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Bacterial persisters, which are slow-growing or non-growing phenotypic variants, complicate the treatment of chronic infections as they are recalcitrant to killing by antibiotics. Two studies now report novel approaches to eradicate persisters, and both of these strategies involve proteolysis by the ClpP protease.

The compound acyldepsipeptide (ADEP) from Streptomyces hawaiiensis kills growing cells by triggering the activation of ClpP, which leads to uncontrolled proteolysis of nascent peptides and eventual cell death. Conlon et al. reasoned that extended incubation with a more potent ADEP derivative, ADEP4, might activate ClpP in dormant persisters and lead to nonspecific degradation of mature proteins. Thus, the authors compared the proteomes of stationary phase Staphylococcus aureus populations with and without ADEP4 treatment and found that the addition of ADEP4 resulted in the degradation of more than 400 proteins. This result indicates that ClpP becomes a nonspecific protease in the presence of ADEP4 and causes uncontrolled proteolysis in non-replicating cells.

To determine whether ADEP4 is effective at killing persisters, Conlon et al. first tested its ability to eradicate S. aureus persisters that survive ciprofloxacin treatment. Unlike rifampicin, which had no killing effect, ADEP4 resulted in the eradication of persisters to the limit of detection. Similarly, ADEP4 showed potent activity against stationary phase S. aureus cultures, resulting in a 4-log reduction in cell count within 2 days. However, after 3 days, the population had regrown owing to the emergence of clpP-null mutants at a high frequency. The authors found that combining rifampicin with ADEP4 led to the eradication of the population to the limit of detection. This killing effect was even more pronounced when the cells were grown in minimal medium, which resulted in complete sterilization of the culture. The combination of ADEP4 and rifampicin was also tested on a range of S. aureus strains, including antibiotic-resistant clinical isolates, and was shown to be highly effective. Importantly, Conlon et al. also show that the combination of ADEP4 and rifampicin results in complete eradication of S. aureus biofilms in vitro and in a deep-seated chronic infection in a mouse model.

In the second study, Kim et al. engineered a dual-control (DUC) inducible genetic switch that combines transcriptional repression of a target gene and regulated proteolysis of the encoded product and show that it can be used to identify and eliminate proteins that are required for Mycobacterium tuberculosis persistence. The DUC switch consists of two tetracycline repressors that suppress transcription at promoters containing tet operator sequences, in addition to the SspB adaptor protein, which targets proteins for degradation by ClpP. Thus, this switch enables silencing at the transcriptional and protein levels, which ensures that complete knockdown of the target protein occurs.

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for numerous biochemical reactions, but its requirement for persistence was unknown. Kim et al. used the DUC switch to block expression of M. tuberculosis NAD synthetase (NadE) in replicating cells and found that NadE was rapidly depleted, which resulted in growth arrest and bacterial death. The authors then assessed the requirement of NadE for persistence during starvation and under hypoxic conditions, and found that depletion of NadE led to the eradication of non-replicating M. tuberculosis persisters. Finally, depletion of NadE also resulted in a substantial reduction in M. tuberculosis colonization during both acute and chronic infection of mice, which confirms that NadE activity is essential for persistence in vivo and validates this protein as a potential target for antibiotics.

Considering the current antibiotic resistance crisis and the major threat that is posed by persisters, these two studies are an important advance for the development of novel antibacterial agents that have the ability to eliminate persisters.

This article is modified from the original in Nature Rev. Microbiol. (http://dx.doi.org/10.1038/nrmicro3181).