Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis

Abstract

Epidemiological studies suggest frequent association of enteropathogenic bacteria with <i>Entamoeba histolytica</i> during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic <i>Escherichia coli</i> (strain DH5α) could modify the virulence of <i>E. histolytica</i> to cause disease in animal models of amebiasis. <i>In vitro</i> studies showed a 2-fold increase in CaCo2 monolayer destruction when <i>E. histolytica</i> interacted with EPEC but not with <i>E. coli</i> DH5α for 2.5 h. This was associated with increased <i>E. histolytica</i> proteolytic activity as revealed by zymogram analysis and degradation of the <i>E. histolytica</i> CP-A1/5 (<i>Eh</i>CP-A1/5) peptide substrate Z-Arg-Arg-pNC and <i>Eh</i>CP4 substrate Z-Val-Val-Arg-AMC. Additionally, <i>E. histolytica</i>-EPEC interaction increased <i>EhCP-A1</i>, -<i>A2</i>, -<i>A4</i>, and -<i>A5</i>, <i>Hgl</i>, <i>Apa</i>, and C<i>ox-1</i> mRNA expression. Despite the marked upregulation of <i>E. histolytica</i> virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either <i>E. histolytica</i>-EPEC or <i>E. histolytica</i>-<i>E. coli</i> DH5α. Histopathology of livers of <i>E. histolytica</i>-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of <i>il-1β</i>, <i>ifn-γ</i>, and <i>tnf-α</i> proinflammatory cytokine genes compared with that in livers of <i>E. histolytica</i>-<i>E. coli</i> DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with <i>E. histolytica</i>-EPEC manifested by downregulation of <i>Math1</i> mRNA with a corresponding increase in the expression of <i>Muc2</i> mucin and proinflammatory cytokine genes <i>il-6</i>, <i>il-12</i>, and <i>mcp-1</i> These results demonstrate that <i>E. histolytica</i>/EPEC interaction enhanced the expression and production of key molecules associated with <i>E. histolytica</i> virulence, critical in pathogenesis and progression of disease.