Activated ClpP kills persisters and eradicates a chronic biofilm infection.

A new addition to the antimicrobial arsenal.

As you will all know by now, antibiotics are becoming less and less effective with resistance increasing in natural populations. Methicillin resistant Staphylococcus aureus (MRSA) is probably the most infamous microbe in medicine as it targets immunocompromised patients and those who require invasive surgery. It’s not true to say that it is resistant to antibiotic action. In fact, in growing populations, antibiotics such as ciprofloxacin produce a biphasic pattern of killing but leave persister cells. These persisters primary function is to survive attack and remain viable until growth can resume.

So how do you kill a dormant persister, especially when they are usually buried deep inside a protective biofilm?

Ribosomal synthesis of proteins is not always perfect. Sometimes peptides are produced that are misfolded. The cell has a mechanism for the removal and recycling of these misfolded proteins, ClpP, a protease dependant on ATP. A known antibiotic for growing cells is Acyldepsipeptide (ADEP). This antibiotic activates the ClpP protease and keeps it’s catalytic chamber open, allowing for proteolysis to occur. It also removes the dependence on ATP. In this study ADEP4, a derivative from Streptomyces hawaiensis was chosen because of it’s previous success in eradicating Gram-positive bacteria.

Using proteomics on treated and untreated samples, ADEP4 was shown to activate the degradation of at least 417 proteins with FtsZ being a major target, hence the microbicidal action in growing populations. In an exponentially growing culture of S. aureus treated with antibiotics the population is reduced to persisters. With the addition of ADEP4 the population if further reduced to the limit of detection, implying that the ADEP4 causes self-digestion within the dormant cells. In a stationary phase culture of S. aureus most of the cells act as persisters and are extremely difficult to treat with antibiotics. ADEP4 here managed to reduce the population by 4 log₁₀ but a rebound in the population then occurred. This is because of the null clpP mutants. S. aureus do not require ClpP so null clpP wild type mutants arise readily. However when ADEP4 was coupled with rifampicin the stationary phase culture was eradicated. It was hypothesized that the null mutants have diminished fitness and are therefore susceptible to antibiotics. This may indicate that the null clpP mutants do not enter persister state, possibly due to their temperature sensitive phenotypic characteristics, which would make for inadequate dormant cells.

The combination of ADEP4 and rifampicin was tested on many known strains of MRSA (SA113, USA300, UAMS-1 & strain 37), none of which showed any colonies after incubation for 72h. One of the major problems with MRSA is its formation of antibiotic resistant biofilms. Using a biofilm produced by the osteomyelitis (infection of the bone) associated strain UAMS-1 the authors, again, showed the complete eradication of all living cells using ADEP4 and rifampicin. This has not been seen before with such low concentrations of antibiotics.

The final and most important question seems obvious. Can this work in an organism? For this they used a deep-seated mouse thigh infection model in which the mouse is made neutropenic (without neutrophils), infected with a large dose of pathogen and allowed 24h for incubation. This was used as a model for hard to treat human chronic infection. Treatment with ADEP4 and rifampicin, remarkably, completely cleared the infection although I doubt the mouse lived to tell the tale.

This discovery, to me, seems like a game changer, opening up more potential avenues of investigation involving self-digestive proteases and other potential activators. Although it does seem naïve to think that microbes will not respond with new strategies to combat this type of microbicidal action, we can still take comfort in the fact that there are always going to be new innovations on both sides.

Conlon, B. P., Nakayasu, E. S., Fleck, L. E., LaFleur, M. D., Isabella, V. M., Coleman, K., … Lewis, K. (2013). Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature, advance on.