Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Abstract

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenicEscherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), andCitrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.

Attaching-effacing Bacteria in Animals

“…Tir binds to the Cterminus of the intimin molecule (Liu et al, 1999;Luo et al, 2000), the amino-acid sequence of which varies between AE organisms. Four principal subtypes of intimin (a, b, g and 3) have been established, based on antigenic, amino-acid and eae sequence differences at the C-terminus domain (Adu-Bobie et al, 1998;Oswald et al, 2000). However, up to seven further subtypes have been proposed, based upon eae sequence variation (Zhang et al, 2002;Jores et al, 2003).…”

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

“…Although the intimin C-terminal portion interacts with Tir, it has been suggested that the different intimin subtypes may drive bacteria to different intestinal regions (Phillips & Frankel, 2000). Thus far, eae variant alleles encoding 33 different intimin subtypes have been established (reviewed in Schmidt, 2010), with subtypes alpha, beta, and gamma being the most common (Adu-Bobie et al, 1998;Beutin et al, 2003;Ramachandran et al, 2003;Blanco et al, 2006a, b;Abe et al, 2009;Horcajo et al, 2012).…”

Comparison of eae, tir, espA and espB genes of bovine and human attaching and effacing Escherichia coli by multiplex polymerase chain reaction

“…In addition, eae from a dog EPEC [13] and a bovine EHEC [14] have likewise been cloned and sequenced. The eae gene variants of human strains have been recently classi¢ed as K, L, Q and O ( [15], Oswald, personal communication). They can be distinguished by PCR or gene probes [15].…”