The antimicrobial peptide DGL13K is active against resistant gram-negative bacteria and subinhibitory concentrations stimulate bacterial growth without causing resistance

Background of preprint

The rise of antimicrobial resistance in the 21^st Century has pushed researchers to seek out alternative solutions to the upcoming antibiotic crisis. Indeed, throughout the past few decades, a growing chorus of voices—most recently manifesting in a 2020 commentary published in Nature Microbiology [1]—has been calling for better stewardship of our rapidly diminishing antibiotic resource.

One potential alternative to antibiotics lies in the use of antimicrobial peptides (AMPs), short and generally positively charged peptides that often form part of the innate immune response [2]. AMPs have risen to prominence due to their ability to reduce or slow bacterial resistance. While resistance to AMPs has been generated under laboratory conditions, leading to concerns that AMP-resistant bacteria may also be resistant to endogenous host-defence peptides, further research and development on AMPs as potential antibiotics have been justified on two counts.

First, AMPs are more likely to exhibit the phenomenon of collateral sensitivity than cross-resistance to traditional antibiotics [3]. This means that the development of resistance to one AMP may decrease, rather than increase, the bacteria’s resistance to another AMP.

Second, closely related peptide enantiomers may exhibit different interactions with bacterial defences, reducing the chance that resistance to both enantiomers develops.

Through their previous work on anti-inflammatory and bacterial agglutinating peptides, Gorr et al. had developed the modified AMP GL13K from the human salivary protein BPIFA2 [4-8]. In this preprint, Gorr et al. expanded on their past work from two angles.

First, they determined the relative activity of the L- and D-enantiomers of GL13K (LGL13K and DGL13K, respectively). Both enantiomers were tested on wild-type and drug-resistant strains of gram-negative bacteria, as well as on bacterial biofilms. Specifically, the authors chose to work on Klebsiella pneumoniae, Acinetobacter baumanii, and Pseudomonas aeruginosa, three of the six ESKAPE pathogens (preprint Table 1). First described in 2008 [9], ESKAPE is a group of six pathogens exhibiting multidrug resistance and virulence that pose a major concern to the World Health Organisation (WHO), which has characterised them as “pathogens for which antibiotics are urgently needed” [10,11].

Gorr et al. next determined the subinhibitory concentrations of GL13K peptides in P. aeruginosa. The preprint authors observed that the GL13K peptides appeared to have a hormesis effect on P. aeruginosa, in which small amounts of GL13K seemed to promote, rather than inhibit, P. aeruginosa metabolism. They also showed that there was no acquired resistance to DGL13K or cross-resistance between the two enantiomers of GL13K.

Key findings of preprint

(A) Relative activity of the L- and D-enantiomers of GL13K

Minimum inhibitory concentrations (MICs) of DGL13K were generally lower than those of LGL13K, corroborating their previous results [12,13]. Moreover, DGL13K was most active against K. pneumoniae and A. baumanii. Both DGL13K and LGL13K killed P. aeruginosa biofilms at similar concentrations. Gorr et al. also found that the mass of biofilms did not change significantly following any peptide treatments, including the negative control peptide, suggesting that dead bacteria were not dislodged by any of these treatments.

(B) Concerns over resistance

To address concerns regarding the development of resistance to the AMPs, Gorr et al. exposed P. aeruginosa to a range of concentrations of both GL13K enantiomers. Doing so led the preprint authors to make an interesting observation: concentrations of GL13K up to 0.5 x MIC seemed to promote P. aeruginosa metabolic activity. This observation describes the pharmacological effect of hormesis: a phenomenon where the organism appears to benefit from being exposed to small quantities or low concentrations of otherwise harmful toxins.

This observation led Gorr et al. to test for resistance arising from repeated exposure to sub-MIC concentrations, by repeatedly exposing P. aeruginosa to 0.5 x MIC of both GL13K enantiomers over 16 days. Repeated exposure to DGL13K did not seem to increase to increase the MIC; in contrast, repeated exposure to LGL13K appeared to increase the MIC slightly. Importantly, however, the latter observation did not reach statistical significance, and cross-resistance between both GL13K enantiomers—a key consideration in the development of AMPs [14]—was not observed. Therefore, even with a conservative approach, these results suggest that the development of resistance is unlikely to be a cause for concern.

What I like about this preprint

Since joining preLights in 2018, many of my posts have been about developments that mitigate the worrying trend of antimicrobial resistance—I wrote about the brain-eating amoeba Balamuthia mandrillaris in my first preLight, and about the ESKAPE pathogens last year.

I chose this preprint for two reasons. First, this preprint advances the authors’ efforts—spanning almost a decade—on developing GL13K (Fig. 1): they had discovered GL13K [5-7,13,15], characterised the in vitro and in vivo activity of GL13K on both gram-positive and gram-negative bacteria [12,16], and characterised its toxicity [12].

Figure 1. Work in preprint in relation to work in the past decade.

Second, I found the preprint authors’ observation of hormesis to be particularly noteworthy. The effect of hormesis in antimicrobial therapies is indeed an uncommon observation, because we generally expect most antibiotics to exhibit a dose-dependent response.

The classical pharmacological dose-dependent response of antibiotics usually manifests in either time- or concentration-dependence. Time-dependent antibiotics become efficacious once they reach concentrations above a certain MIC; concentration-dependent antibiotics become more efficacious as their maximum concentration increases. Gorr et al. attribute the observed hormesis to the possibility that GL13K selectively stimulates certain metabolic pathways of P. aeruginosa through currently unknown mechanisms.

Therefore, future work will likely revolve around figuring out the cellular and physiological mechanisms underlying the observed phenomena. In order to advance the development of GL13K, the in vivo activity and toxicity of both DGL13K and LGL13K will need to be characterised through preclinical work—such as those on mice, dogs, or monkeys. Given that GL13K is an AMP, its pharmacokinetics will also need to be studied to advance it to the next stage of development.

References

[1] Nathan C, Resisting antimicrobial resistance, Nature Reviews Microbiology 18(5) (2020) 259-260.

[2] Mahlapuu M, Håkansson J, Ringstad L, Björn C, Antimicrobial Peptides: An Emerging Category of Therapeutic Agents, Frontiers in Cellular and Infection Microbiology 6(194) (2016).

[3] Lázár V, Martins A, Spohn R, Daruka L, Grézal G, Fekete G, Számel M, Jangir PK, Kintses B, Csörgő B, Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides, Nature microbiology 3(6) (2018) 718-731.

[4] Nandula SR, Huxford I, Wheeler TT, Aparicio C, Gorr S-U, The parotid secretory protein BPIFA2 is a salivary surfactant that affects LPS action, Experimental Physiology n/a(n/a) (2020).

[5] Gorr S-U, Sotsky JB, Shelar AP, Demuth DR, Design of bacteria-agglutinating peptides derived from parotid secretory protein, a member of the bactericidal/permeability increasing-like protein family, Peptides 29(12) (2008) 2118-2127.

[6] Gorr S-U, Abdolhosseini M, Shelar A, Sotsky J Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein,  p^pp, Portland Press Ltd. (2011)

[7] Abdolhosseini M, Sotsky JB, Shelar AP, Joyce PBM, Gorr S-U, Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain, Molecular and Cellular Biochemistry 359(1) (2012) 1-8.

[8] Geetha C, Venkatesh SG, Bingle L, Bingle CD, Gorr S-U, Design and Validation of Anti-inflammatory Peptides from Human Parotid Secretory Protein, Journal of Dental Research 84(2) (2005) 149-153.

[9] Rice LB, Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE, The Journal of Infectious Diseases 197(8) (2008) 1079-1081.

[10] Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N, Aboderin AO, Al-Abri SS, Awang Jalil N, Benzonana N, Bhattacharya S, Brink AJ, Burkert FR, Cars O, Cornaglia G, Dyar OJ, Friedrich AW, Gales AC, Gandra S, Giske CG, Goff DA, Goossens H, Gottlieb T, Guzman Blanco M, Hryniewicz W, Kattula D, Jinks T, Kanj SS, Kerr L, Kieny M-P, Kim YS, Kozlov RS, Labarca J, Laxminarayan R, Leder K, Leibovici L, Levy-Hara G, Littman J, Malhotra-Kumar S, Manchanda V, Moja L, Ndoye B, Pan A, Paterson DL, Paul M, Qiu H, Ramon-Pardo P, Rodríguez-Baño J, Sanguinetti M, Sengupta S, Sharland M, Si-Mehand M, Silver LL, Song W, Steinbakk M, Thomsen J, Thwaites GE, van der Meer JWM, Van Kinh N, Vega S, Villegas MV, Wechsler-Fördös A, Wertheim HFL, Wesangula E, Woodford N, Yilmaz FO, Zorzet A, Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis, The Lancet Infectious Diseases 18(3) (2018) 318-327.

[11] Mulani MS, Kamble EE, Kumkar SN, Tawre MS, Pardesi KR, Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review, Frontiers in microbiology 10 (2019) 539-539.

[12] Gorr S-U, Flory CM, Schumacher RJ, In vivo activity and low toxicity of the second-generation antimicrobial peptide DGL13K, PloS one 14(5) (2019).

[13] Hirt H, Gorr S-U, Antimicrobial Peptide GL13K Is Effective in Reducing Biofilms of <span class=”named-content genus-species” id=”named-content-1″>Pseudomonas aeruginosa</span>, Antimicrobial Agents and Chemotherapy 57(10) (2013) 4903-4910.

[14] Fleitas O, Franco OL, Induced Bacterial Cross-Resistance toward Host Antimicrobial Peptides: A Worrying Phenomenon, Frontiers in Microbiology 7(381) (2016).

[15] Abdolhosseini M, Nandula SR, Song J, Hirt H, Gorr S-U, Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity, Peptides 35(2) (2012) 231-238.

[16] Hirt H, Hall JW, Larson E, Gorr S-U, A D-enantiomer of the antimicrobial peptide GL13K evades antimicrobial resistance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii, PloS one 13(3) (2018).

Tags: acinetobacter baumannii, antimicrobial peptide, antimicrobial resistance, biofilm, eskape, hormesis, klebsiella pneumoniae, pseudomonas aeruginosa

doi: https://doi.org/10.1242/prelights.20722

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