Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.

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.Funding: IMA and XL were supported by Wellcome Trust Project Grant 088266 to IMA and AJB. AKJV was funded by an E.C. Marie Curie postdoctoral fellowship (MEIF-CT-2005-023694) and an EMBO short-term fellowship (244-2007). XL was supported additionally by Wellcome Trust Project Grant 088231 to AJB and KN. DR was supported by a University of Bristol Centenary Postgraduate Studentship. This work was also supported in part by MRC project grants G0701243 and MR-J002097-1 to AJB and AJB and KN, respectively, and by Grants-in-Aid for Scientific Research (22570161 and 23121516 to TM, and 21227006 to KN) and Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Takeda Science Foundation (to TM). YVM is a Research Fellow of the Japanese Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Delivering better outcomes through customer-led project management: the case of the major project BT 21st Century Network in the UK

Traditional approaches of major project management take the strategy of selecting a supplier-led prime/systems integrator. Although this strategy pushes a significant amount of risk to the supplier, project performance may suffer due to lower engagement of the customer in the anticipation of potential issues involving a major project. Thus, this research investigates the implications of the customer, as opposed to a selected external supplier, assuming the role of systems/prime integrator, as a Problem Structuring Method (PSM) to better deal with the soft side and uncertainties of the project. A case study approach is conducted on the major project BT 21st Century Network (BT21CN) to demonstrate that customer-led systems integration projects may provide more balance in the relationship and distribution of risks between supplier and customer, having a positive impact on project performance, accelerating the development of BT’s organisational capabilities, and producing better project outcomes in the long term

A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome

Many bacteria use a type III secretion system to inject effector proteins into host cells. Selection and export of the effectors is controlled by a set of soluble proteins at the cytosolic interface of the membrane spanning type III secretion “injectisome”. Combining fluorescence microscopy, biochemical interaction studies and fluorescence correlation spectroscopy, we show that in live Yersinia enterocolitica bacteria these soluble proteins form complexes both at the injectisome and in the cytosol. Binding to the injectisome stabilizes these cytosolic complexes, whereas the free cytosolic complexes, which include the type III secretion ATPase, constitute a highly dynamic and adaptive network. The extracellular calcium concentration, which triggers activation of the T3SS, directly influences the cytosolic complexes, possibly through the essential component SctK/YscK, revealing a potential mechanism involved in the regulation of type III secretion