The Salmonella enterica virulence : its role in bacterial adaption to mammalian and protozoan cells
Author: Tezcan-Merdol, Dilek
Date: 2004-05-27
Location: Föreläsningssalen, Mikrobiologiskt och Tumörbiologiskt Centrum (MTC), Theorells väg 1
Time: 9.30
Department: Mikrobiologiskt och Tumörbiologiskt Centrum (MTC) / Microbiology and Tumor Biology Center (MTC)
Abstract
Salmonellae are Gram-negative enteric bacteria and facultative
intracellular pathogens responsible for a diversity of illnesses in a
wide range of hosts, including man. Many serovars of Salmonella enterica
harbor a plasmid that enhances bacterial virulence in infection models,
and that seems to promote extraintestinal infection in man. Consequently,
the plasmid has been referred to as the"virulence plasmid". The virulence
plasmid varies in its constitution among different serovars, but all
these plasmids carry in common the highly conserved spv (Salmonella
plasmid virulence) gene cluster. The spv gene cluster consists of five
genes, spvRABCD with main promoters in front of spvR and spvA. The
predicted amino acid sequence of SpvR positions it to the LysR/MetR
family of prokaryotic transcriptional regulators, and functional analyses
have confirmed SpvR to act as a transcriptional activator of spv
expression. While the spv genes represent a major virulence trait of the
plasmid, the function of the SpvABCD proteins and the mechanism by which
they interact with the host are currently not understood. The work
described in this thesis aims at understanding the role of Spv proteins
in infection of mammalian and protozoan cells.
The results presented in this thesis demonstrate that one of the Spv proteins, SpvB, functions as a mono (ADP-ribosyl) transferase. The SpvB protein uses actin as a substrate for ribosylation and thereby hinders actin polymerization in vitro. Microscopic examination of MDCK cells infected with S. enterica revealed morphological changes and disappearance of F- actin.
When the ability of Salmonellae to infect and replicate in axenic Acanthamoeba was studied, electron microscopy of Salmonella-infected A. rhysodes showed the replicating bacteria to be localized within membrane-bound vacuoles. Prolonged incubation of the bacteria-amoebae cultures resulted in a gradual change in amoebae morphology, partially dependent on SpvB, and in subsequent detachment and disintegration of the host cells. In parallel, we demonstrate that in vitro SpvB-mediates label transfer from [ 32p]-NAD to a 43kDa protein that co-migrate with bovine thymus beta/gamma-actin whereas A. rhysodes cell lysate prevent SpvB-mediated [32p] -NAD-dependent labeling of thymus beta/gamma-actin Although we could not demonstrate any in situ SpvB-mediated modification of A. rhysodes proteins during infection, we did observe Spv-independent label transfer from [32p] -NAD to an 80-kDa protein. This labeling was dependent on infection, required permeabilization of the amoebae, and did not occur when infected with a laboratory strain of E. coli or when the mono (ADPribosyl) transferase inhibitor novobiocin was applied.
Our findings show that a facultative intracellular parasite can apply a classical bacterial toxin activity, ADP-ribosylation, to modify mammalian actin during intracellular replication, and that the spv locus is active during bacterial intracellular infection of a protozoan host.
The results presented in this thesis demonstrate that one of the Spv proteins, SpvB, functions as a mono (ADP-ribosyl) transferase. The SpvB protein uses actin as a substrate for ribosylation and thereby hinders actin polymerization in vitro. Microscopic examination of MDCK cells infected with S. enterica revealed morphological changes and disappearance of F- actin.
When the ability of Salmonellae to infect and replicate in axenic Acanthamoeba was studied, electron microscopy of Salmonella-infected A. rhysodes showed the replicating bacteria to be localized within membrane-bound vacuoles. Prolonged incubation of the bacteria-amoebae cultures resulted in a gradual change in amoebae morphology, partially dependent on SpvB, and in subsequent detachment and disintegration of the host cells. In parallel, we demonstrate that in vitro SpvB-mediates label transfer from [ 32p]-NAD to a 43kDa protein that co-migrate with bovine thymus beta/gamma-actin whereas A. rhysodes cell lysate prevent SpvB-mediated [32p] -NAD-dependent labeling of thymus beta/gamma-actin Although we could not demonstrate any in situ SpvB-mediated modification of A. rhysodes proteins during infection, we did observe Spv-independent label transfer from [32p] -NAD to an 80-kDa protein. This labeling was dependent on infection, required permeabilization of the amoebae, and did not occur when infected with a laboratory strain of E. coli or when the mono (ADPribosyl) transferase inhibitor novobiocin was applied.
Our findings show that a facultative intracellular parasite can apply a classical bacterial toxin activity, ADP-ribosylation, to modify mammalian actin during intracellular replication, and that the spv locus is active during bacterial intracellular infection of a protozoan host.
List of papers:
I. Otto H, Tezcan-Merdol D, Girisch R, Haag F, Rhen M, Koch-Nolte F (2000). "The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase. " Mol Microbiol 37(5): 1106-15
Pubmed
II. Tezcan-Merdol D, Nyman T, Lindberg U, Haag F, Koch-Nolte F, Rhen M (2001). "Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB. " Mol Microbiol 39(3): 606-19
Pubmed
III. Tezcan-Merdol D, Ljungstrom M, Winiecka-Krusnell J, Linder E, Engstrand L, Rhen M (2004). "Uptake and replication of Salmonella enterica within Acanthamoeba rhysodes." Environmental Microbiology (In Print)
View record in Web of Science®
IV. Tezcan-Merdol D, Engstrand L, Rhen M (2004). "NAD-dependent modification of Acanthamoeba rhysodes proteins in response to Salmonella enterica." (Manuscript)
I. Otto H, Tezcan-Merdol D, Girisch R, Haag F, Rhen M, Koch-Nolte F (2000). "The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase. " Mol Microbiol 37(5): 1106-15
Pubmed
II. Tezcan-Merdol D, Nyman T, Lindberg U, Haag F, Koch-Nolte F, Rhen M (2001). "Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB. " Mol Microbiol 39(3): 606-19
Pubmed
III. Tezcan-Merdol D, Ljungstrom M, Winiecka-Krusnell J, Linder E, Engstrand L, Rhen M (2004). "Uptake and replication of Salmonella enterica within Acanthamoeba rhysodes." Environmental Microbiology (In Print)
View record in Web of Science®
IV. Tezcan-Merdol D, Engstrand L, Rhen M (2004). "NAD-dependent modification of Acanthamoeba rhysodes proteins in response to Salmonella enterica." (Manuscript)
Issue date: 2004-05-06
Publication year: 2004
ISBN: 91-7349-941-2
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