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.

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