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

Surfactant replacement and open lung concept – Comparison of two treatment strategies in an experimental model of neonatal ARDS

Background: Several concepts of treatment in neonatal ARDS have been proposed in the last years. The present study compared the effects of open lung concept positive pressure ventilation (PPVOLC) with a conventional ventilation strategy combined with administration of two different surfactant preparations on lung function and surfactant homoeostasis. Methods: After repeated whole-lung saline lavage, 16 newborn piglets were assigned to either PPVOLC(n = 5) or surfactant treatment under conventional PPV using a natural bovine (n = 5) or a monomeric protein B based surfactant (n = 6). Results: Comprehensive monitoring showed each treatment strategy to improve gas exchange and lung function, although the effect on PaO2and pulmonary compliance declined over the study period in the surfactant groups. The overall improvement of the ventilation efficiency index (VEI) was significantly greater in the PPVOLCgroup. Phospholipid and protein analyses of the bronchoalveolar lavage fluid showed significant alterations to surfactant homoeostasis in the PPVOLCgroup, whereas IL-10 and SP-C mRNA expression was tendentially increased in the surfactant groups. Conclusion: The different treatment strategies applied could be shown to improve gas exchange and lung function in neonatal ARDS. To which extent differences in maintenance of lung function and surfactant homeostasis may lead to long-term consequences needs to be studied further

Modified Needle-Tip PcrV Proteins Reveal Distinct Phenotypes Relevant to the Control of Type III Secretion and Intoxication by Pseudomonas aeruginosa

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication

Non-linear effects of drought under shade: reconciling physiological and ecological models in plant communities

The combined effects of shade and drought on plant performance and the implications for species interactions are highly debated in plant ecology. Empirical evidence for positive and negative effects of shade on the performance of plants under dry conditions supports two contrasting theoretical models about the role of shade under dry conditions: the trade-off and the facilitation hypotheses. We performed a meta-analysis of field and greenhouse studies evaluating the effects of drought at two or more irradiance levels on nine response variables describing plant physiological condition, growth, and survival. We explored differences in plant response across plant functional types, ecosystem types and methodological approaches. The data were best fit using quadratic models indicating a humped-back shape response to drought along an irradiance gradient for survival, whole plant biomass, maximum photosynthetic capacity, stomatal conductance and maximal photochemical efficiency. Drought effects were ameliorated at intermediate irradiance, becoming more severe at higher or lower light levels. This general pattern was maintained when controlling for potential variations in the strength of the drought treatment among light levels. Our quantitative meta-analysis indicates that dense shade ameliorates drought especially among drought-intolerant and shade-tolerant species. Wet tropical species showed larger negative effects of drought with increasing irradiance than semiarid and cold temperate species. Non-linear responses to irradiance were stronger under field conditions than under controlled greenhouse conditions. Non-linear responses to drought along the irradiance gradient reconciliate opposing views in plant ecology, indicating that facilitation is more likely within certain range of environmental conditions, fading under deep shade, especially for drought-tolerant species

Timing is everything: the regulation of type III secretion

Type Three Secretion Systems (T3SSs) are essential virulence determinants of many Gram-negative bacteria. The T3SS is an injection device that can transfer bacterial virulence proteins directly into host cells. The apparatus is made up of a basal body that spans both bacterial membranes and an extracellular needle that possesses a channel that is thought to act as a conduit for protein secretion. Contact with a host-cell membrane triggers the insertion of a pore into the target membrane, and effectors are translocated through this pore into the host cell. To assemble a functional T3SS, specific substrates must be targeted to the apparatus in the correct order. Recently, there have been many developments in our structural and functional understanding of the proteins involved in the regulation of secretion. Here we review the current understanding of protein components of the system thought to be involved in switching between different stages of secretion

Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds