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



Recombinant attenuated bacterial vector systems based on genetically engineered Salmonella have been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238-239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic ΔaroA mutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroA leads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella. These results suggest that the enhanced immunogenicity of aroA-deficient Salmonella strains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of ΔaroA Salmonella in bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany sebastian.felgner@helmholtz-hzi.de.
Sebastian
S
Felgner
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Michael
M
Frahm
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Dino
D
Kocijancic
Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Manfred
M
Rohde
Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Denitsa
D
Eckweiler
Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Agata
A
Bielecka
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Emilio
E
Bueno
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Felipe
F
Cava
Department of Chemical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Wolf-Rainer
WR
Abraham
Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida, USA.
Roy
R
Curtiss
Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Susanne
S
Häussler
Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Marc
M
Erhardt
Department of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany Institute of Immunology, Medical School Hannover, Hannover, Germany.
Siegfried
S
Weiss
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001835:Body Weight; D004195:Disease Models, Animal; D017353:Gene Deletion; D020869:Gene Expression Profiling; D054884:Host-Pathogen Interactions; D053858:Metabolic Networks and Pathways / Q000235:genetics; D055432:Metabolomics; D051379:Mice; D012480:Salmonella Infections / Q000382:microbiology* / Q000473:pathology*; D012486:Salmonella typhimurium / Q000276:immunology / Q000472:pathogenicity* / Q000502:physiology; D012765:Shikimic Acid / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000097:blood; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics*
D001426:Bacterial Proteins; D014409:Tumor Necrosis Factor-alpha; D037521:Virulence Factors; D012765:Shikimic Acid; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase