Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.


Reviewer 1
The manuscript by Rauter et al describes their intensive investigation of the differential mechanism of action of a suite of 6deoxy lipoglycosides against B. anthracis and related model strains. The authors 1) synthesize a suite of lipoglycoside analogs and test their antibiotic activity and cytotoxicity, 2) examine differential activity against cell wall deficient Grampositive and Gram-negative strains, and 3) test hypothetical mechanism of action by in silico modeling and assays in unilamellar and multilamellar vesicles. Their conclusion, based on the presented data is that the 6-doexy series of compounds selectively targets membrane lipid polymorphism in a phosphatidylethanolamine (PE) dependent manner. Overall, this is some rather nice work and the experiments that are presented have been carried out with a reasonable degree of rigor. I believe that the conclusions, if properly substantiated, would be of significant interest to the journal broad readership, in particular, to those with interests in antibiotic activity and microbial cell biology. Still, I have several scientific concerns that keep me from fully buying the authors conclusions and these would need to be addressed before publication in the journal 1.
First, the authors provide only a slight SAR for their lipoglycosides. They present just one glycoside containing a 6-hydroxyl group as the potential inactive variant. It would be helpful to have a couple such 6-hydroxy containing compounds so as to ascertain the strength of this key pharmacophore position. The current synthesis should be readily amenable to making a couple additional control compounds. This is especially necessary because the SAR on the rest of the molecule is flat -there is negligible difference between the 6-deoxy compounds (difference is within assay error).
Reply: According to this request, we have added a new set of compounds (including their synthesis and characterization) to those previously given in the manuscript, namely the dodecyl 2-deoxy C-glycoside 14 analogue to 13 and the 4-deoxy Oglycoside 15, both of them with the 6-OH group in their structure. We have added the 4,6-dideoxy glycoside 11 and the βanomers of the 2,6-dideoxy and the 4,6-dideoxy glycosides, namely compounds 5 and 12, aiming to give some new examples of active and inactive deoxy glycosides, thus reinforcing the role of the glycone structure to increase antimicrobial activity. Accordingly, Fig. 1 was changed with the introduction of the structure of such compounds, and SI contains now their synthesis and structure characterization. Maria Teresa Blazquéz Sanchez was added as co-author, since she synthesized newly introduced compounds.
The antimicrobial activity of compounds 1, 5, 11, 12, 13, 14 and 15 over B. cereus and B. anthracis strains Sterne, ovine and pathogenic was evaluated using the broth microdilution method on Müller-Hinton medium. Also cytotoxicity over Caco-2-cells was evaluated. So, with the new added structures we followed reviewer 1 requests, by adding inactive structures (e.g. compounds 5 and 12), showing that the anomeric configuration and the deoxygenation pattern are paramount for the bioactivity, and the presence of 6-OH decreases compound bioactivity, as deduced by comparing compound pairs 14/10 and 15/11 (Fig 1), including 13/9 for B. anthracis Sterne and B. cereus. We discovered that the 4,6-dideoxy pattern is the most promising one leading to the most active compound in all bacteria and strains tested. Compound 13 revealed inactive for B. cereus and B. anthracis Sterne, as expected, but active over B. anthracis pathogenic and ovine. This was not detected in our preliminary assays with compound 13, carried out by the paper disc diffusion method over Bacillus cereus, and also not expected because B. cereus genome is known to be very similar to that of B. anthracis, as reported in the literature and cited in the manuscript. Therefore, compound 13 was investigated, together with the lead compound 1, to evaluate the mechanism of action. In fact the battery of biophysical tests carried out demonstrated that compound 13 has a completely different behavior than that of the lead compound, which bioactivity results from targeting membrane lipid polymorphism, namely the propensity of phosphatidylethanolamine to undergo a lamellar to hexagonal phase transition.

2.
Second, the mechanism that the authors put forth based on their anisotropy experiments seems to suggest PE binding, but this is insufficiently explored. The authors mention AMPs that bind PE, such as cinnamycin and duramycin. PE binding by these class I antibiotics has been validated in micellar systems via ITC (see Machaidze et al, Biochemistry, 2003, 42 (43), pp 12570-12576 and citing). Could the authors provide analogous experiments to tightly validate a PE-dependent binding mechanism? Additionally, the cinnamycin binding experiments contradict the authors statements about PE-binding AMPs (lines 296-8) being largely electrostatic (cinnamycin has a net formal charge of 0 at physiological pH). Also, cinnamycin activity results in resistant mutants that down regulate membrane associated PE. How do the authors justify a PE-based mechanism with no analogous mutations? I wonder if the authors might be able to test activity against mutants deficient in PE based on their mechanism. Overall, a secondary experiment to validate the PE mechanism would be heavily preferred.
Reply: We fully agree with this Reviewer 1 comment, and therefore we provide, in the revised version novel data to validate the PE-dependent mechanism of action. We now include two additional types of approaches: 1) membrane permeabilizing activity of compounds 1 and 13 towards PC versus PE membranes and 2) surface pressure experiments in PE and PC monolayers. In both cases the data strongly support the proposed mechanism and previous conclusions (new Figs 7 and 8).
The new results now presented, in particular the membrane permeabilizing activity, show unequivocally that PE membranes are highly susceptible to the active compound 1, but not to compound 13. Moreover, they also show very clearly that the impact on PE membranes of the active compound is not only much stronger, but is also much faster, when compared to its impact on PC membranes, which may also help to explain the lack of resistance development and corroborates the MD simulations in PC membranes. In addition, the effects observed suggest the induction of highly curved membrane regions, which in the extreme situation would correspond to the formation of inverted hexagonal phase as observed by fluorescence anisotropy in PE membranes, but when operating in a more localized fashion can induce e.g., vesiculation. Joaquim T.
Marquês was added as co-author, since he performed the new biophysical experiments.
In the revised version of the manuscript, one can now read: "A membrane passive permeability assay was used to directly assess both the ability of compounds 1 and 13 to interact with POPE versus POPC bilayers, and to evaluate their membrane permeabilizing activity towards these two different glycerophospholipids. LUVs suspensions with encapsulated carboxyfluorescein at a high concentration (40 mM) will have very low fluorescence intensity due to self-quenching. As it crosses the lipid membrane, carboxyfluorescein will be at very low concentration in the outer buffer and, consequently, will be relieved from fluorescence self-quenching. As a result, the kinetics of leakage can be monitored as an increase of fluorescence intensity over time. These membrane permeability curves are shown in Fig. 7A.
From the results obtained for the membrane passive permeability it is clear that compound 1 is the most active one and that POPE membranes are extremely susceptible to this compound, that induces a complete release (~100 % of leakage) of encapsulated carboxyfluorescein, whereas compound 13 only seems to slightly perturb the membrane. In this case only ~10 % leakage was obtained (see also Fig. 7B). Such results show that compounds 1 and 13 interact with the POPE membrane differently. While compound 13 seems to promote a minor perturbation of membrane organization, compound 1 leads to a more drastic reorganization of the lipid membrane with the concomitant full release of carboxyfluorescein, in very good agreement with the previous results. Moreover, POPC LUVs were more resilient than POPE liposomes to the action of compound 1, since a total membrane leakage of ~30 % was observed for POPC membranes during the course of the experiment in opposition to the ~100 % of leakage for POPE liposomes. Even the Lmax value obtained from the fit (Fig.  7B) is less than 50% for POPC. These results strongly suggest that compound 1 interacts differently with PC or PE membranes. The low permeabilizing activity in PC bilayers seems to be in good agreement with the results of the MD simulations. The formation of pores owing to the localized transition from lamellar to an inverted hexagonal-like phase in POPE membranes (or at least of high curvature stress areas) as a result of the interaction with compound 1 would have as outcome the complete release of carboxyfluorescein. On the other hand, all other situations where an incomplete release of carboxyfluorescein was obtained, surely, do not involve a lamellar-to-hexagonal phase transition or a high curvature stress, but most probably only a smaller perturbation on the packing of the lipids within the bilayer. The presence of 1.2 % (v/v) of ethanol only leads to a negligible leakage (1-2 %) during the course of the experiment (14 h). From the analysis of the permeability curves the time of leakage, L, was also obtained (Fig. 7C). The fastest process was the permeabilization of POPE by 1 (L of about 160 min), whereas the slowest activity was also for compound 1, but when added to POPC (L of about 692 min). Thus the interaction of 1 is much faster with PE bilayers than PC, suggesting a higher affinity for the PE bilayer. On another hand, compound 13 had intermediate values of L that were not markedly different for both lipid bilayers, of about 326 min for POPE and 256 min for POPC. Thus the speed of interaction of this compound (and thus possibly its affinity) is similar for both lipids. Both compounds have a hydrophobic dodecyl chain, so it is expected that they present some interaction with both glycerophospholipid bilayers. Overall these results strongly support that compound 1 but not 13 has higher affinity for PE than PC, and also that the mode of action of 1 behind its antimicrobial activity towards microorganisms with high levels of exposed PE indeed involves its specific interaction with PE leading to membrane permeabilization.
To further support the distinctive interaction of compound 1 with PE, surface pressure (π) measurements were carried out using a Langmuir trough. The effect of compounds 1 and 13 on the compression isotherm of POPE molecules at the air/water interface was assessed (Fig. 8, A and B). Compression isotherms of POPE alone showed a liquid expanded-liquid condensed transition around 37 mN/m and collapsed upon reaching surface pressures of ~54 mN/m, which is in good agreement with other reports from literature38,39. However, the compression curves recorded after injection of compound 1 (Fig. 8A) show a clear shift to the left, i.e. lower mean molecular area per lipid (A/ lipid) values. To attain the same A/lipid, the difference (drop in this case) in π can be as high as 15 mN/m. Such shift may be a consequence of altered packing properties of the POPE monolayer and/or a lesser number of POPE molecules available for the formation of the monolayer. The local action of compound 1 may trigger the increase of monolayer curvature, possibly with the formation of invaginations. These per se can justify the shift of the curves towards lower π values. If a fraction of the POPE molecules in these high curvature areas undergo a lamellar-to-hexagonal phase transition they no longer reside at the air-water interface plane, as they will tend to aggregate and possibly precipitate. In opposition, compression isotherms of POPE recorded after the incubation with compound 13 (Fig. 8B) exhibit a slight shift to the right, i.e. higher A/ lipid molecule values. This observation is consistent with the insertion/incorporation of compound 13 into the lipid membrane without triggering any remarkable membrane reorganization.
Moreover, preformed POPE monolayers at ~25 mN/m were incubated either with compounds 1 or 13 and the variation in π was monitored over time until equilibration, i.e., the interaction/incorporation of the compounds on the PE monolayer was followed over time (inset in Fig. 8A). Notably, the addition of compound 1 at a concentration as small as 0.2 µM leads to a marked decreased in π of almost 3 mN/m, especially when compared with the effect of compound 13 at the same concentration, which was unable to promote any obvious change in the final π value. Such a large effect for this concentration range denotes a specific/high affinity interaction between the antimicrobial agent with the lipid molecules rather than a general interaction of an amphiphilic compound, such as a fatty acid or a fatty acid derivative, with the lipid monolayer40-42. Changes induced by compound 13 were smaller than 0.5 mN/m and after equilibration π returned to its initial value.
Compression isotherms of pure POPC monolayers, contrary to POPE monolayer, revealed that POPC remains in the liquid expanded phase during all compression cycle and that it collapses around 44 mN/m, which is in fine agreement with  (Fig. 8C) before and after injection of compound 1 also support a unique interaction between the compound and POPE. As mentioned, upon the incubation of POPE monolayers with compound 1 the compression curves are clearly shifted to lower A/lipid. However, in what concerns POPC, the curves obtained after incubation with compound 1 are practically superimposed with the ones acquired in the absence of the compound. Although cell membranes are more complex than liposomes, these results point to structural tendencies of lipids while interacting with glycoside 1 and support the distinct behavior of compounds 1 and 13 against Bacillus cereus. The bioactivity relates to PE reorganization thereby promoting lamellar to hexagonal phase transition, which emerges as the mechanism underlying membrane disruption and bactericidal activity of compound 1 over B. cereus. The lack of activity of 13 over B. cereus is consistent with the biophysical studies herein presented. Worth mentioning, PE constitutes only 23% of mammalian plasma membrane phospholipids, and is mostly confined to the inner monolayer 44,45 , therefore not directly accessible to the antimicrobial agent, highlighting the importance of this mode of action with therapeutic potential.
We also wish to acknowledge the Reviewer by noting that the importance of electrostatics in the membrane binding and mechanism of action was not stated clearly and contradicts the results with cinammycin. This is now corrected in the revised version. We have also included references Machaidze et al, Biochemistry "Generally, it is assumed that membrane interactions involving charge neutralization play an important role in antimicrobial peptides (AMPs) mode of action, as many of these peptides are cationic or present a highly cationic surface that promotes binding to bacterial membrane anionic phospholipids, such as lipopolysaccharides, phosphatidylglycerol or cardiolipin 45-47 . However, cinammycin, a peptide that targets PE through a very strong binding, while presenting very weak binding to PC membranes, has net formal charge zero at physiological pH. 46,48,49 In agreement, our active compound, bearing no charge, also targets PE. " As the Reviewer pointed out, PE is a nearly zwitterionic lipid at that pH. In agreement, our active compound, also targeting PE, is a neutral molecule. Nonetheless, all the biophysical results presented by us in this revised version, as stated above, highlight that the interaction of compound 1 with PE is not merely of hydrophobic nature, as the outcomes of insertion into