Dimerization of the Pseudomonas aeruginosa Translocator Chaperone PcrH Is Required for Stability, Not Function

Tomalka, Amanda G.; Zmina, Stephanie E.; Stopford, Charles M.; Rietsch, Arne

doi:10.1128/jb.00335-13

Cited by 4 publications

(7 citation statements)

References 22 publications

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“…For each hit, the orthologue proteins, extracted from the Ortholuge DB [30], that were found in DEG are indicated with the abbreviation of the harboring bacterial species. Bacterial species: Ab ( Acinetobacter baylyi ), Bs ( Bacillus subtilis ), Bt ( Bacteroides thetaiotaomicron ), Cc ( Caulobacter crescentus ), Ec ( Escherichia coli ), Fn ( Francisella novicida ), Hi ( Haemophilus influenzae ), Hp ( Helicobacter pylori) , Mt ( Mycobacterium tuberculosis ), Mp ( Mycoplasma pulmonis ), Mg ( Mycoplasma genitalium ), Pg ( Porphyromonas gingivalis ), Pa ( Pseudomonas aeruginosa ), Se ( Salmonella enterica ), St ( Salmonella typhimurium ), Sp ( Streptococcus pneumoniae ), Ss ( Streptococcus sanguinis ), Sa (S taphylococcus aureus ), Vc ( Vibrio cholerae ). d Previous reports [24-26] did not mention growth defects associated to deletion of popD gene. e Hit not present in DEG whose essentiality was experimentally demonstrated in P. aeruginosa [21]. …”

Section: Resultsmentioning

confidence: 99%

“…Among these, PA1709 ( popD ), coding for a subunit of the PopB/D translocon complex of the type III secretion-translocation system (TTSS), is implicated in effector translocation across the host plasma membrane. Previous reports on P. aeruginosa PopD function [ 24 - 26 ] did not mention growth defects associated to deletion of popD gene. Therefore, the growth-impairing effects of S5A10 insert corresponding to PA1709 (Table 1 ) did not seem to match the PopD role characterized so far.…”

Section: Discussionmentioning

confidence: 99%

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A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa

et al. 2014

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BackgroundAntibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics.ResultsWe aimed to identify novel essential genes in P. aeruginosa by shotgun antisense screening. This technique was developed in Staphylococcus aureus and, following a period of limited success in Gram-negative bacteria, has recently been used effectively in Escherichia coli. To also target low expressed essential genes, we included some variant steps that were expected to overcome the non-stringent regulation of the promoter carried by the expression vector used for the shotgun antisense libraries. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 28 “essential-for-growth” genes: five were “classical” essential genes involved in DNA replication, transcription, translation, and cell division; seven were already reported as essential in other bacteria; and 16 were “novel” essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics, namely protein secretion, biosynthesis of cofactors, prosthetic groups and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcription regulation. This study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes, of which 25 have homologs reported as essential in other bacteria. Finally, four multigenic growth-impairing inserts belonged to operons that have never been reported to play an essential role.ConclusionsFor the first time in P. aeruginosa, we applied regulated antisense RNA expression and showed the feasibility of this technology for the identification of novel essential genes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa

et al. 2014

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“…A short peptide derived from the N-terminal anchor of these translocators, however, is unlikely to disrupt the dimeric chaperone. Furthermore, the dimeric conformation of the chaperone is not required for its function but likely contributes to its stability (Tomalka et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Structure of AcrH–AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly

et al. 2015

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Type III secretion systems (T3SSs) are adopted by pathogenic bacteria for the transport of effector proteins into host cells through the translocon pore composed of major and minor translocator proteins. Both translocators require a dedicated chaperone for solubility. Despite tremendous efforts in the past, structural information regarding the chaperone-translocator complex and the topology of the translocon pore have remained elusive. Here, we report the crystal structure of the major translocator, AopB, from Aeromonas hydrophila AH-1 in complex with its chaperone, AcrH. Overall, the structure revealed unique interactions between the various interfaces of AopB and AcrH, with the N-terminal "molecular anchor" of AopB crossing into the "N-terminal arm" of AcrH. AopB adopts a novel fold, and its transmembrane regions form two pairs of helical hairpins. From these structural studies and associated cellular assays, we deduced the topology of the assembled T3SS translocon; both termini remain extracellular after membrane insertion.

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“…The origin of the translocator-effector secretion hierarchy is not understood, but has been proposed to arise from differential affinities and competition for binding sites either between chaperones and their effector or translocator cargo or between chaperone-effector/translocator complexes and cytosolic components of the T3SS [10] , [11] , [29] – [32] . To assess the importance of gatekeeper-translocator chaperone interactions in diverse pathogens, and because adequate tools and reagents for functional analysis of CopN mutants are not available in Chlamydia , we have extended our structural analysis with functional studies of MxiC and IpgC, the gatekeeper and translocator-specific chaperone from Shigella .…”

Section: Introductionmentioning

confidence: 99%

A Gatekeeper Chaperone Complex Directs Translocator Secretion during Type Three Secretion

Archuleta

Spiller

2014

PLoS Pathog

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Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

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Dimerization of the Pseudomonas aeruginosa Translocator Chaperone PcrH Is Required for Stability, Not Function

Cited by 4 publications

References 22 publications

A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa

A shotgun antisense approach to the identification of novel essential genes in Pseudomonas aeruginosa

Structure of AcrH–AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly

A Gatekeeper Chaperone Complex Directs Translocator Secretion during Type Three Secretion

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