Phage-mediated horizontal transfer of Salmonella enterica virulence genes with regulatory feedback from the host

Abstract

Phage-mediated horizontal transfer of virulence genes can enhance the transmission and pathogenicity of Salmonella enterica (S. enterica), a process potentially regulated by its regulatory mechanisms. In this study, we explored the global dynamics of phage-mediated horizontal transfer in S. enterica and investigated the role of its regulatory mechanisms in transduction. A total of 5178 viral sequences encoding 12 S. enterica virulence genes were retrieved from the Integrated Microbial Genomes and Virome (IMG/VR) database, alongside 466,136 S. enterica genomes from EnteroBase. Virulence genes, including iacP (acyl carrier protein), mgtB (P-type Mg²⁺ transporter), misL (autotransporter porin), and fliC (flagellar filament protein), were widely distributed in phages and S. enterica across North America, Europe, and Asia. Phylogenetic analysis revealed close genetic affinity between phage- and bacterial-encoded virulence genes, suggesting shared ancestry and historical horizontal gene transfer events. The global regulator carbon storage regulator A (csrA) was highly conserved and ubiquitous in S. enterica. Overexpression of csrA inhibited prophage cyclization and release by upregulating the prophage cI repressor during horizontal gene transfer. Overall, these findings enhance our understanding of phage-mediated horizontal transfer of virulence genes, explore new areas of bacterial regulators that inhibit gene exchange and evolution by affecting phage life cycles, and offer a novel approach to controlling the transmission of phage-mediated S. enterica virulence genes.

Keywords: Salmonella enterica; horizontal gene transfer; phages; regulators; virulence genes.

FIGURE 1

Global geographical distribution of S. enterica. (A) Patterns of global variation in S. enterica abundance with longitude. The x‐axis represents longitude and corresponds exactly to the longitude of the map. The y‐axis represents the abundance of Salmonella carrying virulence genes. The colors represent habitats. (B) Global distribution of S. enterica in different habitats. The size of the pie chart represents the abundance of S. enterica. And the colors represent habitats. (C) The abundance of four virulence genes (fliC, iacP, mgtB, and misL) of S. enterica in North America, Africa, Oceania, Asia, Europe, and South America of each habitat. The colors represent habitats. Each bar represents the abundance of a specific virulence gene, with segments representing habitats.

FIGURE 2

Global distribution of phages encoding S. enterica virulence genes. (A) The pattern of global variation in phage abundance with longitude. The x‐axis represents longitude and corresponds exactly to the longitude of the map. The y‐axis represents the abundance of phages carrying virulence genes. The colors represent habitats. (B) Global distribution of phages encoding S. enterica virulence genes in different habitats. The size of the pie chart represents the abundance of phages. And the colors represent habitats. (C) Abundance of the four genes of interest (fliC, iacP, mgtB, and misL) in the phages in North America, Africa, Oceania, Asia, Europe, and South America of each habitat. The colors represent habitats. Each bar represents the abundance of a specific virulence gene, with segments representing habitats.

FIGURE 3

Phage host prediction and phylogenetic analysis of virulence genes. (A) Linkage between phages carrying virulence genes with S. enterica, as well as other bacteria. Left: the S. enterica serotypes that can match to phages carrying virulence genes, and phages match to host bacteria. Middle: the phages that can match to host. Right: bacterial hosts at the genus level, and phages only match to S. enterica. (B) Phylogenetic analysis of virulence gene fliC encoded by phages and S. enterica. The branch length represents the tree scale. The green branch represented a phage‐carried fliC, and the blue branch represented a Salmonella‐carried fliC. The color of the leaves name represents the habitat where the fliC is located. The bar next to the phylogenetic tree annotates the geographic location where fliC is located. (C) Genomic structures of phage‐encoded virulence genes. Pink represents virulence genes, yellow represents viral genes assigned V flags by DRAMV, green represents other types of genes identified, and blue represents genes with unidentified functions. (D) Predicted quaternary structures of phage‐encoded virulence genes (fliC, iacP, mgtB, and misL).

FIGURE 4

Global distribution of regulators in S. enterica. (A) Global distribution of virulence regulators encoded by S. enterica. Different colors represent different regulatory genes. And the size of the pie chart represents the abundance of genes. (B) The average copy number of regulatory genes changes with 10 serotypes from left to right: S. Enteritidis, S. Anatum, S. Infantis, S. Montevideo, S. Typhimurium, S. Derby, S. Schwarzengrund, S. Kentucky, S. Heidelberg, and S. Newport. (C) Phylogenetic tree of csrA. The branch length represents the tree scale. The bar and number next to it annotates the count of csrA in each branch.

FIGURE 5

Experiments to validate the effect of csrA on phage cyclization and cI repressor. (A) The complete genomic structure of the prophage PSAP2‐2. Specific genes are indicated with arrows, with cyan for tail protein genes and gray for other genes. (B) Strains with E. coli plasmid PHB20TG carrying csrA and strain with only plasmid PHB20TG as a control were successfully constructed. (C) Phage titers of strains overexpressing csrA (S12::csrA‐1 and S12::csrA‐2) and wild‐type strain (WT) after induction of prophage expression. p < 0.05 indicated significant difference between strains overexpressing csrA and WT strain. The relationship between the expression of the cI repressor and csrA at a dilution gradient of 1:16 (D) and 1:32 (E), respectively. p < 0.001 indicated a significant correlation between the expression of csrA and that of the cI repressor. (F) Schematic representation of the main steps and results of the experiment. The green oval boxes on the left and right represent S. Typhimurium S12 carrying an empty plasmid and a plasmid containing the csrA gene via electroporation method, respectively. The prophage PSAP2‐2 inserted in the host DNA is determined by the expression of its own CI repressor to enter the lysis cycle. Upon induction, the CI repressor is cleaved, the cro gene‐expressed protein binds the RM promoter (P _RM), represses the expression of cI, opens the left and right promoters (P _L and P _R), and the prophage enters the lysis cycle. However, the overexpression of csrA promotes the expression of cI repressor, which activates the RM promoter (P _RM), so that the phage tends to maintain the lysogenic cycle and reduces the release of phages. Released phages were infiltrated with S. Typhimurium S3, and the influence of csrA on prophage cyclization and release was quantified based on the count of phage spots on the petri dishes.