Antiviral Activity of Shiga Toxin Requires Enzymatic Activity and Is Associated with Increased Permeability of the Target Cells
Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, Idaho 83844-3052,1 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 991642[m, http://www.100md.com
Received 10 July 2002/ Returned for modification 9 September 2002/ Accepted 15 October 2002[m, http://www.100md.com
ABSTRACT[m, http://www.100md.com
This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA11 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.
INTRODUCTIONs8, http://www.100md.com
Ruminant animals are a reservoir for Shiga toxin (Stx)-producing Escherichia coli (STEC) that can cause hemorrhagic colitis and life-threatening sequelae in humans (15, 23, 25). STEC are part of the normal ruminant gastrointestinal microbiota and are frequently isolated from cattle, sheep, and deer (8, 38, 41, 45). Surveys of healthy domesticated cattle routinely show a high prevalence of STEC in animals around the world (7, 8, 10, 12, 34-36). The reasons for the wide distribution of STEC in ruminants are not known. The possible benefits arising from gastrointestinal tract colonization by STEC include postulated enhancement of gastrointestinal mucosal architecture (E. D. E. Hoey, C. G. Currie, R. W. Else, A. Nutikka, C. A. Lingwood, D. L. Gally, and D. G. E. Smith, Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001, Abstr. B-222, 2001) and antiviral activity (17).s8, http://www.100md.com
Bovine leukemia virus (BLV) is an oncogenic retrovirus that infects B lymphocytes and induces chronic, benign, mostly subclinical infection in cattle (20). Most BLV-infected animals experience some elevation in peripheral B lymphocyte numbers, and about one-third develop persistent lymphocytosis, a preneoplastic polyclonal expansion of peripheral B lymphocytes containing provirus (16). The majority of these cattle remain clinically normal, but a small percentage develop a B-cell lymphosarcoma (21). Although resistance to BLV disease includes humoral and cellular immunity (21), the presence of STEC flora may constitute another factor limiting the viremia and disease progression. A hallmark of peripheral blood mononuclear cells (PBMC) from cattle in the persistently lymphocytotic stage of BLV infection is spontaneous lymphocyte proliferation (SLP) when placed in culture (55, 56). SLP is initiated by a rapid derepression of viral gene transcription and viral protein synthesis (3, 20, 21), so it is a measure of viral activity. Previous work by Ferens and Hovde shows that Stx specifically blocks BLV-dependent initiation of SLP and does not cause indiscriminate cell death (17). On this basis, we used cultured PBMC from BLV-positive cattle as a model system for investigating the antiviral activity of the Stxs. Although inhibition of SLP is not a direct measure of antiviral activity, we refer to it as an antiviral assay rather than as a cell proliferation inhibition assay because it represents cell division specifically triggered by BLV derepression.
The family of Stxs includes Stx1, Stx2, and Stx2 variants (13). These toxins belong to a larger group of proteins called the ribosome-inactivating proteins (RIPs) (found in a variety of higher plants and some bacteria) that share structural features and have N-glycosidase enzymatic activity. Intoxication of eukaryotic cells by RIPs leads to a rapid inhibition of protein synthesis and cell death (49). Most RIPs are hemitoxins (enzymatically active A chains), and some are holotoxins (one A chain associated with a specific number of B chains). B subunits mediate toxin binding to receptors on eukaryotic cells and receptor-mediated endocytosis (31). Thus, holotoxins are highly toxic to cells expressing receptors for a B subunit(s), but not to receptor-deprived cells, and are not toxic to normal cells as isolated A chains (5, 14, 24). Plant hemitoxins are not toxic to plants that synthesize them and have low cytotoxicity against animal cells, unless the cells have high pinocytic activity (11, 59). However, these hemitoxins can enter and eliminate virally infected plant cells (4) and some are also found to be highly toxic to various virally infected animal cells (24).8z/5(, http://www.100md.com
Stxs have a single enzymatically active(Indira Basu Witold A. Ferens Diana M. Stone and Carolyn J. Hovde)
Received 10 July 2002/ Returned for modification 9 September 2002/ Accepted 15 October 2002[m, http://www.100md.com
ABSTRACT[m, http://www.100md.com
This study expanded our earlier finding that Shiga toxin type 1 (Stx1) has activity against bovine leukemia virus (BLV) (W. A. Ferens and C. J. Hovde, Infect. Immun. 68:4462-4469, 2000). The Stx molecular motifs required for antiviral activity were identified, and a mechanism of Stx action on virally infected cells is suggested. Using inhibition of BLV-dependent spontaneous lymphocyte proliferation as a measure of antiviral activity, we showed that Stx2 had antiviral activity similar to that of Stx1. Enzymatic and antiviral activities of three StxA1 chain mutants deficient in enzymatic activity or aspects of receptor-mediated cytotoxicity were compared. Using protein synthesis inhibition to measure enzymatic activity, the mutant E167D was 300-fold less catalytically active than wild-type StxA1, was minimally active in antiviral assays, and did not inhibit synthesis of viral proteins. Two StxA1 mutants, A231D-G234E and StxA11 (enzymatically active but unable to kill cells via the classical receptor-mediated route), had undiminished antiviral activity. Although binding of radiolabeled StxA1 to bovine blood cells or to free virus was not detected, flow cytometric analysis showed that the number of BLV-expressing cells were specifically reduced in cultures treated with Stx. These unique and rare lymphocytes were highly permeable to 40- and 70-kDa fluorescent dextrans, indicating that direct absorption of toxins by virus-expressing cells is a potential mechanism of target cell intoxication. These results support the hypothesis that Stx-producing Escherichia coli colonization of the gastrointestinal tract may benefit ruminant hosts by the ability of Stxs to exert antiviral activity.
INTRODUCTIONs8, http://www.100md.com
Ruminant animals are a reservoir for Shiga toxin (Stx)-producing Escherichia coli (STEC) that can cause hemorrhagic colitis and life-threatening sequelae in humans (15, 23, 25). STEC are part of the normal ruminant gastrointestinal microbiota and are frequently isolated from cattle, sheep, and deer (8, 38, 41, 45). Surveys of healthy domesticated cattle routinely show a high prevalence of STEC in animals around the world (7, 8, 10, 12, 34-36). The reasons for the wide distribution of STEC in ruminants are not known. The possible benefits arising from gastrointestinal tract colonization by STEC include postulated enhancement of gastrointestinal mucosal architecture (E. D. E. Hoey, C. G. Currie, R. W. Else, A. Nutikka, C. A. Lingwood, D. L. Gally, and D. G. E. Smith, Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001, Abstr. B-222, 2001) and antiviral activity (17).s8, http://www.100md.com
Bovine leukemia virus (BLV) is an oncogenic retrovirus that infects B lymphocytes and induces chronic, benign, mostly subclinical infection in cattle (20). Most BLV-infected animals experience some elevation in peripheral B lymphocyte numbers, and about one-third develop persistent lymphocytosis, a preneoplastic polyclonal expansion of peripheral B lymphocytes containing provirus (16). The majority of these cattle remain clinically normal, but a small percentage develop a B-cell lymphosarcoma (21). Although resistance to BLV disease includes humoral and cellular immunity (21), the presence of STEC flora may constitute another factor limiting the viremia and disease progression. A hallmark of peripheral blood mononuclear cells (PBMC) from cattle in the persistently lymphocytotic stage of BLV infection is spontaneous lymphocyte proliferation (SLP) when placed in culture (55, 56). SLP is initiated by a rapid derepression of viral gene transcription and viral protein synthesis (3, 20, 21), so it is a measure of viral activity. Previous work by Ferens and Hovde shows that Stx specifically blocks BLV-dependent initiation of SLP and does not cause indiscriminate cell death (17). On this basis, we used cultured PBMC from BLV-positive cattle as a model system for investigating the antiviral activity of the Stxs. Although inhibition of SLP is not a direct measure of antiviral activity, we refer to it as an antiviral assay rather than as a cell proliferation inhibition assay because it represents cell division specifically triggered by BLV derepression.
The family of Stxs includes Stx1, Stx2, and Stx2 variants (13). These toxins belong to a larger group of proteins called the ribosome-inactivating proteins (RIPs) (found in a variety of higher plants and some bacteria) that share structural features and have N-glycosidase enzymatic activity. Intoxication of eukaryotic cells by RIPs leads to a rapid inhibition of protein synthesis and cell death (49). Most RIPs are hemitoxins (enzymatically active A chains), and some are holotoxins (one A chain associated with a specific number of B chains). B subunits mediate toxin binding to receptors on eukaryotic cells and receptor-mediated endocytosis (31). Thus, holotoxins are highly toxic to cells expressing receptors for a B subunit(s), but not to receptor-deprived cells, and are not toxic to normal cells as isolated A chains (5, 14, 24). Plant hemitoxins are not toxic to plants that synthesize them and have low cytotoxicity against animal cells, unless the cells have high pinocytic activity (11, 59). However, these hemitoxins can enter and eliminate virally infected plant cells (4) and some are also found to be highly toxic to various virally infected animal cells (24).8z/5(, http://www.100md.com
Stxs have a single enzymatically active(Indira Basu Witold A. Ferens Diana M. Stone and Carolyn J. Hovde)