20240803_membrane digest – Membrane Protein Biochemistry Lab

Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport.

Wotherspoon, P., Johnston, H., Hardy, D.J. et al.

Nat Commun 15, 6394 (2024).

https://doi.org/10.1038/s41467-024-50615-3

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

The Effect of Reactive Oxygen Species on Respiratory Complex I Activity in Liposomes.

Eisermann J, Liang Y, Wright JJ, Clifford E, Wilton-Ely JDET, Kuimova M, Roessler M.

Chemistry. 2024 Jul 26:e202402035.

doi: 10.1002/chem.202402035. Epub ahead of print.

PMID: 39058376.

Respiratory complex I (R-CI) is an essential enzyme in the mitochondrial electron transport chain but also a major source of reactive oxygen species (ROS), which are implicated in neurodegenerative diseases and ageing. While the mechanism of ROS production by R-CI is well-established, the feedback of ROS on R-CI activity is poorly understood. Here, we perform EPR spectroscopy on R-CI incorporated in artificial membrane vesicles to reveal that ROS (particularly hydroxyl radicals) reduce R-CI activity by making the membrane more polar and by increasing its hydrogen bonding capability. Moreover, the mechanism that we have uncovered reveals that the feedback of ROS on R-CI activity via the membrane is transient and not permanent; lipid peroxidation is negligible for the levels of ROS generated under these conditions. Our successful use of modular proteoliposome systems in conjunction with EPR spectroscopy and other biophysical techniques is a powerful approach for investigating ROS effects on other membrane proteins.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Hariharan P, Bakhtiiari A, Liang R, Guan L.

J Biol Chem. 2024 Jul;300(7):107427.

doi: 10.1016/j.jbc.2024.107427. Epub 2024 May 31.

PMID: 38823641.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Role of AcrAB-TolC and Its Components in Influx-Efflux Dynamics of QAC Drugs in Escherichia coli Revealed Using SHG Spectroscopy.

Singh D, Kumar D, Gayen A, Chandra M.

J Phys Chem Lett. 2024 Jul 25:7832-7839.

doi: 10.1021/acs.jpclett.4c01189. Epub ahead of print.

PMID: 39052610.

Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

Raicu V, Stoneman M, Yokoi K, Biener G, Killeen T, Adhikari D, Rahman S, Harikumar K, Miller L.

Res Sq [Preprint]. 2024 Jul 19:rs.3.rs-4683780.

doi: 10.21203/rs.3.rs-4683780/v1.

PMID: 39070646.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Divergent folding-mediated epistasis among unstable membrane protein variants.

Chamness LM, Kuntz CP, McKee AG, Penn WD, Hemmerich CM, Rusch DB, Woods H, Dyotima, Meiler J, Schlebach JP.

Elife. 2024 Jul 30;12:RP92406.

doi: 10.7554/eLife.92406.

PMID: 39078397.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Transport and inhibition mechanisms of the human noradrenaline transporter.

Hu T, Yu Z, Zhao J, Meng Y, Salomon K, Bai Q, Wei Y, Zhang J, Xu S, Dai Q, Yu R, Yang B, Loland CJ, Zhao Y.

Nature. 2024 Jul 31.

doi: 10.1038/s41586-024-07638-z. Epub ahead of print.

PMID: 39085602.

The noradrenaline transporter (also known as norepinephrine transporter) (NET) has a critical role in terminating noradrenergic transmission by utilizing sodium and chloride gradients to drive the reuptake of noradrenaline (also known as norepinephrine) into presynaptic neurons. It is a pharmacological target for various antidepressants and analgesic drugs. Despite decades of research, its structure and the molecular mechanisms underpinning noradrenaline transport, coupling to ion gradients and non-competitive inhibition remain unknown. Here we present high-resolution complex structures of NET in two fundamental conformations: in the apo state, and bound to the substrate noradrenaline, an analogue of the χ-conotoxin MrlA (χ-MrlAEM), bupropion or ziprasidone. The noradrenaline-bound structure clearly demonstrates the binding modes of noradrenaline. The coordination of Na+ and Cl- undergoes notable alterations during conformational changes. Analysis of the structure of NET bound to χ-MrlAEM provides insight into how conotoxin binds allosterically and inhibits NET. Additionally, bupropion and ziprasidone stabilize NET in its inward-facing state, but they have distinct binding pockets. These structures define the mechanisms governing neurotransmitter transport and non-competitive inhibition in NET, providing a blueprint for future drug design.

Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.

Harikumar KG, Zhao P, Cary BP, Xu X, Desai AJ, Dong M, Mobbs JI, Toufaily C, Furness SGB, Christopoulos A, Belousoff MJ, Wootten D, Sexton PM, Miller LJ.

PLoS Biol. 2024 Jul 31;22(7):e3002673.

doi: 10.1371/journal.pbio.3002673.

PMID: 39083706.

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms. Static, low energy, high-resolution structures of the mutant CCK1R constructs, stabilized in complex with G protein, were not substantially different, suggesting that alterations to receptor dynamics were key to altered function. We reveal that cholesterol-dependent dynamic changes in the conformation of the helical bundle of CCK receptors affects both ligand binding at the extracellular surface and G protein coupling at the cytosolic surface, as well as their interrelationships involved in stimulus-response coupling. This provides an ideal setting for potential allosteric modulators to correct the negative impact of membrane cholesterol on CCK1R.

Membrane

Tetraspanin proteins in membrane remodeling processes.

Dharan R, Sorkin R.

J Cell Sci. 2024 Jul 15;137(14):jcs261532.

doi: 10.1242/jcs.261532. Epub 2024 Jul 25.

PMID: 39051897.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Influence of archaeal lipids isolated from Aeropyrum pernix K1 on physicochemical properties of sphingomyelin-cholesterol liposomes.

Kejžar J, Mrak P, Osojnik Črnivec IG, Poklar Ulrih N.

Biochim Biophys Acta Biomembr. 2024 Jul 23;1866(7):184374.

doi: 10.1016/j.bbamem.2024.184374. Epub ahead of print.

PMID: 39053569.

We investigated the influence of archaeal lipids (C25,25) isolated from thermophilic archaeon Aeropyrum pernix K1 on physicochemical properties of liposomes comprised of egg sphingomyelin (SM) and cholesterol (CH) using fluorescence emission anisotropy, calcein release studies, dynamic light scattering, transmission electron microscopy and phase analysis light scattering. The 2 mol% addition of archaeal lipids enabled formation of small unilamellar vesicles by sonication while also having significant effect on reducing mean size, polydispersity index and zeta potential of C25,25/SM/CH vesicles. Increasing the ratio of C25,25 lipids in mixture of C25,25/SM/CH decreased lipid ordering parameter in dose dependent manner at different temperatures. We also demonstrated that adding 15 mol% C25,25 to SM/CH mixture will cause it to notably interact with fetal bovine serum which could make them a viable alternative adjuvant to synthetic ether-linked lipids in development of advanced liposomal vaccine delivery systems. The prospect of combining the proven strengths of SM/CH mixtures with the unique properties of C25,25 opens exciting possibilities for advancing drug delivery technologies, promising to yield formulations that are both highly effective and adaptable to a range of therapeutic applications.

Visualizing intermediate stages of viral membrane fusion by cryo-electron tomography.

Kephart SM, Hom N, Lee KK.

Trends Biochem Sci. 2024 Jul 24:S0968-0004(24)00160-9.

doi: 10.1016/j.tibs.2024.06.012. Epub ahead of print.

PMID: 39054240.

Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion.

Aqueous Ionic Liquid Mixtures as Minimal Models of Lipid Bilayer Membranes.

Volmer J, Cerajewski U, Alfes M, Bender J, Abert J, Schmidt C, Ott M, Hinderberger D.

ACS Biomater Sci Eng. 2024 Jul 27.

doi: 10.1021/acsbiomaterials.4c00740. Epub ahead of print.

PMID: 39066733.

We introduce aqueous ionic liquid (IL) mixtures, specifically mixtures of 1-butyl-3-imidazoliumtetrafluoroborate (BMImBF4), with water as a minimal model of lipid bilayer membranes. Imidazolium-based ILs are known to form clustered nanoscale structures in which local inhomogeneities, micellar or lamellar structures, are formed to shield hydrophobic parts of the cation from the polar cosolvent (water). To investigate these nanostructures, dynamic light scattering (DLS) on samples with different mixing ratios of water and BMImBF4 was performed. At mixing ratios of 50% and 45% (v/v), small and homogeneous nanostructures can indeed be detected. To test whether, in particular, these stable nanostructures in aqueous mixtures may mimic the effects of phospholipid bilayer membranes, we further investigated their interaction with myelin basic protein (MBP), a peripheral, intrinsically disordered membrane protein of the myelin sheath. Using dynamic light scattering (DLS), continuous wave (CW) and pulse electron paramagnetic resonance (EPR), and small-angle X-ray scattering (SAXS) on recombinantly produced, “healthy” charge variants rmC1WT and double cysteine variant C1S17CH85C, we find that the size and the shape of the determined nanostructures in an optimum mixture offer model membranes in which the protein exhibits native behavior. SAXS measurements illuminate the size and shape of the nanostructures and indicate IL-rich “beads” clipped together by functional MBP, one of the in vivo roles of the protein in the myelin sheath. All the gathered data combined indicate that the 50% and 45% aqueous IL mixtures can be described as offering minimal models of a lipid mono- or bilayer that allow native processing and potential study of at least peripheral membrane proteins like MBP.

Key contributions of a glycolipid to membrane protein integration.

Shimamoto K, Fujikawa K, Osawa T, Mori S, Nomura K, Nishiyama KI.

Proc Jpn Acad Ser B Phys Biol Sci. 2024;100(7):387-413.

doi: 10.2183/pjab.100.026.

PMID: 39085064.

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome.

Rathore D, Marino MJ, Issara-Amphorn J, Hwan Yoon S, Manes NP, Nita-Lazar A.

J Proteome Res. 2024 Aug 2;23(8):3280-3293.

doi: 10.1021/acs.jproteome.3c00838. Epub 2024 Mar 25.

PMID: 38527097.

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins’ (RBPs’) effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.

Methods

Protein Engineering and High-Throughput Screening by Yeast Surface Display: Survey of Current Methods.

Lopez-Morales J, Vanella R, Appelt EA, Whillock S, Paulk AM, Shusta EV, Hackel BJ, Liu CC, Nash MA.

Small Sci. 2023 Dec;3(12):2300095.

doi: 10.1002/smsc.202300095. Epub 2023 Nov 8.

PMID: 39071103.

Yeast surface display (YSD) is a powerful tool in biotechnology that links genotype to phenotype. In this review, the latest advancements in protein engineering and high-throughput screening based on YSD are covered. The focus is on innovative methods for overcoming challenges in YSD in the context of biotherapeutic drug discovery and diagnostics. Topics ranging from titrating avidity in YSD using transcriptional control to the development of serological diagnostic assays relying on serum biopanning and mitigation of unspecific binding are covered. Screening techniques against nontraditional cellular antigens, such as cell lysates, membrane proteins, and extracellular matrices are summarized and techniques are further delved into for expansion of the chemical repertoire, considering protein-small molecule hybrids and noncanonical amino acid incorporation. Additionally, in vivo gene diversification and continuous evolution in yeast is discussed. Collectively, these techniques enhance the diversity and functionality of engineered proteins isolated via YSD, broadening the scope of applications that can be addressed. The review concludes with future perspectives and potential impact of these advancements on protein engineering. The goal is to provide a focused summary of recent progress in the field.

Making the cut: Multiscale simulation of membrane remodeling.

Beiter J, Voth GA.

Curr Opin Struct Biol. 2024 Aug;87:102831.

doi: 10.1016/j.sbi.2024.102831. Epub 2024 May 12.

PMID: 38740001.

Biological membranes are dynamic heterogeneous materials, and their shape and organization are tightly coupled to the properties of the proteins in and around them. However, the length scales of lipid and protein dynamics are far below the size of membrane-bound organelles, much less an entire cell. Therefore, multiscale modeling approaches are often necessary to build a comprehensive picture of the interplay of these factors, and have provided critical insights into our understanding of membrane dynamics. Here, we review computational methods for studying membrane remodeling, as well as passive and active examples of protein-driven membrane remodeling. As the field advances towards the modeling of key aspects of organelles and whole cells – an increasingly accessible regime of study – we summarize here recent successes and offer comments on future trends.

Formation of a planar biomimetic membrane with a novel zwitterionic polymer for nanopore sequencing.

Yang X, Yang J, Wei L, Zhang Y, Yang J, Ni M, Dong Y.

J Mater Chem B. 2024 Jul 31.

doi: 10.1039/d4tb01007h. Epub ahead of print.

PMID: 39082061.

Biological membranes containing transmembrane channels play a crucial role in numerous cellular processes, and mimicking of cell membranes has garnered significant interest in various biomedical applications, particularly nanopore sequencing technology, where remarkable progress has been made with nanopore membranes. Considering the fragility of biomimetic membranes formed by artificial lipids and the limited mimicry of those formed by common block copolymers, this study developed a novel amphiphilic polymer by covalently linking hydrophilic heads of phospholipids to the ends of hydrophobic poly(dimethyl siloxane) (PDMS) chains. The absence of hydrophilic blocks allowed for good control over the polydispersity of this polymer within a narrow range. The high flexibility of PDMS chains, combined with relatively uniform molecular weights, would confer enhanced stability and robustness to polymeric membranes. Dynamic light scattering measurements and microdroplet formation tests demonstrated good amphipathic properties of these novel polymers when maintaining an appropriate hydrophilic-hydrophobic ratio. Moreover, the high similarity between the hydrophilic heads and natural phospholipids makes this polymer more compatible with biomolecules. A successful protein insertion experiment confirmed both the stability of this polymeric membrane and its compatibility with membrane proteins. As a result, this novel amphiphilic polymer exhibits great potential for biomembrane mimicking and paves a new path for material design in biomedical applications.

Just passing through: Deploying aquaporins in microbial cell factories.

Jenkins Sánchez LR, Sips LM, Van Bogaert INA.

Biotechnol Prog. 2024 Jul 25:e3497.

doi: 10.1002/btpr.3497. Epub ahead of print. PMID: 39051848.

As microbial membranes are naturally impermeable to even the smallest biomolecules, transporter proteins are physiologically essential for normal cell functioning. This makes transporters a key target area for engineering enhanced cell factories. As part of the wider cellular transportome, aquaporins (AQPs) are responsible for transporting small polar solutes, encompassing many compounds which are of great interest for industrial biotechnology, including cell feedstocks, numerous commercially relevant polyols and even weak organic acids. In this review, examples of cell factory engineering by targeting AQPs are presented. These AQP modifications aid in redirecting carbon fluxes and boosting bioconversions either by enhanced feedstock uptake, improved intermediate retention, increasing product export into the media or superior cell viability against stressors with applications in both bacterial and yeast production platforms. Additionally, the future potential for AQP deployment and targeting is discussed, showcasing hurdles and considerations of this strategy as well as recent advances and future directions in the field. By leveraging the natural diversity of AQPs and breakthroughs in channel protein engineering, these transporters are poised to be promising tools capable of enhancing a wide variety of biotechnological processes.

Microbio

Non-interchangeable functions of efflux transporters of Pseudomonas aeruginosa in survival under infection-associated stress.

Adamiak JW, Ajmal L, Zgurskaya HI.

J Bacteriol. 2024 Jul 25;206(7):e0005424.

doi: 10.1128/jb.00054-24. Epub 2024 Jun 14. PMID: 38874367.

The role of multidrug efflux pumps in the intrinsic and clinical levels of antibiotic resistance in Pseudomonas aeruginosa and other gram-negative bacteria is well-established. Their functions in bacterial physiology, however, remain unclear. The P. aeruginosa genome comprises an arsenal of efflux pumps from different protein families, the substrate specificities of which are typically assessed by measuring their impact on susceptibility to antibiotics. In this study, we analyzed how deletions and overproductions of efflux pumps affect P. aeruginosa growth under human-infection-induced stresses. Our results show that the physiological functions of multidrug efflux pumps are non-redundant and essential for the survival of this important human pathogen under stress.

Bacterial efflux pump modulators prevent bacterial growth in macrophages and under broth conditions that mimic the host environment.

Allgood SC, Su C-C, Crooks AL, Meyer CT, Zhou B, Betterton MD, Barbachyn MR, Yu EW, Detweiler CS.

mBio. 2023 Dec 19;14(6):e0249223.

doi: 10.1128/mbio.02492-23. Epub 2023 Nov 3.

PMID: 37921493.

Bacterial efflux pumps are critical for resistance to antibiotics and for virulence. We previously identified small molecules that inhibit efflux pumps (efflux pump modulators, EPMs) and prevent pathogen replication in host cells. Here, we used medicinal chemistry to increase the activity of the EPMs against pathogens in cells into the nanomolar range. We show by cryo-electron microscopy that these EPMs bind an efflux pump subunit. In broth culture, the EPMs increase the potency (activity), but not the efficacy (maximum effect), of antibiotics. We also found that bacterial exposure to the EPMs appear to enable the accumulation of a toxic metabolite that would otherwise be exported by efflux pumps. Thus, inhibitors of bacterial efflux pumps could interfere with infection not only by potentiating antibiotics, but also by allowing toxic waste products to accumulate within bacteria, providing an explanation for why efflux pumps are needed for virulence in the absence of antibiotics.

Blood culture-free ultra-rapid antimicrobial susceptibility testing.

Kim TH, Kang J, Jang H, Joo H, Lee GY, Kim H, Cho U, Bang H, Jang J, Han S, Kim DY, Lee CM, Kang CK, Choe PG, Kim NJ, Oh MD, Kim TS, Kim I, Park WB, Kwon S.

Nature. 2024 Jul 24.

doi: 10.1038/s41586-024-07725-1. Epub ahead of print.

PMID: 39048820.

A method to quickly identify the bacteria involved in life-threatening sepsis — and which antibiotics will kill them— could save patient lives.