Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.

TMEM16 scramblases thin the membrane to enable lipid scrambling

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. It was proposed that lipid headgroups move between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryoEM structure of the Ca2+-bound afTMEM16 scramblase in nanodiscs showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove’s intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not entail specific protein-lipid interactions with the extracellular vestibule. Further, we find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes despite an open pathway. Our results show how afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.

Structure and lipid dynamics in the maintenance of lipid asymmetry inner membrane complex of A. baumannii

Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

Peptides affecting outer membrane lipid asymmetry (MlaA-OmpC/F) system reduce avian pathogenic Escherichia coli (APEC) colonization in chickens

Avian pathogenic E. coli (APEC), an extra-intestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of Lactobacillus rhamnosus GG (LGG) derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR: P1; PDENK: P2, and VHTAPK: P3) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 logs) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membrane either by causing membrane shedding, rupturing or flaccidity. Further, gene expression analysis revealed that peptides downregulated the expression of omp C (>13.0 folds), omp F (>11.3 folds) and mla A (>4.9 folds) genes responsible for maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1: N, E, R and P; P2: D and E; P3: T, P, and K) for their activity. Overall, our study identified peptides with new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. Importance APEC is a subgroup of ExPEC and considered as a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Our study identified LGG-derived peptides (P1: NPSRQERR, P2: PDENK, and P3: VHTAPK) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt APEC membrane by affecting MlaA-OmpC/F system responsible for maintenance of OM lipid asymmetry, a promising new druggable target to overcome resistance problem in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs and other related Gram-negative pathogens. Further, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.

LOSS OF PLASMA MEMBRANE LIPID ASYMMETRY CAN INDUCE ORDERED DOMAIN (RAFT) FORMATION

The lipids in one leaflet of an asymmetric artificial lipid vesicle can induce or suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether suppression of domain formation might occur in plasma membranes was studied by using plasma membrane vesicles (PMVs) from RBL-2H3 cells. Ordered domain formation was assessed by FRET and fluorescence anisotropy. Ordered domains in PMV prepared by N-ethyl maleimide (NEM) treatment formed to some extent up to about 37oC. In contrast, ordered domains in symmetric vesicles formed from extracted PMV lipids were stable up to 55oC. This indicates that the stability of ordered domains was substantially less in the intact PMV. A similar decrease in ordered domain stability was observed in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles. This suggested either that the intact PMV have a significant degree of lipid asymmetry or that PMV proteins suppress domain formation. Additional experiments ruled out the latter explanation. First, stabilization of ordered domain formation relative to intact PMV was observed in protein-containing symmetric vesicles prepared by detergent solubilization of intact PMV, followed by rapid dilution of detergent. Second, ordered domain stability in intact PMV was not altered after extensively removing PMV proteins with proteinase K. We conclude that intact NEM-induced PMV preserve a significant amount of the lipid asymmetry of plasma membranes, and that loss of PMV lipid asymmetry can induce ordered domain formation, consistent with the possibility that dynamic control of lipid asymmetry can regulate ordered domain formation in the plasma membrane.

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.