RNase III in Salmonella Enteritidis enhances bacterial virulence by reducing host immune responses

Reviewer #1 (Public review):

Summary:

The central question of this manuscript is the role of RNase III in supporting Salmonella infection. The authors begin with an RNAseq analysis of a collection of food or clinical Salmonella isolates from China, identifying RNase III (encoded by rnc) as an upregulated gene in clinical ("high virulence") isolates. Based on follow-up studies with knockout and complemented strains, the authors propose that RNase III has two roles - one in the upregulation of sodA expression to counter host-derived ROS, and the other in general degradation of dsRNA to dampen host immune responses. Overall, the manuscript is logical and the authors make largely reasonable interpretations of their data. However, the depth of supporting evidence limits the breadth of the authors' conclusions in their current form. Thus, this manuscript will be useful to researchers in directly related fields of study, but more work is required to understand how these proposed mechanisms function during infection.

Strengths:

(1) The use of comparative RNAseq between different isolates to identify potential virulence mechanisms is a powerful approach to understanding what makes certain strains more likely to cause infection over others.

(2) The experiments identifying dsRNA as the factor contributing to increased innate immune induction in the rnc knockout strain are particularly thorough.

(3) The authors observed an in vivo mammalian infection defect for RNase III-deficient Salmonella, a novel finding for the field and strong evidence that this protein is required to support pathogen fitness.

Weaknesses:

(1) The strengths of the manuscript are in places obscured by a lack of clarity and justification in the manuscript about strain selection and rationale for using some backgrounds over others. Moreover, several aspects of the organization and flow of the manuscript could be improved, as data is described out of order and the text description of results does not always align with the data presented.

(2) The specific claim that the relatively modest increase in expression of RNase III in some isolates (Figure 1A) accounts for their "virulence" is not well-supported, since the only comparisons in the study are between total knockouts or wild-type (and not overexpression) and the actual protein levels of RNase III are not quantified.

(3) Although the experiments on dsRNA are strong, they would have benefited from measurements of cytokine production/immune responses during infection with the actual knockout strains instead of transfected RNA along with quantification of Salmonella burdens.

(4) The contribution of RNase III catalytic activity (i.e., through the use of a catalytically dead mutant) was not assessed, which means that a role for general RNA binding or protein-protein interactions cannot be ruled out from this study.

(5) The in vivo work was limited to survival analysis, so whether the proposed mechanisms account for the defects observed could not be resolved.

(6) Statistical analysis throughout the manuscript is inconsistently applied, making it hard in places to determine whether the differences seen in phenotypes are biologically significant.

Reviewer #2 (Public review):

Summary:

This work attempted to investigate how the gene rnc, which showed higher expression in clinical strains of Salmonella Enteritidis compared to those isolated from food, affects the virulence of this bacteria through modulating dsRNA levels and the immune response of host cells.

Strengths:

The authors clearly demonstrated that the deletion of rnc Salmonella Enteritidis leads to an accumulation of dsRNA inside the cells, which further activates the immune response of host cells. It is also well demonstrated that the rnc gene deletion results in an increased ROS level through regulating the SodA protein.

Weaknesses:

(1) It is unclear whether the higher rnc expression in clinical strains of Salmonella Enteritidis is universal or just specific to several strains, because of the inadequate data provided and different strains used for different tests in this study.

(2) A lot of specific information is missing in the Figure legends and Method section, which makes it hard to understand some of the key results in the manuscript.

Reviewer #3 (Public review):

Summary:

Chan et al. evaluated the role of RNase III, encoded by the rnc gene, in Salmonella virulence. Chan et al. first identified rnc among the genes with upregulated mRNA levels in virulent Salmonella isolates. The authors further showed that deletion of rnc resulted in increased double-stranded RNA (dsRNA) and reduced invasion rate and replication rate in an in vitro macrophage model. The authors then showed that transfection of total RNA of rnc knock-out strains upregulates (with respect to a WT Salmonella strain) expression levels of immune-related genes (e.g., TNF-a, IL-1B, etc.) in a dsRNA-dependent manner. The authors reported reduced SodA protein accumulation in the rnc knock-out strains, despite higher levels of sodA mRNA, suggesting a role of SodA in the protection against reactive oxygen species. Finally, the authors showed, using a mice model, the partial contribution of sodA in the restoration of virulence levels in the rnc knock-out strains.

Strengths:

(1) The manuscript is well written.

(2) The authors evaluated the impact of rnc deletion in both in vitro and mice infection models. Both experiment setups supported the contribution of rnc to Salmonella virulence.

(3) The authors tested the effect of rnc deletion in different genetic backgrounds (i.e., different bacterial isolates) offering additional support to their claims.

(4) Measurement of SodA protein levels nicely complemented and informed initial findings at the mRNA level.

Weaknesses:

(1) The authors failed to discuss how their work differentiates from recent studies of rnc deletion strains in Salmonella (NIH PMID: 38182942) and Escherichia coli (NIH PMID: 35456749). Remarkably, the first publication performed genome-wide transcriptional profiling of a rnc deletion Salmonella strain. The second publication explored the link between rnc and sodA in E. coli.

(2) The authors should explain what the criteria for selecting food and clinical isolates for molecular characterization were. This information is valuable for the reader as they may wonder about the impact of isolate selection in the study's conclusions. Similarly, the authors need to explain how they selected their controls for baseline gene expression, virulence, etc.. Furthermore, I wondered if they could use an avirulent Salmonella strain as an additional control.

(3) The authors do not perform any analysis of the differentially expressed genes (DEGs) identified in their study. They should leverage DEGs to expand their mechanistic insights of other genes or functional processes putatively linked to rnc activity and virulence. Additionally, authors should make transcriptional data and the output of their differential expression analysis (and the list of differentially expressed genes-DEGs) available to the readers. In fact, it is not clear how the DEGS were defined.