Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

Abstract

We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia coli strains able to invade epithelial cells by the expression of the inv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomal sodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid-and oxidative stress-sensitive E. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality of E. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.Until a few years ago Cu,Zn superoxide dismutase (Cu, ZnSOD) was considered almost exclusively a eukaryotic enzyme, protecting the cytosol and the extracellular environment of higher organisms from damage by oxygen free radicals (1). Recently, Cu,ZnSOD has been identified in the periplasmic space of a wide range of gram-negative bacteria, including Brucella abortus (6), Haemophilus spp., Actinobacillus spp., Pasteurella spp., Neisseria meningitidis (24-26), Escherichia coli K-12 (7), Legionella pneumophila (40), Salmonella spp. (9), and Mycobacterium tuberculosis (45). This enzyme is thought to protect bacteria from toxic oxygen-free radicals generated outside the cell or in the periplasm itself, since superoxide is unable to cross the cytoplasmic membrane (21). Therefore, Cu,ZnSOD has been proposed to be a determinant of virulence in bacteria potentially exposed to toxic free radicals produced by the host in response to bacterial infection. In vivo experiments have demonstrated the role of bacterial Cu,ZnSOD in the virulence and pathogenicity of infecting microorganisms (15,18,19,36,42,43), while in vitro models have provided conflicting data concerning Cu,ZnSOD involvement in bacterial resistance to macrophage killing (19, 42) or survival within nonprofessional phagocytes (42). However, more recent results have shown that this enzyme protects Salmonella enterica serovar Typhimurium (15) and an overproducing strain of E. coli (4) from macrophage killing and that neutropenia restores virulence to an attenuated C...

The role of two periplasmic copper- and zinc-cofactored superoxide dismutases in the virulence of Salmonella choleraesuis

“…With two functional sodC genes, S. choleraesuis achieves enhanced survival under the conditions of oxidative stress encountered during experimental murine infection by the oral or parenteral route, reproduced ex vivo in experimental infection of activated macrophages. This observation finds a correlate in the observations of Battistoni et al (2000). An E. coli strain engineered to increase [Cu,Zn]-SOD production was found to be much more resistant to intracellular killing than the wild-type strain, while there was very little difference in survival between the wild-type and a sodCnull mutant.…”

Involvement of Reactive Oxygen Species in Bacterial Killing within Epithelial Cells

“…2). In fact, 24 h after the infection nearly all E. coli HB 101 (pRI203) cells appeared damaged within the endosome-lysosome ( Fig.2A), as already described (17). On the contrary, at the same time post-infection, most of the E. coli HB 101(pRI203) cells observed within HeLa cells treated with DPI were still intact (Fig.…”

“…Production of oxygen radicals might be involved in the response to bacterial invasion either by inducing cytokine production or by inducing oxidative damage to intracellular bacteria. This latter possibility is supported by the observation that phagocytic stimuli induce superoxide release by epithelial cell (22) and by our recent demonstration that increased expression of the sodC gene enhances survival of invasive E. coli strains within HeLa and Caco-2 cells (17). These two cell lines express homologs of gp91phox, the catalytic subunit of the phagocytic NADPH oxidase (23).…”

Regulatory and Structural Differences in the Cu,Zn-Superoxide Dismutases of Salmonella enterica and Their Significance for Virulence

“…Previous studies in cultured macrophages (11) and mice (8,9,16,18,21,23,24) have shown a consistent contribution of SodCI to Salmonella virulence. However, some of these studies have found a substantial role of SodCII in virulence as well (8,12,16,18), whereas others have not (21,23,24).…”