Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport.

Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

The therapeutic potential of bacteriocins as protein antibiotics.

The growing incidence of antibiotic-resistant Gram-negative bacterial infections poses a serious threat to public health. Molecules that have yet to be exploited as antibiotics are potent protein toxins called bacteriocins that are produced by Gram-negative bacteria during competition for ecological niches. This review discusses the state of the art regarding the use for therapeutic purposes of two types of Gram-negative bacteriocins: colicin-like bacteriocins (CLBs) and tailocins. In addition to in vitro data, the potency of eight identified CLBs or tailocins has been demonstrated in diverse animal models of infection with no adverse effects for the host. Although the characteristics of bacteriocins will need further study, results obtained thus far regarding their in vivo potency, immunogenicity and low levels of resistance are encouraging. This leads the way for the development of novel treatments using bacteriocins as protein antibiotics.