Functional analyses reveal an important role for tyrosine residues in the staphylococcal multidrug efflux protein QacA

<p>Abstract</p> <p>Background</p> <p>The staphylococcal QacA multidrug efflux protein confers resistance to an exceptional number of structurally unrelated antimicrobial compounds. Aromatic amino acid residues have been shown to be highly important for the transport function of several multidrug transporters and are intimately involved in multidrug binding. This study investigated the structural and functional importance of the seven tyrosine residues in QacA by examining the phenotypic effect of incorporating conservative (aromatic) and non-conservative (non-aromatic) substitutions for these residues.</p> <p>Results</p> <p>Determination of the resistance profiles and analysis of drug transport assays revealed that non-conservative substitutions for most tyrosine residues influenced the QacA drug recognition spectrum. However, an aromatic residue at three tyrosine positions, 63, 410 and 429, was of importance for QacA-mediated transport and resistance to the majority of substrates tested.</p> <p>Conclusion</p> <p>A tyrosine or phenylalanine residue at amino acid positions corresponding to 63 of QacA in related drug efflux proteins is found to be highly conserved. Therefore, an aromatic side chain at this position is likely to partake in a function common to these drug transporters, such as proton translocation or essential intramolecular contacts, whereas aromatic residues at the non-conserved 410 and 429 positions are expected to mediate a QacA-specific function, possibly forming or stabilising part of the QacA drug binding region.</p

Africa's largest long-lasting insecticide-treated net producer: lessons from A to Z Textiles

Abstract Background Field trials have demonstrated the efficacy of insecticide-treated nets, and the WHO has recently endorsed a shift toward Long-Lasting Insecticide Treated nets (LLINs) due to factors such as reduced distribution costs. However, the need for LLINs poses several challenges. Is it possible to manufacture LLINs in large quantities in the African continent, where malaria is most endemic? When production is located in low-income countries, what role is played by local funding and employment, scaling up manufacturing, and partnerships? What factors influence availability and pricing? Discussion A case study of A to Z Textiles was undertaken to answer the question of how large-scale production of LLINs can occur in a low income setting. One of the largest sources of bed nets for Africa, A to Z Textiles is Africa-based, and its Tanzanian operations have a production capacity of 30 million LLINs per year, along with full WHO recommendation for its nets. Our analysis is based on semi-structured interviews with key informants familiar with A to Z, site visits in Tanzania, and literature reviews. This paper discusses the history and current status of A to Z Textiles, identifies the factors that led to its success, and suggests policy considerations that could support similar initiatives in the future. Local funding, scaling up manufacturing, technology transfer, and partnerships all played important roles in A to Z’s ascent, as did perceived benefits of local employment and capacity-building. Regulatory issues and procurement rules acted as barriers. A to Z cost-effectively manufactures high-quality LLINs where malaria is most endemic. Summary With a production capacity of 30 million LLINs per year, and full WHOPES (WHO Pesticide Evaluation Scheme) certification, A to Z Textiles demonstrates how key health goods can be successfully produced in the low-income countries that use them. Its example may be instructive and of high interest to readers in the malaria community, especially in developing countries, and to those who wish to support or partner with efforts by developing countries to build their health innovation capacity.</p

Structural and Functional Analysis of the Type III Secretion System from \u3ci\u3ePseudomonas fluorescens\u3c/i\u3e Q8r1-96

Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae. We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1-1 and hopM1, as well as a novel type III effector gene designated ropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses

Short chain diamines are the physiological substrates of PACE family efflux pumps

Acinetobacter baumannii has rapidly emerged as a major cause of gram-negative hospital infections worldwide. A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. AceI is also the prototype for the recently discovered proteobacterial antimicrobial compound efflux (PACE) family of transport proteins that confer resistance to a range of antibiotics and antiseptics in many gram-negative bacteria, including pathogens. The gene encoding AceI is conserved in the core genome of A. baumannii, suggesting that it has an important primordial function. This is incongruous with the sole characterized substrate of AceI, chlorhexidine, an entirely synthetic biocide produced only during the last century. Here we investigated a potential primordial function of AceI and other members of the PACE family in the transport of naturally occurring polyamines. Polyamines are abundant in living cells, where they have physiologically important functions and play multifaceted roles in bacterial infection. Gene expression studies revealed that the aceI gene is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines when energized by an electrochemical gradient. Assays conducted using 8 additional diverse PACE family proteins identified 3 that also catalyze cadaverine transport. Taken together, these results demonstrate that short-chain diamines are common substrates for the PACE family of transport proteins, adding to their broad significance as a novel family of efflux pumps

Effect of Tannic Acid on the Transcriptome of the Soil Bacterium Pseudomonas protegens Pf-5

Tannins are a diverse group of plant-produced, polyphenolic compounds with metal-chelating and antimicrobial properties that are prevalent in many soils. Using transcriptomics, we determined that tannic acid, a form of hydrolysable tannin, broadly affects the expression of genes involved in iron and zinc homeostases, sulfur metabolism, biofilm formation, motility, and secondary metabolite biosynthesis in the soil-and rhizosphere-inhabiting bacterium Pseudomonas protegens Pf-5.Keywords: Fluorescens PF-5, Condensed tannins, Biofilm formation, Escherichia Coli, Oxidative stress, Staphylococcus aureus, Binding, Aeruginosa, Radical formation, IronKeywords: Fluorescens PF-5, Condensed tannins, Biofilm formation, Escherichia Coli, Oxidative stress, Staphylococcus aureus, Binding, Aeruginosa, Radical formation, Iro

The Effect of Iron Limitation on the Transcriptome and Proteome of Pseudomonas fluorescens Pf-5

One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation

Root Exudates Alter the Expression of Diverse Metabolic, Transport, Regulatory, and Stress Response Genes In Rhizosphere \u3ci\u3ePseudomonas\u3c/i\u3e

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialog between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates of B. distachyon and demonstrated the presence of multiple carbohydrates, amino acids, organic acids, and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for the Pseudomonas strains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains’ pangenome, reflecting the importance of the variable gene content in the adaptation of P. fluorescens to the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts

Lessons from the development process of the Afghanistan integrated package of essential health services

In 2017, in the middle of the armed conflict with the Taliban, the Ministry of Public Health decided that the Afghan health system needed a well-defined priority package of health services taking into account the increasing burden of non-communicable diseases and injuries and benefiting from the latest evidence published by DCP3. This leads to a 2-year process involving data analysis, modelling and national consultations, which produce this Integrated Package of Essential health Services (IPEHS). The IPEHS was finalised just before the takeover by the Taliban and could not be implemented. The Afghanistan experience has highlighted the need to address not only the content of a more comprehensive benefit package, but also its implementation and financing. The IPEHS could be used as a basis to help professionals and the new authorities to define their priorities

Support for UNRWA's survival

The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) provides life-saving humanitarian aid for 5·4 million Palestine refugees now entering their eighth decade of statelessness and conflict. About a third of Palestine refugees still live in 58 recognised camps. UNRWA operates 702 schools and 144 health centres, some of which are affected by the ongoing humanitarian disasters in Syria and the Gaza Strip. It has dramatically reduced the prevalence of infectious diseases, mortality, and illiteracy. Its social services include rebuilding infrastructure and homes that have been destroyed by conflict and providing cash assistance and micro-finance loans for Palestinians whose rights are curtailed and who are denied the right of return to their homeland