aroA -Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant

Abstract

Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine.

The immunogenic potential of bacterial flagella for Salmonella‐mediated tumor therapy

“…In general, the number of differentially secreted cytokines was lower than with the flagella mutants in the Wt background. This was expected as the response was normalized to SF102, which itself is already highly immunogenic . Again, the nonflagellated Salmonella variants harboring a ΔflhDC , ΔfliF or ΔfliHIJ deletion exhibited the strongest pattern of cytokine induction.…”

“…As most of the above‐mentioned phenotypes still represented a serious health burden for the mice, the most promising flagella mutations were inserted into our previously established attenuated and immunogenic background strain SF102 ( ΔlpxR ΔpagP ΔpagL ΔaroA ) . As a control, we inserted a deletion of the flagellar master regulatory operon flhDC ( ΔflhDC) , which completely abolishes flagella synthesis .…”

“…Salmonella typhimurium usually encodes two antigenically distinct filament proteins (the flagellins FliC or FljB), whose mutually exclusive expression is regulated by phase switching . Of note, strains constitutively expressing the phase‐2 flagellin protein FljB were found to be more potent in targeting tumors in a murine tumor model compared to strains expressing phase‐1 FliC . In line with these observations, Eom and colleagues observed an enhanced adjuvanticity of Salmonella that co‐expressed both flagellin proteins FliC and FljB .…”

“…RNA isolation was performed as described previously . Library preparation of planktonic cultures grown to mid‐log phase was done using the ScriptSeqTM v2 RNA‐Seq Library Prep Kit (Illumina) and the vendor's protocol.…”

Identification and Characterization of Serotype-Specific Variation in Group A Streptococcus Pilus Expression

“…B acterial pathogens often show significant intraspecies phenotypic variation, with colony morphology, growth requirements, adherence, and disease manifestations being just a few of the variable phenotypes described (1,2). In some instances, phenotypic variation can be divided along type-specific (e.g., serotype, clonal type, haplotype) lines, indicating that there are type-specific genetic components responsible for the observed phenotypic differences (3,4). For example, Staphylococcus aureus isolates from pulsed-field type USA300 show enhanced virulence relative to isolates from pulsed-field type USA200, and this is in part explained by differences in the Agr regulatory system, which is a major regulator of virulence factor expression (5,6).…”