The very hungry bacterium: moving toward a holistic understanding of the bacterial sugar transport network

By Aritha Dornau

Twitter: @FalseUnit

In many bacteria the Phosphotransferase System (PTS) is the main route for importing key sugars such as glucose, maltose and mannitol from the environment. Sugars transported via the PTS become phosphorylated by their associated transporter: glucose, for example, is converted to glucose-6-phosphate and can then enter directly into glycolysis. Several key PTS proteins operate upstream of transport, executing a cascade of phosphorylation reactions that ultimately prime transporters for phospho-transfer. PTS activity has also been shown to have substantial downstream affects, regulating major cellular processes such as chemotaxis.

Previous studies have largely focused on characterising individual branches of the PTS network and so far the global architecture of this complex system has been difficult to piece together. In order to gain a more holistic view of the PTS, Victor Sourjvik’s group at the Max Plank Institute for Terrestrial Microbiology are exploring the interactions between a range of proteins within the PTS network. Using Förster resonance energy transfer (FRET), a technique that can detect when fluorescently labelled proteins come into close proximity with one another, they were able to investigate the dynamics of protein interactions within the PTS of Escherichia coli.

Intracellular FRET was used to test interactions between more than sixty protein pairs within the PTS network, revealing nine pairs that responded to PTS sugars, four of which responded in a stimulation-dependent manner. PTS activity was also observed upon stimulation with non-PTS sugars and compounds such as serine, pyruvate, glycerol and oxaloacetate, while compounds that were previously hypothesised to play a role in PTS regulation, such as glucose-6-phosphate, did not elicit a measurable response.

The study also demonstrated that all FRET pairs with a cytoplasmic PTS component were recruited to membrane transporters upon stimulation with PTS sugars. Converging at the site of transport is likely advantageous to the cell as the reduction in diffusion time increases reaction efficiency. Interestingly, recruitment did not require stimulation by a transporter’s cognate sugar, and was even observed with non-PTS sugars. If a cell detects one type of sugar in its environment it is possible that others are close by, so it would make sense to immediately prepare for uptake of other sugars. Furthermore, for several FRET pairs, the amplitude of the measured response was the same regardless of which PTS sugar was used to stimulate the cell, as illustrated in the figure below. This indicates that the PTS is not intrinsically biased toward any particular sugar and that cells use the PTS network to sense sugar influx on a global scale.

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Figure 1: Activity of three FRET pairs in response to saturating levels of PTS sugars The amplitude of the FRET response is an average of measurements for three replicate experiments. Data are normalised to the response for glucose.

All FRET experiments were carried out in buffer using naïve E. coli cells that were initially grown on amino acids and had never been exposed to sugars. The observed FRET responses therefore reflect the basal state of the PTS network. Exposure to a sugar initiates transcription of catabolic enzymes and transporters to maximise exploitation of the new carbon source, resulting in the sugar-specific preferences observed in many microorganisms. Thus the uptake rates measured in these experiments do not necessarily reflect rates observed in actively growing cells. Surprisingly, upon measuring the basal uptake rates of PTS sugars, the group found that sugars with a higher metabolic efficiency had higher basal uptake rates – indicating that E. coli may be evolutionarily optimised for growth on different carbon sources.

To investigate this further the authors used mathematical modelling to explore how the rate of biomass production correlates with the basal rate of sugar uptake. The model demonstrated that, in accordance with the experimental data, the basal sugar uptake rate increases linearly with metabolic efficiency. Energy expended on sugar catabolism must be delicately balanced with energy used for sugar transport, therefore metabolic pre-optimisation may help E. coli to rapidly maximise its growth rate when encountering a mixture of sugars.

The E. coli chemotactic response to glucose is known to be stimulated by both the glucose chemoreceptor and the PTS. To get a better idea of how PTS signals propagate through to the chemotaxis network, the group looked at the FRET response of PTS network proteins and chemotaxis regulatory proteins in an E. coli strain lacking a glucose-inducible chemoreceptor. The measured responses showed a linear correlation between the FRET signals with increasing glucose levels, providing further evidence that the PTS plays a role in chemotaxis and showing that PTS-induced chemotaxis operates autonomously from chemoreceptor-induced responses.

This research has provided significant insights into the dynamic nature of the PTS and its integrated role in bacterial physiology. Further work in this fundamental area will be highly impactful, as a better understanding of the mechanisms that allow hungry bacteria to sense sugars in the environment and respond optimally is valuable for the myriad of biotechnological applications that require efficient sugar exploitation to facilitate industrial scalability.

 

Source: Somavanshi, R., et al. (2016). “Sugar Influx Sensing by the Phosphotransferase System of Escherichia coli.” PLoS Biol 14(8): e2000074.

Using a periplasmic binding protein as a biosensor for thiamine

By Sophie Rugg

Twitter: @sophiejrugg

Thiamine, also known as vitamin B1, is an essential micronutrient with an important role in metabolism for all life forms. Thiamine can’t be synthesised by animals, and so has to be obtained from their diet. Until recently, detecting thiamine was limited to either the use of expensive high-performance liquid chromatography (HPLC), or a slow assay involving microbial growth. This is because there is no antibody available that is specific for thiamine, making commonly used high throughput detection techniques such as enzyme-linked immunosorbent assay (ELISA) impossible.

Periplasmic binding proteins are components of the ABC transporters of Gram-negative bacteria, and bind their substrates with high affinity and specificity to enable them to be transported into the bacterial cell. These properties of high affinity binding and specificity make periplasmic binding proteins ideal for use as the recognition element in a biosensor. As bacterial ABC transporters are used for the import of nutrients, including a transporter for thiamine, many of these binding proteins have evolved to recognise small molecules which it may be difficult to raise an antibody against.

Edwards et al., (2016) developed a biosensor for thiamine based on the periplasmic binding protein for thiamine from Escherichia coli. This binding protein was incorporated into dye-encapsulating liposomes in order to amplify the signal. Immobilised to the surface of a streptavidin coated plate is biotin conjugated thiamine analogue. The thiamine analogue is connected to the biotin via a long polyethylene glycol (PEG)linker, so that the thiamine analogue doesn’t get in the way of the biotin binding the streptavidin coating. The immobilised thiamine analogue binds to the periplasmic binding protein with lower affinity than thiamine. After the thiamine containing sample has been added, any material not bound to the surface is removed. Any liposomes still stuck to the surface of the plate are lysed and the resulting dye concentration is inversely proportional to the thiamine concentration.

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Overview of assay for thiamine detection taken from Edwards et al., (2016). Competitive assay with biotin conjugated thiamine analogue immobilized via streptavidin in microtiter plates and detected via periplasmic binding protein for thiamine conjugated to the lipid bilayer of dye encapsulating liposomes (left). After competition with sample thiamine, unbound materials are removed (middle) and liposomes remaining bound are lysed to release dye yielding a signal inversely proportional to thiamine concentration (right).

This work shows that periplasmic binding proteins can be used effectively in biosensors , particularly where there is no antibody available. With the wide range of periplasmic binding proteins evolved by bacteria to be able to transport nutrients into their cells, this technique is open to use across a wide range of applications provided that a suitable analogue of the substrate can be immobilised to the surface of a plate.

Source: Edwards et al., 2016. High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition. Analytical  Chemisty88 (16), pp 8248–8256. DOI: 10.1021/acs.analchem.6b02092

ABC system for dietary oligosaccharides: a weapon of Bifidobacterium sp. in the ‘metabolic’ war of the gut

By: Constantinos Drousiotis

Twitter: @Ecolinnit

The human gut microbiota (HGM) is the community of microbes that thrive in the gastrointestinal tract. Lately, it has become evident that HGM has a profound impact on human health which has now attracted a great interest from the scientific community. Current research is aiming to understand the complicated metabolic interactions that exist in the microbiota which can reveal the type of imbalances in diet that could potentially lead to disease.

A rich diet in legumes and seeds leads to an increased population of Bifidobacteria in the HGM. This is ought to the fact that the latter can metabolise raffinose family oligosaccharides (RFO) as opposed to the human gut cells which are unable to. The specialised transport machinery enabling these bacteria to transport and utilise RFOs hasn’t been characterised previously.

The study carried out by Morten et al. aimed to characterise the substrate binding protein (SBP) of the ABC transport system that was expressed in response to growth on RFOs, ie. BlG16BP. The group solved the structure of the BlG16BP and showed that the binding pocket of the protein accommodates oligosaccharides with a glucosyl or galactosyl C4-OH at position 1(non-reducing glycosyl unit) and an α-glycosidic bond to a glucosyl moiety at position 2. Additionally, they showed that the fructosyl or glucosyl groups are tolerated well at position 3 because of the lack of direct polar contacts of the protein with the sugar at this position and additionally, the cleft’s open architecture. These are all features of the sugar structures of raffinose and panose.

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Crystal structure of BlG16BP in complex with panose (A) and raffinose (B). The SBPs consist of an N-terminal domain (Domain 1, brown) and a larger C-terminal domain (Domain 2, green). The two domains are linked by hinge regions shown in light blue. Shown on right-hand side, is a close-up of the binding sites of BlG16BP in complex with panose (D) and raffinose (E). The non-reducing end glycosyl unit of both ligands (galactosyl in raffinose and glucosyl in panose) stacks onto Phe-392 (defined as position 1) and makes polar contacts to Asp-394, Asn-109, and His-395. Asp-394 is able to form hydrogen bonds to both the equatorial C4-OH of the non-reducing end glucosyl in panose and the axial C4-OH of the galactosyl in raffinose. The glucosyl moiety of raffinose and panose at position 2 stacks onto Tyr-291 and makes polar contacts to Lys-58, Glu-60, and Asp-326. The position 3 is tolerated well, as the glucosyl moiety of panose stacks onto Trp-216 with almost parallel planes of the sugar rings as opposed to the fructosyl group of raffinose which sits orthogonal against Trp216 due to a smaller area of Van der Walls contacts. As a point of reference, the oligosaccharide structures are provided on the bottom of the figure.

 

Growth assays with a mixture of RFOs as the carbon source revealed that raffinose and melibiose were utilised first in order throughout the course of growth assays of B. animalis subsp. lactis Bl-04, indicating that are preferentially recognised over tetra- and pentasaccharides. The group suggests that the preferential binding by this transporter could potentially reflect the levels of the respective sugars in the gut or reveal the ability of bifidobacteria to further process the tetra- and pentasaccharides extracellularly. Nonetheless, BlG16BP SBP has a lower affinity to the only two previously characterised oligosaccharide binding proteins from bifidobacteria; the lower affinity could indicate that RFOs are found in higher concentrations than the ligands of the other two oligosaccharide binding proteins.  Also, it could point out to the low level of competition for RFOs which would be agreeable with the fact that this ABC system is not phylogenetically diverse.

Notably, phylogenetic analysis showed, that as far as Lactobacillus species are concerned, this α-galactoside transporter is only found in the human-gut adapted clade of thereof. The lack of this system in Lactobacillus which thrive in other ecological niches suggests that the transporter was acquired by horizontal gene transfer as a survival strategy in response to the fierce microbial competition in the gut. This is in accordance with previous studies which point towards horizontal gene transfer viewed as an adaptation strategy to gut niche.

The study reports the first biochemical and structural insight into an ABC-associated glycoside transport protein and provides evidence that ABC-mediated uptake may confer a competitive edge in the fierce competition for metabolic resources in the human gut niche. Altogether, the findings improved our understanding of the impact of oligosaccharide uptake in preferential glycan utilization.

 

Source: Morten et. al., (2016) , An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates. The Journal of Biological Chemistry, 291(38), 20220-231.

Eating the Poison

By Bryony Ackroyd

Twitter: @BryonyAckroyd

The oligopeptide permease system (Opp) is an ABC transporter that commonly transports peptides into Gram-positive and Gram-negative bacterial cells. However, it has been demonstrated that Opp can also transport the antibiotic GE81112. This means the bacteria are effectively “eating the poison” that will eventually kill them.

GE81112 belongs to a structurally novel class of antibiotics and is key in the fight against antibiotic resistance and “super-bugs”. The tetrapeptide antibiotic GE81112 binds the 30S ribosomal subunit and interferes with the binding of initiator fMet-tRNA to the 30S subunit therefore inhibiting protein synthesis.

When Maio et al., began testing the microbiological activity of GE81112 on a series of microorganisms they obtained a number of unusual results. For example, the same bacteria (S. aureus, B. subtilis and E. coli) that in complete media are insensitive to GE81112 were sensitive to GE81112 in minimal or chemically defined rich media. One explanation for these results could be that GE81112, once in the cytoplasm, was disrupting 30S subunit with a different efficiency, however in vitro studies disproved this theory.

It was then hypothesised that a possible inhibitory or inactivating molecule was present in the rich media, causing the discrepancies in antibiotic sensitivity between rich media and minimal media. It was also noted that in chemically defined complete medium the activity of GE81112 is only slightly reduced compared to minimal media, indicating that the ineffectiveness of GE81112 in complete medium is not due to the concentration of nutrients.

To test the above hypothesis a series of experiments were conducted. The activity of GE81112 was measured by the change in the minimum inhibitory concentration in different growth medias. Whereas addition of individual amino acids to the growth media did not have any influence on GE81112 activity, the addition of casamino acids resulted in an increase in the minimum inhibitory concentration of GE81112. The difference between these two results was put down to the presence of di-, tri- and oligopeptides in casamino acids that may compete with GE81112 for an import system.

Due to GE81112 being a tetrapeptide the dipeptide and tripeptide transport systems were ruled out and instead the oligopeptide transport system, Opp, was investigated. An E. coli opp- mutant and wild-type were grown on minimal medium agar plates with the addition of the GE81112 antibiotic. The opp- mutants were not inhibited by GE81112, whereas the wild-type cells produced a large halo of inhibition indicating that Opp is the means of import for GE81112. Further experiments were carried out showing that presence of the whole Opp transporter was necessary for transport and sensitivity to GE81112.

Although the antimicrobial activity of GE81112 is not very efficient on bacteria growing in rich media, due to the competition for the Opp transport systems by other oligopeptides, it is important for antimicrobial resistance as it has been shown to be effective against methicillin resistant bacteria. Evidence suggests that mutations altering the cytoplasmic antibiotic target of GE81112 are few and far between, indicating that bacterial resistance to GE81112 could be slowed if entry into the bacterial cell is not blocked by oligopeptides. Could it then be possible to modify GE81112 to enter the bacterial cell without the aid of Opp to improve GE81112 efficiency and reduce resistance?

 

Source: Gualerzi et al., (2016). The Oligopeptide Permease Opp Mediates Illicit Transport of the Bacterial P-site Decoding Inhibitor GE81112. Antibiotics, 5(2): 17.

A food poisoning bacterium could aid in the fight against multidrug resistant cancers

By Caroline Pearson

Twitter: @CarolineRosePea

Salmonella enterica serovar Typhimurium is a food borne bacterial pathogen that commonly causes gastroenteritis in humans. However, it has been found that this pathogen can selectively grow inside tumours and modulate many biochemical pathways. This resulted in its recognition as a possible tool in the treatment of cancer to deliver therapeutic agents directly to the source of the cancer following systemic infection. Although many applications for this surprisingly therapeutic pathogen have been suggested, translating them into clinical use has been a stumbling point due to the possibility of systemic infections or immune mediated toxic responses to the invading bacteria.

An alternative approach to delivering the live salmonella bacteria to a cancer patient is to identify the therapeutic agents produced by S.Typhimurium which allow it to modulate biochemical pathways and administer these directly to the patient without the risk of systemic Salmonella infection. This approach has been taken by Mercado-Lubo et al., who have identified the molecule responsible for reducing the levels of multidrug resistance (MDR) transporter P-glycoprotein (P-gp) in tumour cells which increases their susceptibility to chemotherapeutic drugs.

Upregulated P-gp expression is associated with poor prognosis in several types of cancer.  The P-gp protein is encoded by MDR1, and is a MDR ABC transporter responsible for one aspect of the MDR phenotype in cancer cells. Recent studies have found that S. Typhimurium was able to reduce levels of P-gp in cancer cells and that the Salmonella type III secretory system was essential for this modulation. Therefore, S. Typhimurium type III secreted effector proteins were screened for their ability to modulate P-gp resulting in the identification of SipA.

SipA is able to modulate P-gp by activation of caspase 3 which then cleaves the P-gp protein so that it can no longer be presented at the cell surface to function as a drug efflux pump.

caroline blog post
Working model of SipA downregulation of P-gp taken from Mercado-Lubo et la., 2016. (a) Cancer cells express different types of ABC transporters, especially P-gp, to gain multidrug resistance. This allows tumour cells to extrude cytotoxic drugs from the intracellular space. (b) The SipA-AuNP may act extracellularly, by interacting with a transmembrane receptor to induce a CASP3 dependent cleavage of P-gp. The activation of caspase-3 also results in apoptosis; a cell death process. (c) Cleavage of P-gp results in the appearance of two protein fragments of about 90 and 60 kDa. Such cleavage destroys the P-gp scaffold essentially removing this transporter from the plasma membrane thereby preventing the active efflux of doxorubicin and enhancing its cytotoxic activity.

To harness the therapeutic potential of this effector protein without having to infect patients with potentially pathogenic S. Typhimurium, Mercado-Lubo et al., built a Salmonella nanoparticle mimic by fusing an inert gold nanoparticle with multiple copies of the SipA protein.  In vitro and in vivo studies both showed that the SipA nanoparticle possessed the ability to reduce P-gp levels in multiple cancer cell lines and increase their susceptibility to treatment with doxorubicin (a chemotherapeutic drug). The nanoparticle structure also enhanced SipA functionality in comparison to free SipA, presumably due to the nanoparticle complex stabilising SipA and preventing its degradation before reaching its target.

The writers suggest that this semi-synthetic Salmonella nanoparticle mimic could be applied to various chemotherapeutic drugs to overcome MDR in tumours and that the findings represent an important step forward in demonstrating the potential of this strategy as a ‘stand alone’ approach to increase cancer cell sensitivity to conventional chemotherapeutics.

 

Source: Mercado-Lubo, R., Zhang, Y., Zhao, L., Rossi, K., Wu, X., Zou, Y., Castillo, A., Leonard, J., Bortell, R. & other authors. (2016). A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours. Nat Commun 7, 12225. Nature Research.

ABC Transporter is a Key Component in Bacitracin Resistance

In the past few years a number of bacterial transport proteins have been shown to act as co-sensors for signal transduction pathways. This process generally occurs via a protein-protein interaction between the membrane bound sensor domain, which binds specific substrates, and the signalling domain, which transfers the signal information into the cytoplasm of the cell.

In this paper by Dintner et al., as well as in previously published studies, it has been shown that in the absence of the transporter component these signal transduction pathways are rendered inactive. This is due to signalling activation being entirely dependent on a sensory transporter sensing its specific substrate. All currently known examples of these systems are involved in resistance to antibiotics and the role of a transporter in signalling is conserved.

The system used in this paper to investigate this phenomenon in greater detail was the BceRS-BceAB system from the Bacillus subtilis bacterium, which confers resistance against the antibiotic bacitracin. The BceRS component of the system is a two-component regulatory system (TCS) and the signal transduction domain, whereas BceAB is an ABC transporter and the sensor domain. It is not known exactly how BceRS-BceAB confers resistance to bacitracin, however it is possible that it’s sequestered into the cytoplasm via the BceAB ABC transporter. Bacitracin is known to inhibit both cell wall and peptidoglycan synthesis in bacteria.

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Schematic model diagram showing BceAB and BceRS. BceAB constitutes the ABC transporter sensory domain, whilst BceRS constitutes the TCS signal tansduction domain. Double headed arrows indicate direct interactions between domains. Dotted arrows indicate transcription events. BceAB and BceS interact within the membrane. ATP hydrolysis by BceAB causes activation of BceS which allows phosphorylation of BceR. BceR then triggers increased production of BceAB. Taken from Dintner et al., 2014.

It has previously been shown that BceS, the histidine kinase component, is unable to detect the presence of bacitracin without BceAB, the ABC transporter component. This therefore lead to the assumption that BceAB is the sensory part of the system.

Initial experiments showed clear interactions between BceS and BceB or BceAB, however BceA was not observed to interact with any components of the TCS (BceS, BceR or BceRS). Dintner et al., also showed that BceR production in the absence of BceS resulted in a lack of interaction with the transporter (BceA, BceB or BceAB). This lead to the conclusion that BceS and BceAB form a scaffold that allows BceR to interact with the complex. Addition of the bacitracin antibiotic did not appear to have an effect on complex formation.

Following on from these discoveries the group wanted to identify whether BceAB, the ABC transporter, interacted directly with the substrate bacitracin or not. They investigated this via surface plasmon resonance (SPR) spectroscopy. This technique uses light diffracted off the underside of a surface containing the molecule of interest to create a spectrum. The change in this spectrum as a substrate is added to the surface, and possibly binds the molecule of interest, can be measured accurately along with the association and dissociation rates.

Unfortunately the BceAB complex was unstable under the SPR conditions and so BceB alone was used in the studies. Zn2+-bacitracin, the active form of the antibiotic, was used as the substrate along with the peptide nisin as a nonsubstrate control. The KD of Zn2+-bacitracin under steady state was calculated to be 60nM, whilst nisin showed no binding to the BceB. Interestingly the absence of Zn2+ prevented bacitracin binding BceB, giving further evidence of the specificity of BceB to the active peptide, Zn2+-bacitracin. The data obtained from these experiments show that the transporter, BceAB, binds free Zn2+-bacitracin specifically and with high affinity.

Dintner et al., conclude by stating that they have proposed a “working model for the mechanism of signal transduction within Bce-like models”. Bce-like systems “represent widely spread resistance determinants against peptide antibiotics in Firmicutes bacteria” and therefore make this study important in the war against antibiotic resistance.

 

Source: Dintner et al., (2014). A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis. The Journal of Biological Chemistry, 289(40)27899-910.

Bryony Ackroyd

Twitter: @BryonyAckroyd

 

 

ABC transporter implicated in parasite drug resistance

An ABC transporter in Leishmania potentially confers resistance to the antimony used in leishmanicidal drugs by sequestering the compound in vesicles and exporting them via the parasite’s flagellar pocket.

Leishmaniasis is a neglected tropical disease (NTD) caused by the protozoan parasite Leishmania. It is responsible for 20 000 – 30 000 deaths every year in countries including India, Bangladesh and South Sudan. The World Health Organisation (WHO) estimates that 310 million are at risk of developing visceral leishmaniasis.

Leishmania has two distinct life cycles, one in its mammalian host and one in its sandfly vector.  The sandfly injects promastigotes into the skin during a blood meal. These promastigotes are then taken up by macrophages where they transform into amastigotes and multiply. They are eventually released from the infected cell into the bloodstream from where they may be taken up by another sandfly during its next blood meal.

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Leishmania have two distinct life cycle stages, one within their mammalian hosts and one within the sand fly vector. The parasites are taken up during an infected blood meal and replicate within mammalian cells before being transferred to the sand fly during the next meal. Adapted from CDC (http://www.cdc.gov/parasites/leishmaniasis/biology.html).

Current treatments for leishmaniasis, including amphotericin B, miltefosine and pentavalent antimonials, can be both toxic and expensive. This coupled with the ever-increasing issue of drug resistance means that the disease is in danger of reaching crisis point. Scientists have been attempting to elucidate the various ways in which resistance could arise in the hope of curtailing some of the problems facing Leishmania control.

A team from Spain have done just that, identifying an ATP-binding cassette (ABC) transporter in Leishmania which they believe might be involved in resistance to antimony. Leishmania has 42 ABC genes yet few have been characterised. The team led by Ana Perea looked at SbV, an antimony-based drug which is taken up by the amastigote (intracellular) form of the parasite. It becomes reduced to SbIII and activated once inside the macrophages. Leishmania encodes enzymes that are capable of reducing SbV to SbIII, which then combines with thiols that are effluxed from the parasite.

The transporter in question is LABCG2. It was chosen as related transporters LABCG4 and LABCG6 had previously been implicated in resistance to the drug miltefosine. LABCG2 is involved in phosphatidylserine (PS) externalisation during infection of the host macrophages. They found that overexpressing LABCG2 resulting in the promastigotes becoming 7-fold more resistant to the antimony-based compound. This resistance was however not seen in other leishmanicidal drugs such as miltefosine.

The team then delved into exactly what was behind the resistance to SbIII. The parasites were incubated in antimony and after 60 minutes the accumulation of the compound was measured. The mutants which overexpressed LABCG2 were found to have accumulated 76% of the total amount of SbIII that the controls had. They interpreted this as an indication that the LABCG2 transporter mediates the elimination of antimony from the parasite.

Finally, they looked to establish whether thiols, which bind to and export heavy metals, could play a role in Leishmania antimony resistance. They found that thiol efflux from the parasites was greater in the presence of antimony and, following tagging by green fluorescent protein (GFP) discovered that the transporter does localise at the plasma membrane.

Overexpressing the LABCG2 ABC transporter might therefore protect Leishmania against otherwise toxic antimonic drugs by effluxing them as a complex bound to thiols. They believe that this could be a mechanism by which Leishmania may become drug resistant, although emphasise the need for LABCG2 knockout mutants to really establish what role the transporter plays in the parasite.

 

Source: Perea et al. (2016). The LABCG2 transporter from the protozoan parasite Leishmania is involved in antimony resistance. Antimicrobial Agents and Chemotherapy, 60, 3489 – 3496.

Rebecca Hall

Twitter: @RebeccaJHall13

Blog https://ananxiousscientist.wordpress.com/