowledge, this study is the first to report the use of this model for assessing virulence of Shigella.…”