Genome-Wide Transposon Screen of a Pseudomonas syringae mexB Mutant Reveals the Substrates of Efflux Transporters.

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The resistance-nodulation-division (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and ΔmexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a ΔmexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants.IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization

Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen

Insights on Mycobacterium leprae Efflux Pumps and Their Implications in Drug Resistance and Virulence

Drug resistance in Mycobacterium leprae is assumed to be due to genetic alterations in the drug targets and reduced cell wall permeability. However, as observed in Mycobacterium tuberculosis, drug resistance may also result from the overactivity of efflux systems, which is mostly unexplored. In this perspective, we discuss known efflux pumps involved in M. tuberculosis drug resistance and virulence and investigate similar regions in the genome of M. leprae. In silico analysis reveals that the major M. tuberculosis efflux pumps known to be associated with drug resistance and virulence have been retained during the reductive evolutionary process that M. leprae underwent, e.g., RND superfamily, the ABC transporter BacA, and the MFS P55. However, some are absent (DinF, MATE) while others are derepressed (Mmr, SMR) in M. leprae reflecting the specific environment where M. leprae may live. The occurrence of several multidrug resistance efflux transporters shared between M. leprae and M. tuberculosis reveals potential implications in drug resistance and virulence. The conservation of the described efflux systems in M. leprae upon genome reduction indicates that these systems are potentially required for its intracellular survival and lifestyle. They potentially are involved in M. leprae drug resistance, which could hamper leprosy treatment success. Studying M. leprae efflux pumps as new drug targets is useful for future leprosy therapeutics, enhancing the global efforts to eradicate endemic leprosy, and prevent the emergence of drug resistance in afflicted countries

Characterization of the l-arabinofuranose-specific GafABCD ABC transporter essential for l-arabinose-dependent growth of the lignocellulose-degrading bacterium Shewanella sp. ANA-3

Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology

Targeted CRISPR activation and knockout screenings identify novel doxorubicin transporters

PurposeTissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been suggested that many drugs need active or defined transporters to pass the cell membrane. In contrast to efflux components induced after anti-cancer drugs reach the intracellular compartment, drug importers are required for initial drug responses. Furthermore, tissue-specific uptake of anti-cancer drugs may directly impact the side effects of many drugs when they accumulate in healthy tissues. Therefore, linking anti-cancer drugs to their respective drug import transporters would directly help to predict drug responses, whilst minimizing side effects.MethodsTo identify drug transporters of the commonly used anti-cancer drug doxorubicin, we performed focused CRISPR activation and knockout genetic screens targeting all potential membrane-associated transporters and proteins. We monitored the direct uptake of doxorubicin by fluorescence-activated cell sorting (FACS) as the screening readout for identifying transporters/proteins directly involved in doxorubicin uptake.ResultsIntegrating the data from these comprehensive CRISPR screenings, we confirmed previously indicated doxorubicin exporters such as ABCB1 and ABCG2 genes, and identified novel doxorubicin importer gene SLC2A3 (GLUT3). Upregulation of SLC2A3 led to higher doxorubicin uptake and better cell killing, indicating SLC2A3 could be a new marker to predict doxorubicin drug response and minimize side effects for the personalized application of this conventional chemotherapeutic drug.ConclusionsOur study provides a comprehensive way for identifying drug transporters, as exemplified by the commonly used anti-cancer drug doxorubicin. The newly identified importers may have direct clinical implications for the personalized application of doxorubicin in treating distinct tumors. Our results also highlight the necessity of combining both CRISPR knockout and CRISPR activation genetic screens to identify drug transporters.Chemical Immunolog

Investigation of microbial community interactions between Lake Washington methanotrophs using genome-scale metabolic modeling

Background. The role of methane in global warming has become paramount to the environment and the human society, especially in the past few decades. Methane cycling microbial communities play an important role in the global methane cycle, which is why the characterization of these communities is critical to understand and manipulate their behavior. Methanotrophs are a major player in these communities and are able to oxidize methane as their primary carbon source. Results. Lake Washington is a freshwater lake characterized by a methane-oxygen countergradient that contains a methane cycling microbial community. Methanotrophs are a major part of this community involved in assimilating methane from lake water. Two significant methanotrophic species in this community are Methylobacter and Methylomonas. In this work, these methanotrophs are computationally studied via developing highly curated genome-scale metabolic models. Each model was then integrated to form a community model with a multi-level optimization framework. The competitive and mutualistic metabolic interactions among Methylobacter and Methylomonas were also characterized. The community model was next tested under carbon, oxygen, and nitrogen limited conditions in addition to a nutrient-rich condition to observe the systematic shifts in the internal metabolic pathways and extracellular metabolite exchanges. Each condition showed variations in the methane oxidation pathway, pyruvate metabolism, and the TCA cycle as well as the excretion of formaldehyde and carbon di-oxide in the community. Finally, the community model was simulated under fixed ratios of these two members to reflect the opposing behavior in the two-member synthetic community and in sediment-incubated communities. The community simulations predicted a noticeable switch in intracellular carbon metabolism and formaldehyde transfer between community members in sediment- incubated vs. synthetic condition. Conclusion. In this work, we attempted to predict the response of a simplified methane cycling microbial community from Lake Washington to varying environments and also provide an insight into the difference of dynamics in sediment-incubated microcosm community and synthetic co-cultures. Overall, this study lays the ground for in silico systems-level studies of freshwater lake ecosystems, which can drive future efforts of understanding, engineering, and modifying these communities for dealing with global warming issues

Into the Wild: Parallel Transcriptomics of the Tsetse-Wigglesworthia Mutualism within Kenyan Populations

Tsetse flies (Diptera: Glossinidae) have medical significance as the obligate vectors of African trypanosomes. In addition, tsetse harbor a simple gut microbiota. A predominant gut microbiota member, the Gammaproteobacterium Wigglesworthia spp., has coevolved with tsetse for a significant portion of Glossina radiation proving critical to tsetse fitness. Although multiple roles have been described for Wigglesworthia within colony flies, little research has been dedicated towards functional characterization within wild tsetse. Here, dual RNA-Seq was performed to characterize the tsetse-Wigglesworthia symbiosis within flies captured in Nguruman, Kenya. A significant correlation in Gene Ontology (GO) distribution between tsetse and Wigglesworthia was observed, with homogeneous enrichment in metabolic and transport categories, likely supporting a hallmark of the symbiosis-bidirectional metabolic exchange. Within field flies, highly transcribed Wigglesworthia loci included those involved in B vitamin synthesis and in substrate translocation, including amino acid transporters and multidrug efflux pumps, providing a molecular means for interaction. The universal expression of several Wigglesworthiaand G. pallidipes orthologs, putatively involved in nutrient provisioning and resource allocation, was confirmed in sister tsetse species. These transcriptional profiles varied through host age and mating status likely addressing varying symbiont demands and also confirming their global importance within Glossina. This study, not only supports symbiont nutrient provisioning roles, but also serves as a foundation for insight into novel roles and molecular mechanisms associated with vector–microbiota interactions. The role of symbiont B vitamin provisioning towards impacting host epigenetics is discussed. Knowledge of vector–microbiota interactions may lead to the discovery of novel targets in pest control

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate

BACKGROUND: Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. RESULTS: The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. CONCLUSIONS: In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals

Addressing recall bias in (post-)conflict data collection and analysis : lessons from a large-scale health survey in Colombia

BACKGROUND: Much applied research on the consequences of conflicts for health suffers from data limitations, particularly the absence of longitudinal data spanning pre-, during- and post-conflict periods for affected individuals. Such limitations often hinder reliable measurement of the causal effects of conflict and their pathways, hampering also the design of effective post-conflict health policies. Researchers have sought to overcome these data limitations by conducting ex-post surveys, asking participants to recall their health and living standards before (or during) conflict. These questions may introduce important analytical biases due to recall error and misreporting. METHODS: We investigate how to implement ex-post health surveys that collect recall data, for conflict-affected populations, which is reliable for empirical analysis via standard quantitative methods. We propose two complementary strategies based on methods developed in the psychology and psychometric literatures-the Flashbulb and test-retest approaches-to identify and address recall bias in ex-post health survey data. We apply these strategies to the case study of a large-scale health survey which we implemented in Colombia in the post-peace agreement period, but that included recall questions referring to the conflict period. RESULTS: We demonstrate how adapted versions of the Flashbulb and test-retest strategies can be used to test for recall bias in (post-)conflict survey responses. We also show how these test strategies can be incorporated into post-conflict health surveys in their design phase, accompanied by further ex-ante mitigation strategies for recall bias, to increase the reliability of survey data analysis-including by identifying the survey modules, and sub-populations, for which empirical analysis is likely to yield more reliable causal inference about the health consequences of conflict. CONCLUSIONS: Our study makes a novel contribution to the field of applied health research in humanitarian settings, by providing practical methodological guidance for the implementation of data collection efforts in humanitarian contexts where recall information, collected from primary surveys, is required to allow assessments of changes in health and wellbeing. Key lessons include the importance of embedding appropriate strategies to test and address recall bias into the design of any relevant data collection tools in post-conflict or humanitarian contexts