Self-association motifs in the enteroaggregative Escherichia coli heat-resistant agglutinin 1

Abstract

The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of Escherichia coli that are exceptional colonizers. Hra1 from enteroaggregative E. coli strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four in silico-predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surfaceexposed region of Rck, a Salmonella enterica serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1-Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.

An Updated View on the Rck Invasin of Salmonella: Still Much to Discover

“…An auto-aggregation motif spanning 80-89 amino acids has recently been identified in the second cell surface-exposed loop and is 100% conserved in all Rck proteins. This motif is not present in the other members of the Ail/Lom family (Glaubman et al, 2016 ). Moreover, the third loop and more precisely the 46 amino-acid region from G114 to V159 is necessary and sufficient to induce the invasion process.…”

Bacterial autoaggregation

“…Small β-barrel proteins such as Hra1 from E. coli or Ail from Yersinia pestis are small (<30 kDa) integral transmembrane proteins of the outer membranes of Gram-negative cells. These proteins consist of a β-barrel transmembrane domain, the loops of which extend into the extracellular space and can thus interact with loops from proteins on the surface of a neighbouring bacterium [65]. Examples are listed in Table 1, and we describe selected examples in detail in later sections.…”

The Not so Good, the Bad and the Ugly: Differential Bacterial Adhesion and Invasion Mediated by Salmonella PagN Allelic Variants

“…Rck has several adhesive properties: it binds to factor H to mediate bacterial resistance to complement and adheres to laminin and interacts with the epidermal growth factor receptor for a zipper mechanism of cell invasion [13,[15][16][17]. Rck also contains a self-association motif that has the potential to mediate interbacterial attachment, as described for Hra1/Hek, an integral outer membrane protein of enteroaggregative Escherichia coli (E. coli) [18,19]. The adhesins/invasins Hra1/Hek, Hra2 and Tia of enteroaggregative or enterotoxigenic E. coli share homologies with membrane spanning domains of PagN, but their surface-exposed loops are less similar, alluding to variable binding affinities for different host receptors [18,[20][21][22].…”