Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer

Abstract

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving the products of at least two operons, exeAB (gspAB) and exeC-N (gspC-N). In the studies reported here, aerolysin secretion was restored to C5.84, an exeA::Tn5-751 mutant, by overexpression of exeD alone in trans. Expression studies indicated that these results did not reflect a role of ExeAB in the regulation of the exeC-N operon. Instead, immunoblot analysis showed that ExeD did not multimerize in C5.84, and fractionation of the membranes showed that the monomeric ExeD remained in the inner membrane. Expression of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion. Pulse-chase analysis also suggested that the induction of ExeAB allowed multimerization of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized even in the absence of ExeAB and, although most remained in the inner membrane, an amount similar to that in wild-type outer membranes fractionated with the outer membrane of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.

Nucleotide Sequence and Evolution of the Five-Plasmid Complement of the Phytopathogen Pseudomonas syringae pv. maculicola ES4326

“…P. syringae pv. maculicola ES4326 carries five plasmids, named sequentially pPMA4326A to pPMA4326E, with sizes of 46,697,40,110,8,244,4,833, and 4,217 bp, respectively (Fig. 1).…”

“…The integrase and resolvase genes of this cassette may be integral components for mobility, while the function of the exeA gene is unclear. ExeA proteins have been implicated in type II secretion, and more specifically, in the secretion of the virulence-related aerolysin toxin by Aeromonas hydrophila; however, this secretion is dependent on a second factor, encoded by exeB (4,36,40,68), which is not present within the P. syringae pv. maculicola ES4326 cassette.…”

Agrobacterium VirB10, an ATP energy sensor required for type IV secretion

“…For example, in Aeromonas hydrophila, the ExeA ATP-binding and the ExeB bitopic subunits of a type II secretion system (T2SS) form a complex that is altered in its conformation by mutation of nucleotide triphosphate binding motifs (31,32). Moreover, formation of the ExeAB complex was shown to promote multimerization of the secretin ExeD, leading to the suggestion that the ExeAB complex energizes assembly of the secretin ring required for aerolysin secretion across the OM (33). In other T2SS, the TonB-like GspC proteins have been shown to interact both with an IM platform composed of VirB11-like GspE and proteins GspF, -L, and -M, and an OM complex composed of GspS lipoprotein and GspD secretin (34)(35)(36)(37).…”

Role of phages in the pathogenesis of Burkholderia, or ‘Where are the toxin genes in Burkholderia phages?’

“…Based solely on primary structure similarity, BcepMu gp8 is related to ExeA of Aeromonas hydrophila. The ExeAB complex of A. hydrophila is required for the localization and assembly of the oligomeric ring structure of ExeD, shown in other systems to be a secretin involved in secretion of toxins such as aerolysin [49]. However, another model for the function of BcepMu gp8 has been proposed that does not implicate a role in pathogenicity.…”