https://doi.org/10.1126/scitranslmed.abj0716

Paper summary

Multidrug-resistant bacteria represent an increaseingly healthy and environmental problem worldwide. The central objective of Zhao et al. (2021) study is to investigate metabolic mechanisms underlying multidrug resistance in Uropathogenic Escherichia coli (UPEC), which are the dominant cause of urinary tract infections (UTI) and are prevalently insensitive to ampicillin. While it is well acknowledged that the active efflux that pumps out antibiotcs is a dominant mechanism of antibiotic resistance, little is known how to enhance cell permiability to antibiotics to counteract the drug resistance mechanism. An earlier study from the same group showed that exogenous amino acids promote aminoglycoside-mediated antibiotic killing. Therefore, the authors hypothesize that exogenous amino acids enhance cell membrane permeability allowing greater antibiotic uptake. The authors first identified notable differences in the metablites between antibiotic-sensitve (E. coli-S) and multidrug-resistant (E. coli-R) E. coli strains isolated from patients suffering from urinary tract infections. The following experiments focused on glutamine, which is the most supressed metabolite in E. coli-R compared to E. coli-S. Next, they showed that exogeous administration of glutamine dramatically enhance the killing effiency of antibotics including AMP (ampicillin) against multiple clinical multidrug-resistant bacteria both in vitro and in the UTI mouse model. The authors further demonstrated that exogenous glutamine significantly increased intracellular AMP concentrations, implying glutamine metabolism could affect bacterial cell membrane permiability to AMP. Subsequently, they used all kinds of knock-out E. coli strains to pinpoint the purine metabolism as the downstream effects of glutamine because defects in purD, purL, purE, or purH resulted in loss of sensitivity to AMP plus glutamine. The authors concluded that glutamine and inosine activate CpxA, which dephosphorylates CpxR-P and in turn ablate the suppression of OmpF, allowing more AMP fluxes into bacterial cell. The outcome of this study would promote the development noval strategy in restoring antibiotic killing effiency.

Interpretation of the study and future directions

In my opinion, the Zhao et al. (2021) study is sound and solid by showing dozens of to hundreds of folds of augmentation in AMP killing efficiency when supplemented with a simple amino acid glutamine. The study provides a new solution to disarm the antibiotic resistance mechanism among a broad spectrum of Gram-negative pathogens by tweaking the amino acid and nucleotide metabolism. In terms of safety, the AMP dose used in this study is equivalent to 26mg/kg in human, which is much lower than current usage dose (200 to 400 mg/kg). On the other hand, glutamine is a non-essential amino acid that human can synthesize and is conserved and essential in eukaryotes and prokaryotes. Therefore, the synergistic efficiency of glutamine plus AMP could be broad, and bacteria are unlikely to develop resistance to glutamine. Nevertheless, there are a few things that can be improved in this paper. In figure 2A, 5A, and 6A, non-linear scale of y-axis was used for visualizing the survival rate of bacteria. First, the bar plot is less preferred because the readers are not able to see each individual data point. Second, the up-side-down bar plot does not provide any help for better understanding the data. Third, non-linear scale makes the plots harder to compare between different treatments. At certain points, the non-linear y-axis is misleading. For example, the figure6A plot tested the strains with defects in glutamine transporters such as glnQ, glnP, and glnH. Their conclusion is that “elevated glutamine mainly depended on glutamine transporters”. However, all the three glutamine transporter mutants were still very sensitive to AMP because more than 90% of bacteria died. My interpretation is that the three tested glutamine transporters are replaceable and independent on each other for the influx of glutamine. The glutamine transporters are not discussed in the discussion section.

Future direction: Incorporate histone to the synergy between AMP and glutamine

While the primary function of histone is considered as condensing eukaryotic DNA, its antimicrobial function is underappreciated until increasing number of studies showed its antimicrobial synergy with antimicrobial peptides. Histones colocalize with antimicrobial peptides in neutrophil extracellular traps. A recent study demonstrated the synergetic effects between antimicrobial peptides and histones, by showing that human-derived antimicrobial peptide LL-37 enhances the cell membrane permeability and thus allows histone H2A to enter bacterial cells. H2A rearranges the bacterial chromosome and suppresses global bacterial transcription (Doolin et al., 2020). Antimicrobial peptides-histones self-amplifying mechanism is general in nature and is targeting bacterial cell membrane, which is reminiscent of the glutamine-inosine-CpxA/R-OmpF pathway that allows more ampicillin entering the bacterial cells (Zhao et al., 2021). It is interesting that cell membrane porin OmpA was downregulated in response to H2A binding on bacterial chromosome, signifying a defense mechanism from the bacterium. H2A and antimicrobial peptides are 14-18 KDa. It is possible their entry is independent on Omp porin family. Ampicillin is larger with 31 KDa and requires OmpF to be on to enter bacterial cells. Enhanced uptake of histone and antibiotics in the two studies seem both require achieving the electrical depolarization that causes loss of bacterial proton motive force (PMF). In Doolin et al. (2020), LL-37 was shown to depolarize the membrane and disrupts the PMF. Future experiments should determine if glutamine-mediated cell membrane permeability has synergistic effects with antimicrobial peptides, which allows more influx of antimicrobial agents such as ampicillin and H2A. The potential antimicrobial synergy among antimicrobial peptides, histones, glutamine, and antibiotics can greatly improve the killing efficacy because of their self-amplifying mechanism to increase the concentration in bacterial cells. Future clinical trial should improve safety and lower the administration dose by focusing on the pharmacological kinetics of these antimicrobial agents.

References

Doolin, T., Amir, H.M., Duong, L., Rosenzweig, R., Urban, L.A., Bosch, M., Pol, A., Gross, S.P., and Siryaporn, A. (2020). Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization. Nat. Commun. 11, 3888. https://doi.org/10.1038/s41467-020-17699-z. Zhao, X., Chen, Z., Yang, T., Jiang, M., Wang, J., Cheng, Z., Yang, M., Zhu, J., Zhang, T., Li, H., et al. (2021) Glutamine promotes antibiotic uptake to kill multidrug-resistant uropathogenic bacteria. Sci. Transl. Med. 13, eabj0716. https://doi.org/10.1126/scitranslmed.abj0716.