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.

Slide1
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.

Slide1
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/

Ivacaftor, the Miracle Drug?

 

Ivacaftor has been hailed as a miracle drug for the treatment of Cystic fibrosis (CF). Although Ivacaftor is not designed for every CF sufferer, it is life changing for those who benefit from it.

CF is a genetic disease that affects 70,000 people worldwide and is characterised by an overly viscous mucus lining of the airways, resulting in difficulty in clearing the airways by coughing, and an increase in infections from opportunistic pathogens.  CF is caused by different mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR), an ABC transporter ion channel, which results in an imbalance in ion concentration and the observed phenotype of highly viscous mucus. The CFTR conducts chloride and as well as regulating other ion channels, such as chloride channels and glutathione transport. There are approximately 1900 known mutations within the CFTR, which is primarily expressed within the airway submucosal glands in the lungs.

Until now all CF care has been supportive rather than curative. However a recent breakthrough with the drug Ivacaftor, which treats the underlying problem rather than just the symptoms of CF, could change the CF care landscape. Ivacaftor is used to treat CF patients with one of a set of specific mutations including G551D, which affects approximately 4% of CF patients and is the third most common mutation. The G551D mutation is characterised by correct positioning of the CFTR on the epithelial cell surface but incorrect function, the CFTR is unable to transport chloride ions.

Slide1
Predicted structure of the CFTR. TMD; Transmembrane Domain, NBD; Nucleotide Binding Domain. The G551D mutation occurs in the NBD2 region and prevents ATP dependent gating. Adapted from Kim and Skach, 2012 (http://dx.doi.org/10.3389/fphar.2012.00201).

Ivacaftor, developed by Vertex Pharmaceuticals together with the Cystic fibrosis Foundation, assists with the function of the mutant CFTR by directly binding the CFTR channel. Binding increases the probability of channel opening by inducing opening of the ion channel independent of ATP binding and hydrolysis. This reduces the imbalance in the ion concentration and lowers the viscosity of the mucus in the airways allowing easier breathing of the CF patient. CF sufferers who have had access to Ivacaftor have been able to “run without coughing” and “take deep breaths”.

The FDA approved Ivacaftor in January 2012 and the combination drug with lumacaftor gained FDA approval in July 2015. The inclusion of lumacaftor in the combination drug enables treatment of CF patients with a slightly different mutation in addition to those treated by Ivacaftor.

However there is a catch, Ivacaftor treatment costs $300,000 per patient per year. Patients are likely to be taking Ivacaftor for the rest of their lives and so the cost becomes astronomical. On the other hand Vertex has stated that they will make the drug available free of charge for those patients in the US without medical insurance and with a household income of less than $150,000 a year.

The real question surrounding Ivacaftor is; how to pay for the miracle?

 

Source: Kotha and Clancy (2013). Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence. Therapeutic Advances in Respiratory Disease, 7(5)288-296.

Bryony Ackroyd

Twitter: @BryonyAckroyd