ABSTRACT
Helicokinin I, a diuretic neuropeptide of the relevant cotton pest Helicoverpa zea represents a promising target for the design of insect neuropeptide mimetics. Using a ring-closing metathesis reaction, N-terminal bridged macrocyclic helicokinin I analogues with different rigidity were prepared and tested in a helicokinin receptor assay. A partially peptidomimetic helicokinin analogue, containing two structural modifications provides a deeper insight into the structural-requirements for receptor-binding.
Subject(s)
Diuretics/chemical synthesis , Insect Proteins/antagonists & inhibitors , Macrocyclic Compounds/chemical synthesis , Neuropeptides/chemistry , Peptidomimetics/chemical synthesis , Receptors, Cell Surface/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , Biological Assay , Diuretics/pharmacology , Insect Proteins/chemistry , Insect Proteins/metabolism , Macrocyclic Compounds/pharmacology , Molecular Sequence Data , Moths , Neuropeptides/metabolism , Peptidomimetics/pharmacology , Protein Binding/drug effects , Receptors, Cell Surface/metabolism , Structure-Activity RelationshipABSTRACT
Type III secretion systems (TTSSs) are specialized protein transport systems in gram-negative bacteria which target effector proteins into the host cell. The TTSS of the plant pathogen Xanthomonas campestris pv. vesicatoria, encoded by the hrp (hypersensitive reaction and pathogenicity) gene cluster, is essential for the interaction with the plant. One of the secreted proteins is HrpF, which is required for pathogenicity but dispensable for type III secretion of effector proteins in vitro, suggesting a role in translocation. In this study, complementation analyses of an hrpF null mutant strain using various deletion derivatives revealed the functional importance of the C-terminal hydrophobic protein region. Deletion of the N terminus abolished type III secretion of HrpF. Employing the type III effector AvrBs3 as a reporter, we show that the N terminus of HrpF contains a signal for secretion but not a functional translocation signal. Experiments with lipid bilayers revealed a lipid-binding activity of HrpF as well as HrpF-dependent pore formation. These data indicate that HrpF presumably plays a role at the bacterial-plant interface as part of a bacterial translocon which mediates effector protein delivery across the host cell membrane.
Subject(s)
Bacterial Proteins/physiology , Plants/microbiology , Xanthomonas campestris/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport, Active , Cell Membrane/metabolism , Cell Membrane/microbiology , Gene Deletion , Genes, Bacterial , Genetic Complementation Test , Lipid Metabolism , Multigene Family , Protein Binding , Protein Transport/physiology , Transcription Activator-Like Effectors , Xanthomonas campestris/pathogenicityABSTRACT
The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key hrp regulator HrpG, in the regions flanking the hrp gene cluster. Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeat-containing protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from Xanthomonas oryzae pv. oryzae and PsvA from Pseudomonas syringae, respectively. XopA and XopD (Xanthomonas outer proteins) are secreted by the Xanthomonas Hrp TTSS and thus represent putative effector proteins. Mutations in xopA, but not in xopD, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants. Since the xopD promoter contains a putative hrp box, which is characteristic of hrpL-regulated genes in P. syringae and Erwinia spp., the gene was probably acquired by horizontal gene transfer. Interestingly, the regions flanking the hrp gene cluster also contain insertion sequences and genes for a putative transposase and a tRNA(Arg). These features suggest that the hrp gene cluster of X. campestris pv. vesicatoria is part of a pathogenicity island.
