A Large Toxin from Pathogenic Escherichia coli Strains That Inhibits Lymphocyte Activation

Abstract

The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown. We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli. Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins. A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation. Enterohemorrhagic E. coli strains of serotype O157:H7 possess a similar gene located on a large plasmid. Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E. coli is associated with gain of this activity. Among 89 strains of E. coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity. Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.Bacteria have evolved a number of mechanisms, including antiphagocytic factors, leukotoxins, and systems for iron chelation, to resist the nonspecific (innate) immune response of vertebrate hosts (25). In addition, several mechanisms to circumvent humoral immunity have been described, such as immunoglobulin A proteases and immunoglobulin-binding proteins. However, few bacterial factors that specifically interfere with cellular immune responses have been described (69). In addition, the mechanisms that allow certain bacterial pathogens to colonize hosts for prolonged periods remain obscure.Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea among infants in developing countries. EPEC is one of the few known bacterial causes of chronic diarrhea (24,34,55). EPEC strains are characterized by their ability to induce profound cytoskeletal rearrangements in host cells that result in the formation of adhesion pedestals upon which the bacteria rest (47). This phenomenon is known as the attaching and effacing effect. Enterohemorrhagic E. coli (EHEC) strains, a subgroup of Shiga-toxin-producing E. coli, also have attaching and effacing activity (26, 64). We previously reported that EPEC produce and secrete a high-molecular-weight, proteasesensitive factor that selectively inhibits production of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon by human peripheral and lamina propria mononuclear cells and inhibits proliferation of these cells (35,36,41). This inhibitory effect was observed regardless of whether the cells were stimulated by phorbol esters, mitogens, CD3 cross-linking, or antigen. The effect was also seen in macrophage-depleted T-cell populations and in Jurkat cells, indicating that this activity did not require participation of cells other than lymphocytes. ...

Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli

“…Although lymphocytes exposed to the factor are nonresponsive, there is no evidence that they undergo apoptosis or are killed. When the gene encoding this factor, lymphostatin, was cloned and mutated, the resulting strain could no longer inhibit lymphocyte function (29). A relatively short stretch of the sequence from this very large protein is homologous to the enzymatic domain of the large Clostridial cytotoxins, which covalently inactivate members of the Rho family of small mammalian GTPases.…”

“…EHEC 2 includes several widespread clones, including, for example, a common nonmotile O111 clone that occurs in both North and South America (29). Members of this clone have eae and produce both Stx1 and enterohemolysin (31).…”

Efa-1/LifA mediates intestinal colonization of calves by enterohaemorrhagic Escherichia coli O26 : H– in a manner independent of glycosyltransferase and cysteine protease motifs or effects on type III secretion

“…The mode of action of Efa-1 is further complicated by the fact that it appears to be bifunctional. EHEC O111 : H-Efa-1 is 97.4 % identical to EPEC O127 : H6 LifA (lymphostatin), which confers upon EPEC an ability to inhibit the mitogen-activated proliferation of peripheral blood and intraepithelial lymphocytes and the synthesis of pro-inflammatory cytokines (Klapproth et al, 1995(Klapproth et al, , 1996Malstrom & James, 1998;Klapproth et al, 2000). Such activity has been reported for Efa-1/LifA in EHEC O103 : H2 against bovine peripheral blood lymphocytes (Abu-Median et al, 2006).…”

“…Strains used in the present study are listed in Table 1; they were amplified in LuriaBertani (LB) medium supplemented as appropriate with nalidixic acid (25 mg ml 21 ), kanamycin (50 mg ml 21 ), ampicillin (100 mg ml 21 ) or chloramphenicol (25 mg ml 21 ). The pHC79-based cosmid containing the EPEC O127 : H6 strain E2348/29 lifA gene (pIV-8-A) has been described (Klapproth et al, 1995(Klapproth et al, , 2000. Plasmid pDM4 is a positiveselection suicide replicon (oriR6K mobRP4 cat sacBR; Milton et al, 1996) and was used to create chromosomal deletions by double homologous recombination.…”

“…Mutation of efa-1 in rabbit EPEC has not been reported to affect type III secretion, and Efa-1-specific antibodies labelled the bacterial surface and inhibited adherence to cultured cells, implying a direct role in adherence (Badea et al, 2003). However, in other studies, actin nucleation under adherent bacteria was taken as a measure of normal LEE function in efa-1 mutants (Nicholls et al, 2000;Klapproth et al, 2000), even though such activity can occur when type III secretion is reduced (Stevens et al, 2002b). The mode of action of Efa-1 is further complicated by the fact that it appears to be bifunctional.…”