Extracellular Vesicles in Pathophysiology: A Prudent Target That Requires Careful Consideration.

Extracellular vesicles (EVs) are membrane-bound particles released by cells to perform multitudes of biological functions. Owing to their significant implications in diseases, the pathophysiological role of EVs continues to be extensively studied, leading research to neglect the need to explore their role in normal physiology. Despite this, many identified physiological functions of EVs, including, but not limited to, tissue repair, early development and aging, are attributed to their modulatory role in various signaling pathways via intercellular communication. EVs are widely perceived as a potential therapeutic strategy for better prognosis, primarily through utilization as a mode of delivery vehicle. Moreover, disease-associated EVs serve as candidates for the targeted inhibition by pharmacological or genetic means. However, these attempts are often accompanied by major challenges, such as off-target effects, which may result in adverse phenotypes. This renders the clinical efficacy of EVs elusive, indicating that further understanding of the specific role of EVs in physiology may enhance their utility. This review highlights the essential role of EVs in maintaining cellular homeostasis under different physiological settings, and also discusses the various aspects that may potentially hinder the robust utility of EV-based therapeutics.

Vesiclepedia 2024: an extracellular vesicles and extracellular particles repository.

Vesiclepedia (http://www.microvesicles.org) is a free web-based compendium of DNA, RNA, proteins, lipids and metabolites that are detected or associated with extracellular vesicles (EVs) and extracellular particles (EPs). EVs are membranous vesicles that are secreted ubiquitously by cells from all domains of life from archaea to eukaryotes. In addition to EVs, it was reported recently that EPs like exomeres and supermeres are secreted by some mammalian cells. Both EVs and EPs contain proteins, nucleic acids, lipids and metabolites and has been proposed to be implicated in several key biological functions. Vesiclepedia catalogues proteins, DNA, RNA, lipids and metabolites from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 3533 EV studies, 50 550 RNA entries, 566 911 protein entries, 3839 lipid entries, 192 metabolite and 167 DNA entries. Quantitative data for 62 822 entries from 47 EV studies is available in Vesiclepedia. The datasets available in Vesiclepedia can be downloaded as tab-delimited files or accessible through the FunRich-based Vesiclepedia plugin.

Proteomic analysis of the small extracellular vesicles and soluble secretory proteins from cachexia inducing and non-inducing cancer cells.

Cancer cachexia is a wasting syndrome characterised by the loss of fat and/or muscle mass in advanced cancer patients. It has been well-established that cancer cells themselves can induce cachexia via the release of several pro-cachectic and pro-inflammatory factors. However, it is unclear how this process is regulated and the key cachexins that are involved. In this study, we validated C26 and EL4 as cachexic and non-cachexic cell models, respectively. Treatment of adipocytes and myotubes with C26 conditioned medium induced lipolysis and atrophy, respectively. We profiled soluble secreted proteins (secretome) as well as small extracellular vesicles (sEVs) released from cachexia-inducing (C26) and non-inducing (EL4) cancer cells by label-free quantitative proteomics. A total of 1268 and 1022 proteins were identified in the secretome of C26 and EL4, respectively. Furthermore, proteomic analysis of sEVs derived from C26 and EL4 cancer cells revealed a distinct difference in the protein cargo. Functional enrichment analysis using FunRich highlighted the enrichment of proteins that are implicated in biological processes such as muscle atrophy, lipolysis, and inflammation in both the secretome and sEVs derived from C26 cancer cells. Overall, our characterisation of the proteomic profiles of the secretory factors and sEVs from cachexia-inducing and non-inducing cancer cells provides insights into tumour factors that promote weight loss by mediating protein and lipid loss in various organs and tissues. Further investigation of these proteins may assist in highlighting potential therapeutic targets and biomarkers of cancer cachexia.

Repurposing of Antibiotic Sulfisoxazole Inhibits Lipolysis in Pre-Clinical Model of Cancer-Associated Cachexia.

Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment that directly targets cancer-induced wasting is desperately needed to improve the quality of life and the survival of cancer patients. Recently, an antibiotic SFX was shown to have anti-tumour and anti-metastatic effects in mouse models of breast cancer. Hence, in this study, we examined the efficacy of SFX in the treatment of cancer-induced cachexia. C26 cachexic mice models were administered with SFX, and the tumour volume and body weight were regularly measured. Blood glucose, skeletal muscles, and adipose tissue were examined at the endpoint. Contrary to a previous study, SFX did not reduce the tumour volume in mice bearing C26 cells. Administration of SFX neither revealed any survival benefit nor rescued C26 cachectic mice from muscle wasting. Interestingly, SFX administration partially rescued (~10%) tumour-induced weight loss by preserving both the subcutaneous and intestinal fat mass. Together, these results suggest that the administration of SFX could partially rescue cancer-induced weight loss by inhibiting lipolysis. As anti-cachexia therapies are scarce, the results could facilitate the design of combinatorial therapies involving SFX, standard-of-care chemotherapeutics, and drugs that inhibit muscle atrophy for the treatment of cancer cachexia.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

Extracellular Vesicles Regulate Cancer Metastasis.

Metastatic cancer is a complex disease associated with poor prognosis and accounts for the majority of cancer related deaths. To date, many of the molecular mechanisms driving metastatic disease remain elusive and require further investigation for the development of effective treatment strategies. Recent studies have shown that extracellular vesicles (EVs) can be exploited by tumors to assist in cancer cell growth, proliferation, migration, invasion and metastasis. Cancer cells have proven efficient in educating fibroblasts, within their microenvironment, to secrete EVs as communicative vessels for mediating phenotypic changes in recipient cells. Using this vesicular delivery system, cancer cells can establish a new metastatic niche within distant sites, away from the primary tumor, thus favoring cancer progression. These findings demonstrate the availability of a new route for therapeutic intervention in the inhibition of cancer dissemination. Although, several approaches to target cancer cell secretion of EVs are detailed in the literature, there is still no defined way to currently apply them in clinical settings. Hence, further studies are required to unravel the molecular mechanisms of metastasis - governed by the establishment and release of cancer associated EVs.

A High-Resolution Mass Spectrometry-Based Quantitative Metabolomic Workflow Highlights Defects in 5-Fluorouracil Metabolism in Cancer Cells with Acquired Chemoresistance.

Currently, 5-fluorouracil (5-FU)-based combination chemotherapy is the mainstay in the treatment of metastatic colorectal cancer (CRC), which benefits approximately 50% of the patients. However, these tumors inevitably acquire chemoresistance resulting in treatment failure. The molecular mechanisms driving acquired chemotherapeutic drug resistance in CRC is fundamental for the development of novel strategies for circumventing resistance. However, the specific phenomenon that drives the cancer cells to acquire resistance is poorly understood. Understanding the molecular mechanisms that regulate chemoresistance will uncover new avenues for the treatment of CRC. Among the various mechanisms of acquired chemoresistance, defects in the drug metabolism pathways could play a major role. In the case of 5-FU, it gets converted into various active metabolites, which, directly or indirectly, interferes with the replication and transcription of dividing cells causing DNA and RNA damage. In this project, we developed a high-resolution mass spectrometry-based method to effectively extract and quantify levels of the 5-FU metabolites in cell lysates and media of parental and 5-FU resistant LIM1215 CRC cells. The analysis highlighted that the levels of 5-FU metabolites are significantly reduced in 5-FU resistant cells. Specifically, the level of the nucleotide fluorodeoxyuridine monophosphate (FdUMP) is reduced with treatment of 5-FU clarifying the compromised 5-FU metabolism in resistant cells. Corroborating the metabolomic analysis, treatment of the resistant cells with FdUMP, an active metabolite of 5-FU, resulted in effective killing of the resistant cells. Overall, in this study, an effective protocol was developed for comparative quantitation of polar metabolites and nucleotide analogues from the adherent cells efficiently. Furthermore, the utility of FdUMP as an alternative for CRC therapy is highlighted.

BCL-2 family protein BOK is a positive regulator of uridine metabolism in mammals.

BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.

A Pilot Study on Screening of BRCA1 Mutations (185delAG, 1294del40) in Nepalese Breast Cancer Patients.

BACKGROUND: Breast cancer is the second most common malignancy among Nepalese women, accounting for 60% of the total cancer cases in females. Women diagnosed with germline mutations in BRCA1 like 185delAG, 1294del40 develop breast and/or ovarian cancer with a lifelong likelihood of up to 85% whereas presence of a mutation increases the risk for mutations to occur in other genes. The major objective of this study was to find the prevalence of these mutations in Nepalese cancer patients. MATERIALS AND METHODS: This prospective study was carried out at two cancer hospitals in the Kathmandu valley over a period of 11 months. Irrespective of age group and stage of canceran appropriate amount of blood was withdrawn from 50 breast cancer patients and 20 controls. DNA was extracted manually and subjected to PCR using primers for 185delAG and 1294del40 mutations. PCR products were then digested with restriction enzyme (DdeII) followed by electrophoresis. RESULTS: Prevalence of 185delAG in reference breast cancer patients was found to be 4/50 (8%) but no 1294del40 was apparent. CONCLUSIONS: Several mutations occurring in different exons of BRCA1 as well as mutations in other genes like BRCA2, for example, should also be taken in account.

Genome-wide transcriptomic analysis of the response to nitrogen limitation in Streptomyces coelicolor A3(2).

BACKGROUND: The present study represents a genome-wide transcriptomic analysis of the response of the model streptomycete Streptomyces coelicolor A3(2) M145 to fermentor culture in Modified Evans Media limited, respectively, for nitrogen, phosphate and carbon undertaken as part of the ActinoGEN consortium to provide a publicly available reference microarray dataset. FINDINGS: A microarray dataset using samples from two replicate cultures for each nutrient limitation was generated. In this report our analysis has focused on the genes which are significantly differentially expressed, as determined by Rank Products Analysis, between samples from matched time points correlated by growth phase for the three pairs of differently limited culture datasets. With a few exceptions, genes are only significantly differentially expressed between the N6/N7 time points and their corresponding time points in the C and P-limited cultures, with the vast majority of the differentially expressed genes being more highly expressed in the N-limited cultures. Our analysis of these genes indicated expression of several members of the GlnR regulon are induced upon nitrogen limitation, as assayed for by [NH4+] measurements, and we are able to identify several additional genes not present in the GlnR regulon whose expression is induced in response to nitrogen limitation. We also note SCO3327 which encodes a small protein (32 amino acid residues) unusually rich in the basic amino acids lysine (31.25%) and arginine (25%) is significantly differentially expressed in the nitrogen limited cultures. Additionally, we investigate the expression of known members of the GlnR regulon and the relationship between gene organization and expression for the SCO2486-SCO2487 and SCO5583-SCO5585 operons. CONCLUSIONS: We provide a list of genes whose expression is differentially expressed in low nitrogen culture conditions, including a putative nitrogen storage protein encoded by SCO3327. Our list includes several genes whose expression patterns are similar to up-regulated members of the GlnR regulon and are induced in response to nitrogen limitation. These genes represent likely targets for future studies into the nitrogen starvation response in Streptomyces coelicolor.

A functional steroid-binding element in an ATP-binding cassette multidrug transporter.

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.

Metabolites of ginsenosides as novel BCRP inhibitors.

We have previously shown ginsenosides derived from Panax ginseng exert opposing effects on angiogenesis. Here, we examined protopanaxadiol-containing ginsenosides (Rg3, Rh2, and PPD) and protopanaxatriol-containing ginsenosides (Rg1, Rh1, and PPT) as potential inhibitors of breast cancer resistance protein (BCRP). Among these ginsenosides, metabolites Rh2, PPD, and PPT significantly enhanced the cytotoxicity of mitoxantrone (MX) to human breast carcinoma MCF-7/MX cells which overexpress BCRP. PPD was the most potent followed by Rh2 and PPT. This effect was not seen in sensitive MCF-7 cells. Rg3, Rg1, and Rh1 were ineffective in either MCF-7 or MCF-7/MX cells. PPD, Rh2, and PPT were able to inhibit MX efflux in MCF-7/MX cells. PPD and Rh2 also increased MX uptake. In inside out membrane vesicles from Lactococcus lactis cells expressing BCRP, only PPD was found to significantly inhibit BCRP-associated vanadate sensitive ATPase activity. These results indicate that metabolites PPD, Rh2, and PPT were inhibitors of BCRP.

New light on multidrug binding by an ATP-binding-cassette transporter.

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

Modulation of p-glycoprotein and breast cancer resistance protein by some prescribed corticosteroids.

Efflux transporters, p-glycoprotein and breast cancer resistance protein (BCRP), located at barrier sites such as the blood-brain barrier may affect distribution of steroids used for treating chronic inflammatory conditions and thus the extent to which they may perturb the hypothalamic-pituitary-adrenal axis. In the present study, six different glucocorticoids were investigated for their possible interactions with these efflux transporters. Beclomethasone dipropionate, mometasone furoate and ciclesonide active principle but not fluticasone propionate or triamcinolone, (all at 0.1 to 10 microM) caused inhibition of efflux, resulting in increased accumulation of mitoxantrone in BCRP-expressing MCF7/MR breast cancer cells and of calcein in p-glycoprotein-expressing SW620/R colon carcinoma cell. At 5 microM the same three increased sensitivity of p-glycoprotein-expressing SW620/R to doxorubicin and stimulated ATPase activity associated with BCRP expressed in bacterial membrane vesicles. Budesonide also stimulated ATPase activity. These data demonstrate the capacity of some clinically used glucocorticoids to interact with efflux transporters.

The role of Trypanosoma brucei MRPA in melarsoprol susceptibility.

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione-spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA.

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 microM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 microM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2).

The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg482-->Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg482 is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg482 and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg482.

A new dimer interface for an ABC transporter.

The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli, provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 A. The E. coli crystal structure of MsbA reveals a dimer. Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface. We considered the interface in a two-armed MsbA dimer, named spiral. Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E. coli MsbA represents a crystallization artefact. A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described.

ABC transporters and drug resistance in parasitic protozoa.

Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATP-binding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium, Leishmania, Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included.

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities.

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.