Function of the Bacterial Cytoskeleton in the Pathogenicity of Salmonella
Lead Research Organisation:
Newcastle University
Department Name: Inst for Cell and Molecular Biosciences
Abstract
Salmonella are bacteria of global medical importance and cause a broad range of diseases in humans from gastroenteritis to typhoid fever. Understanding the mechanisms by which salmonella cause disease may help in developing novel drugs and vaccines to combat disease.
Salmonella possess an armoury of weapons or virulence factors which enable them to cause disease. However the assembly of these complex macromolecular virulence structures remains to be fully elucidated. It is conceivable that a scaffold or cytoskeleton may be required to provide support and organisation for assembling these structures.
The proposal builds upon our unpublished preliminary data on the Salmonella Mre operon which contains seven genes, the first of which mreB encodes a homologue of the cytoskeletal protein actin found in mammalian cells. We have constructed Mre operon mutants and will determine the impact of these mutations on the functionality of important macromolecular virulence factors. We will be studying how the Mre operon encoded proteins function within Salmonella, and modulate their interactions with host tissues. This approach will provide important insights in the contribution of the bacterial cytoskeleton to virulence.
Salmonella possess an armoury of weapons or virulence factors which enable them to cause disease. However the assembly of these complex macromolecular virulence structures remains to be fully elucidated. It is conceivable that a scaffold or cytoskeleton may be required to provide support and organisation for assembling these structures.
The proposal builds upon our unpublished preliminary data on the Salmonella Mre operon which contains seven genes, the first of which mreB encodes a homologue of the cytoskeletal protein actin found in mammalian cells. We have constructed Mre operon mutants and will determine the impact of these mutations on the functionality of important macromolecular virulence factors. We will be studying how the Mre operon encoded proteins function within Salmonella, and modulate their interactions with host tissues. This approach will provide important insights in the contribution of the bacterial cytoskeleton to virulence.
Technical Summary
We hypothesise that the bacterial cytoskeleton plays an important role in the pathogenicity of bacteria by providing an organising scaffold for key virulence structures. We have generated a significant body of unpublished preliminary data to support this hypothesis.
The Salmonella Mre operon encodes a functional homologue of actin, MreB, together with MreC, MreD, and four additional genes of undefined function named maf, cafA, yhdP, and tldD. We have already begun to determine the function of these genes by constructing precise deletion and depletion mutants, and examining the resultant phenotypes. These phenotypic screens reveal an important impact on key virulence determinants including type 3 secretion (T3S), flagella, lipopolysaccharide, and have a significant effect upon virulence in vivo.
We wish to gain mechanistic insights into these observations by using a range of complementary experimental approaches. These include examining the transcriptomes of selected Mre depletion mutants which may provide mechanistic clues on the observed phenotypes. To investigate whether the bacterial cytoskeleton directly interacts with key virulence structures, we will examine the subcellular co-localisation of proteins encoded by the Mre operon and the T3S apparatus, flagella, and lipopolysaccharide, using fluorescent and immunogold electron microscopy. We will also study Mre protein-protein interactions by pulldown assays and the bacterial two-hybrid system. The ability of selected depletion mutants to invade and survive in epithelial and macrophage-like cells will be analysed. Finally, we wish to examine the impact of selected Mre operon depletion mutants on virulence and colonisation in the mouse model.
In summary, this proposal will provide important mechanistic insights into the contribution of the Salmonella cytoskeleton to pathogenicity. This research is timely and will fill a significant gap in our knowledge of this under-studied area of research. The study will have broad implications for other bacterial pathogens and the development of new antimicrobial agents.
The Salmonella Mre operon encodes a functional homologue of actin, MreB, together with MreC, MreD, and four additional genes of undefined function named maf, cafA, yhdP, and tldD. We have already begun to determine the function of these genes by constructing precise deletion and depletion mutants, and examining the resultant phenotypes. These phenotypic screens reveal an important impact on key virulence determinants including type 3 secretion (T3S), flagella, lipopolysaccharide, and have a significant effect upon virulence in vivo.
We wish to gain mechanistic insights into these observations by using a range of complementary experimental approaches. These include examining the transcriptomes of selected Mre depletion mutants which may provide mechanistic clues on the observed phenotypes. To investigate whether the bacterial cytoskeleton directly interacts with key virulence structures, we will examine the subcellular co-localisation of proteins encoded by the Mre operon and the T3S apparatus, flagella, and lipopolysaccharide, using fluorescent and immunogold electron microscopy. We will also study Mre protein-protein interactions by pulldown assays and the bacterial two-hybrid system. The ability of selected depletion mutants to invade and survive in epithelial and macrophage-like cells will be analysed. Finally, we wish to examine the impact of selected Mre operon depletion mutants on virulence and colonisation in the mouse model.
In summary, this proposal will provide important mechanistic insights into the contribution of the Salmonella cytoskeleton to pathogenicity. This research is timely and will fill a significant gap in our knowledge of this under-studied area of research. The study will have broad implications for other bacterial pathogens and the development of new antimicrobial agents.