title: Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.
abstract: Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.
authors.affiliation: Schools of Cellular & Molecular Medicine and Biochemistry, University of Bristol, Bristol, United Kingdom.
authors.forename: Isabel
authors.initials: I
authors.lastname: Martinez-Argudo
authors.forename: Andreas K J
authors.initials: AK
authors.lastname: Veenendaal
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
authors.forename: A Dorothea
authors.initials: AD
authors.lastname: Roehrich
authors.forename: Maria C
authors.initials: MC
authors.lastname: Ronessen
authors.forename: Giulia
authors.initials: G
authors.lastname: Franzoni
authors.forename: Katerine N
authors.initials: KN
authors.lastname: van Rietschoten
authors.forename: Yusuke V
authors.initials: YV
authors.lastname: Morimoto
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Saijo-Hamano
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Avison
authors.forename: David J
authors.initials: DJ
authors.lastname: Studholme
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Tohru
authors.initials: T
authors.lastname: Minamino
authors.forename: Ariel J
authors.initials: AJ
authors.lastname: Blocker
mesh_terms: D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism; D002875:Chromosomes / Q000648:ultrastructure; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005407:Flagella / Q000187:drug effects / Q000378:metabolism*; D017353:Gene Deletion; D006834:Hydrazines / Q000494:pharmacology*; D009068:Movement; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D020156:Salicylic Acid / Q000494:pharmacology*; D019779:Salmonella enterica / Q000187:drug effects* / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D006834:Hydrazines; D020156:Salicylic Acid