Internalization of Escherichia Coli O157:H7 by Bovine Rectal Epithelial Cells

Abstract

Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent.

Invasion of differentiated intestinal Caco-2 cells is a sporadic property among atypical enteropathogenicEscherichia colistrains carrying common intimin subtypes

“…In addition, Sheng et al . () demonstrated that intimin–Tir interaction was not required for efficient EHEC O157:H7 invasion of primary bovine epithelial cells. Furthermore, other effector proteins were associated with EPEC invasion in vitro , such as Map (Jepson et al ., ), EspF (Weflen et al ., ), and EspT (Bulgin et al ., , b), that manipulate Rho‐GTPase signaling pathways (reviewed in Wong et al ., ).…”

Cell invasion and survival of Shiga toxin-producing Escherichia coli within cultured human intestinal epithelial cells

“…Although LEE-encoded effector proteins might play a role in gut colonization (Naylor et al, 2005;Vlisidou et al, 2006;Dziva et al, 2007), LEE-negative STEC strains can colonize cattle efficiently, showing that other factors influence the persistence of STEC in the bovine intestine. Sheng et al (2011) working with rectal biopsies and with primary bovine rectal epithelial cell (PBRE) cultures, showed that a subpopulation of STEC O157 : H7 was internalized, and that deletion of the tir gene, although impairing the intimate attachment and production of A/E lesions, did not affect bacterial internalization of the strain EDL933. Electron micrographs taken from infected PBRE cells showed microvilli-like membrane extensions surrounding the internalizing bacteria.…”

“…Recently, work seeking to explain the bacterial colonization of the bovine intestinal mucosa by STEC moved from the role played by bacterial adhesins to the investigation of STEC invasiveness (Sheng et al, 2011).…”

Strain-Dependent Cellular Immune Responses in Cattle following Escherichia coli O157:H7 Colonization

“…However, to our knowledge, only two studies have considered the role of cellular immunity during EHEC O157:H7 colonization in ruminants; these identified lymphoproliferative cellular responses to heat-killed EHEC O157:H7 (40) and recombinant antigens (41) in bovine peripheral blood mononuclear cells and ovine rectal lymph node cells, respectively. The case for a protective cellular immune response is further strengthened by the observations that colonized bovine epithelial cells are exfoliated in vivo (42) and that some strains are efficiently internalized by bovine epithelial cells both in vivo and in vitro (43).…”