Bacterial Transporter Research Articles

Root cell-type specific expressions of bacterial mercury transporter MerC and plant SNARE SYP121 fusion protein differentially affect cadmium accumulation patterns of Arabidopsis

ABSTRACT There is increasing demand for solutions against cadmium pollution to secure food safety, and phytoremediation is one of the potential tools. We previously found that a bacterial mercury transporter MerC possesses cadmium uptake activity and its overexpression as a fusion protein with a plasma-membrane resident SNARE protein, SYP121 enhances the cadmium uptake ability of Arabidopsis plants. In this study, we examined whether two different root cell-type specific expression systems of MerC-SYP121 fusion protein could efficiently enhance cadmium accumulation of Arabidopsis plants, compared to the p35S-driven ubiquitous expression system. Representative transgenic lines expressing MerC-SYP121 in root surface cells (pEpi lines) or root endodermal cells (pSCR lines), established in our previous studies, were subjected to different cadmium treatments along with the p35S line. A vertical agar plate assay showed that root surface-specific line pEpi, as well as the p35S line, showed about 15% higher cadmium accumulation in shoots after one-day 10 µM cadmium treatment, compared to the wild-type Col-0. On the other hand, the endodermis-specific line pSCR accumulated 30% less cadmium in its shoots. A similar cadmium accumulation pattern in shoots was observed under the environmentally relevant much lower cadmium treatment of 0.1 µM for 4 d, using the hydroponic culture system. To further examine the potential of the MerC-SYP121 expression system for cadmium phytoremediation, the transgenic plants were hydroponically exposed to 0.1 µM for 4 weeks. The cadmium accumulation after the 4 weeks of treatment was again 16% higher in the pEpi shoots compared to that of Col-0, whereas the p35S line only showed 6% higher Cd concentration. Shoots of the pSCR line accumulated slightly less cadmium compared to Col-0. Ionomic profiles in these plants were analyzed, however, pSCR-specific patterns were not evident. Nevertheless, in our previous studies, pEpi and pSCR lines both efficiently accumulated more mercury in shoots than in the wild-type. The presented results suggest that the effects of cell-type-specific MerC-SYP121 expression differ by the target metals and its expression in root surface cells rather than that in endodermis is suitable for enhancing root cadmium uptake and subsequent shoot accumulation.

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids.

Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP-independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T-component, or TatT, is predicted to be a periplasmic-facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long-chain fatty acids. Our bioinformatics, quantitative analyses of protein-ligand interactions, and high-resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.

Tackling Hysteresis in Conformational Sampling: How to Be Forgetful with MEMENTO.

The structure of proteins has long been recognized to hold the key to understanding and engineering their function, and rapid advances in structural biology and protein structure prediction are now supplying researchers with an ever-increasing wealth of structural information. Most of the time, however, structures can only be determined in free energy minima, one at a time. While conformational flexibility may thus be inferred from static end-state structures, their interconversion mechanisms─a central ambition of structural biology─are often beyond the scope of direct experimentation. Given the dynamical nature of the processes in question, many studies have attempted to explore conformational transitions using molecular dynamics (MD). However, ensuring proper convergence and reversibility in the predicted transitions is extremely challenging. In particular, a commonly used technique to map out a path from a starting to a target conformation called steered MD (SMD) can suffer from starting-state dependence (hysteresis) when combined with techniques such as umbrella sampling (US) to compute the free energy profile of a transition. Here, we study this problem in detail on conformational changes of increasing complexity. We also present a new, history-independent approach that we term "MEMENTO" (Morphing End states by Modelling Ensembles with iNdependent TOpologies) to generate paths that alleviate hysteresis in the construction of conformational free energy profiles. MEMENTO utilizes template-based structure modelling to restore physically reasonable protein conformations based on coordinate interpolation (morphing) as an ensemble of plausible intermediates, from which a smooth path is picked. We compare SMD and MEMENTO on well-characterized test cases (the toy peptide deca-alanine and the enzyme adenylate kinase) before discussing its use in more complicated systems (the kinase P38α and the bacterial leucine transporter LeuT). Our work shows that for all but the simplest systems SMD paths should not in general be used to seed umbrella sampling or related techniques, unless the paths are validated by consistent results from biased runs in opposite directions. MEMENTO, on the other hand, performs well as a flexible tool to generate intermediate structures for umbrella sampling. We also demonstrate that extended end-state sampling combined with MEMENTO can aid the discovery of collective variables on a case-by-case basis.

The Potassium Efflux System Kef: Bacterial Protection against Toxic Electrophilic Compounds.

Kef couples the potassium efflux with proton influx in gram-negative bacteria. The resulting acidification of the cytosol efficiently prevents the killing of the bacteria by reactive electrophilic compounds. While other degradation pathways for electrophiles exist, Kef is a short-term response that is crucial for survival. It requires tight regulation since its activation comes with the burden of disturbed homeostasis. Electrophiles, entering the cell, react spontaneously or catalytically with glutathione, which is present at high concentrations in the cytosol. The resulting glutathione conjugates bind to the cytosolic regulatory domain of Kef and trigger activation while the binding of glutathione keeps the system closed. Furthermore, nucleotides can bind to this domain for stabilization or inhibition. The binding of an additional ancillary subunit, called KefF or KefG, to the cytosolic domain is required for full activation. The regulatory domain is termed K+ transport-nucleotide binding (KTN) or regulator of potassium conductance (RCK) domain, and it is also found in potassium uptake systems or channels in other oligomeric arrangements. Bacterial RosB-like transporters and K+ efflux antiporters (KEA) of plants are homologs of Kef but fulfill different functions. In summary, Kef provides an interesting and well-studied example of a highly regulated bacterial transport system.

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

Functional annotation and importance of marine bacterial transporters of plankton exometabolites

Metabolite exchange within marine microbial communities transfers carbon and other major elements through global cycles and forms the basis of microbial interactions. Yet lack of gene annotations and concern about the quality of existing ones remain major impediments to revealing currencies of carbon flux. We employed an arrayed mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their cognate substrates. Mutant experiments verified substrates for thirteen R. pomeroyi transporters. Four were previously hypothesized based on gene expression data (taurine, glucose/xylose, isethionate, and cadaverine/putrescine/spermidine); five were previously hypothesized based on homology to experimentally annotated transporters in other bacteria (citrate, glycerol, N-acetylglucosamine, fumarate/malate/succinate, and dimethylsulfoniopropionate); and four had no previous annotations (thymidine, carnitine, cysteate, and 3-hydroxybutyrate). These bring the total number of experimentally-verified organic carbon influx transporters to 18 of 126 in the R. pomeroyi genome. In a longitudinal study of a coastal phytoplankton bloom, expression patterns of the experimentally annotated transporters linked them to different stages of the bloom, and also led to the hypothesis that citrate and 3-hydroxybutyrate were among the most highly available bacterial substrates. Improved functional annotation of the gatekeepers of organic carbon uptake is critical for deciphering carbon flux and fate in microbial ecosystems.

Dissecting the conformational complexity and mechanism of a bacterial heme transporter

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

Unfolding Individual Domains of BmrA, a Bacterial ABC Transporter Involved in Multidrug Resistance.

The folding and stability of proteins are often studied via unfolding (and refolding) a protein with urea. Yet, in the case of membrane integral protein domains, which are shielded by a membrane or a membrane mimetic, urea generally does not induce unfolding. However, the unfolding of α-helical membrane proteins may be induced by the addition of sodium dodecyl sulfate (SDS). When protein unfolding is followed via monitoring changes in Trp fluorescence characteristics, the contributions of individual Trp residues often cannot be disentangled, and, consequently, the folding and stability of the individual domains of a multi-domain membrane protein cannot be studied. In this study, the unfolding of the homodimeric bacterial ATP-binding cassette (ABC) transporter Bacillus multidrug resistance ATP (BmrA), which comprises a transmembrane domain and a cytosolic nucleotide-binding domain, was investigated. To study the stability of individual BmrA domains in the context of the full-length protein, the individual domains were silenced by mutating the existent Trps. The SDS-induced unfolding of the corresponding constructs was compared to the (un)folding characteristics of the wild-type (wt) protein and isolated domains. The full-length variants BmrAW413Y and BmrAW104YW164A were able to mirror the changes observed with the isolated domains; thus, these variants allowed for the study of the unfolding and thermodynamic stability of mutated domains in the context of full-length BmrA.

Overview of the structure and function of the dopamine transporter and its protein interactions.

The dopamine transporter (DAT) plays an integral role in dopamine neurotransmission through the clearance of dopamine from the extracellular space. Dysregulation of DAT is central to the pathophysiology of numerous neuropsychiatric disorders and as such is an attractive therapeutic target. DAT belongs to the solute carrier family 6 (SLC6) class of Na+/Cl- dependent transporters that move various cargo into neurons against their concentration gradient. This review focuses on DAT (SCL6A3 protein) while extending the narrative to the closely related transporters for serotonin and norepinephrine where needed for comparison or functional relevance. Cloning and site-directed mutagenesis experiments provided early structural knowledge of DAT but our contemporary understanding was achieved through a combination of crystallization of the related bacterial transporter LeuT, homology modeling, and subsequently the crystallization of drosophila DAT. These seminal findings enabled a better understanding of the conformational states involved in the transport of substrate, subsequently aiding state-specific drug design. Post-translational modifications to DAT such as phosphorylation, palmitoylation, ubiquitination also influence the plasma membrane localization and kinetics. Substrates and drugs can interact with multiple sites within DAT including the primary S1 and S2 sites involved in dopamine binding and novel allosteric sites. Major research has centered around the question what determines the substrate and inhibitor selectivity of DAT in comparison to serotonin and norepinephrine transporters. DAT has been implicated in many neurological disorders and may play a role in the pathology of HIV and Parkinson's disease via direct physical interaction with HIV-1 Tat and α-synuclein proteins respectively.

A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion.

Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues.

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen-plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.

Distinct Energy-Coupling Factor Transporter Subunits Enable Flavin Acquisition and Extracytosolic Trafficking for Extracellular Electron Transfer in Listeria monocytogenes.

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A' subunits (RibU, EcfT, EcfA, and EcfA') to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the EetB and FmnA proteins are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrate a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B2 cofactors and the extracytosolic trafficking of a vitamin B2-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.

Mapping the determinants of substrate selectivity in a bacterial metal transporter with model-guided deep mutagenesis.

Membrane transporter proteins control what molecules are able to enter and exit the cell based on their ability to discriminate between substrates. The model bacterial metal transporter Deinococcus radiodurans (Dra)Nramp is responsible for importing rare divalent transition metals such as manganese and iron while excluding common alkaline earth metals such as magnesium and calcium. While recent high-resolution structures of DraNramp have mapped the conformational cycle underlying transport and identified key residues involved in metal binding and conformational gating, exactly which features of DraNramp control selectivity remains unclear, especially given the recent discovery of homologous proteins with altered substrate selectivity. We are simultaneously profiling the effect of thousands of DraNramp variants on import of manganese and magnesium, with the hypothesis that specificity may involve epistatic interactions between key active site positions and residues distal to the active site. To probe beyond single mutants, we have designed a strategy to bias our variant library using evolutionary sequence modeling to test a set of single- to octuple-mutants of DraNramp at positions likely to be involved in controlling substrate selectivity and unlikely to dramatically destabilize the protein. We have also designed novel assays to couple manganese and magnesium import to fluorescence in E. coli, and are testing our biased library in high-throughput with these assays to map the mutational landscape of substrate selectivity in DraNramp. This study will advance our understanding of how proteins bind metals as well as broadly what features of membrane transporter proteins are key to selecting between substrates. The methods for developing and assessing model-guided deep mutagenesis strategies also provide a framework bridging sequencing modeling and experimental biochemistry for future researchers.

Investigating the regulation and function of bacterial quaternary ammonium compound exporters.

Quaternary ammonium cations were introduced as commonly used disinfectant compounds in the 1930s. Today, they are often used in disinfectant solutions and wipes. Bacterial resistance to these antiseptic compounds is mediated by transporters from the small multidrug resistance (SMR) family of proteins. SMR transporters are bacterial proton-coupled transporters that export cationic substrates including quaternary ammonium compounds. Here we examine the genetic regulation of SMR expression. In addition, we use solid supported membrane (SSM) electrophysiology to observe transport of quaternary ammonium compounds by purified, reconstituted QacE proteins. Our findings allow us to functionally characterize and obtain insight into the evolution of these quaternary ammonium antiseptic transporters so that we can further investigate how to mitigate the spread of antiseptic resistance among bacterial populations.

Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.

Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter

The Zrt-/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. Here, we present a study on a prokaryotic ZIP from Bordetella bronchiseptica (BbZIP) by combining structural biology, evolutionary covariance, computational modeling, and a variety of biochemical assays to tackle the issue of the transport mechanism which has not been established for the ZIP family. The apo state structure in an inward-facing conformation revealed a disassembled transport site, altered inter-helical interactions, and importantly, a rigid body movement of a 4-transmembrane helix (TM) bundle relative to the other TMs. The computationally generated and biochemically validated outward-facing conformation model revealed a slide of the 4-TM bundle, which carries the transport site(s), by approximately 8 Å toward the extracellular side against the static TMs which mediate dimerization. These findings allow us to conclude that BbZIP is an elevator-type transporter.

Substrate space analysis of the bacterial proton-coupled oligopeptide transporter YdgR by cheminformatics.

Proton-dependent oligopeptide transporters (POTs) are recognized for their substrate promiscuity due to their ability to transport a wide range of substrates. POTs are conserved in all forms of life ranging from bacteria to humans. A dipeptide-fluorophore conjugate, H-(β-Ala)-Lys(AMCA)-OH, is a well-known substrate of the transporter YdgR that is commonly used as a fluorescent reporter. In order to understand the substrate space of YdgR, we used this dipeptide as a bait reference, when screening an ensemble of compounds (previously tested in PEPT/PTR/NPF space) via a cheminformatic analysis based on the Tanimoto similarity index. Eight compounds (sinalbin, abscisic acid, carnosine, jasmonic acid, N-acetyl-aspartate, N-acetyl-lysine, aspartame, and N-acetyl-aspartylglutamate), covering a wide range on the Tanimoto scale, were tested for YdgR-mediated transport. Carnosine was the only compound observed to be a YdgR substrate based on cell-based transport assays and molecular docking. The other compounds tested were neither inhibitors nor substrates. Thus, we found that neither the Tanimoto similarity index nor ADME (absorption, distribution, metabolism, and excretion) properties appear useful for the identification of substrates (e.g., dipeptides) in YdgR-mediated drug transport.

MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

Bactericidal Properties of Proline-Rich Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF).

The antimicrobial properties of proline-rich Aedes aegypti decapeptide TMOF (AeaTMOF) and oncocin112 (1-13) were compared. Incubations with multidrug-resistant Escherichia coli cells showed that AeaTMOF (5 mM) was able to completely inhibit bacterial cell growth, whereas oncocin112 (1-13) (20 mM) partially inhibited bacterial growth as compared with bacterial cells that were not multidrug-resistant cells. AeaTMOF (5 mM) was very effective against Acinetobacter baumannii and Pseudomonas aeruginosa, completely inhibiting cell growth during 15 h incubations. AeaTMOF (5 mM) completely inhibited the Gram-positive bacteria Staphylococcus aureus and Bacillus thurengiensis sups. Israelensis cell growth, whereas oncocin112 (1-13) (10 and 20 mM) failed to affect bacterial cell growth. E. coli cells that lack the SbmA transporter were inhibited by AeaTMOF (5 mM) and not by oncocin112 (1-13) (10 to 20 mM), indicating that AeaTMOF can use other bacterial transporters than SbmA that is mainly used by proline-rich antimicrobial peptides. Incubation of E. coli cells with NaAzide showed that AeaTMOF does not use ABC-like transporters that use ATP hydrolysis to import molecules into bacterial cells. Three-dimensional modeling and docking of AeaTMOF to SbmA and MdtM transporters showed that AeaTMOF can bind these proteins, and the binding location of AeaTMOF inside these protein transporters allows AeaTMOF to be transported into the bacterial cytosol. These results show that AeaTMOF can be used as a future antibacterial agent against both multidrug-resistant Gram-positive and -negative bacteria.