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.

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