Glyoxylate Shunt and Pyruvate-to-Acetoin Shift Are Specific Stress Responses Induced by Colistin and Ceragenin CSA-13 in Enterobacter hormaechei ST89

ABSTRACT Ceragenins, including CSA-13, are cationic antimicrobials that target the bacterial cell envelope differently than colistin. However, the molecular basis of their action is not fully understood. Here, we examined the genomic and transcriptome responses by Enterobacter hormaechei after prolonged exposure to either CSA-13 or colistin. Resistance of the E. hormaechei 4236 strain (sequence type 89 [ST89]) to colistin and CSA-13 was induced in vitro during serial passages with sublethal doses of tested agents. The genomic and metabolic profiles of the tested isolates were characterized using a combination of whole-genome sequencing (WGS) and transcriptome sequencing (RNA-seq), followed by metabolic mapping of differentially expressed genes using Pathway Tools software. The exposure of E. hormaechei to colistin resulted in the deletion of the mgrB gene, whereas CSA-13 disrupted the genes encoding an outer membrane protein C and transcriptional regulator SmvR. Both compounds upregulated several colistin-resistant genes, such as the arnABCDEF operon and pagE, including genes coding for DedA proteins. The latter proteins, along with beta-barrel protein YfaZ and VirK/YbjX family proteins, were the top overexpressed cell envelope proteins. Furthermore, the l-arginine biosynthesis pathway and putrescine-ornithine antiporter PotE were downregulated in both transcriptomes. In contrast, the expression of two pyruvate transporters (YhjX and YjiY) and genes involved in pyruvate metabolism, as well as genes involved in generating proton motive force (PMF), was antimicrobial specific. Despite the similarity of the cell envelope transcriptomes, distinctly remodeled carbon metabolism (i.e., toward fermentation of pyruvate to acetoin [colistin] and to the glyoxylate pathway [CSA-13]) distinguished both antimicrobials, which possibly reflects the intensity of the stress exerted by both agents. IMPORTANCE Colistin and ceragenins, like CSA-13, are cationic antimicrobials that disrupt the bacterial cell envelope through different mechanisms. Here, we examined the genomic and transcriptome changes in Enterobacter hormaechei ST89, an emerging hospital pathogen, after prolonged exposure to these agents to identify potential resistance mechanisms. Interestingly, we observed downregulation of genes associated with acid stress response as well as distinct dysregulation of genes involved in carbon metabolism, resulting in a switch from pyruvate fermentation to acetoin (colistin) and the glyoxylate pathway (CSA-13). Therefore, we hypothesize that repression of the acid stress response, which alkalinizes cytoplasmic pH and, in turn, suppresses resistance to cationic antimicrobials, could be interpreted as an adaptation that prevents alkalinization of cytoplasmic pH in emergencies induced by colistin and CSA-13. Consequently, this alteration critical for cell physiology must be compensated via remodeling carbon and/or amino acid metabolism to limit acidic by-product production.

It is unclear what the authors intended to mean: Did it mean to say, for example, that "none of the genes in this region were previously known to be associated with colistin resistance"?
Ln 138: -The title of this section is misleading: "Impact of colistin and CSA-13 at the gene expression level." Reading this title, I thought about gene expression in E. hormaechei cells grown in media containing either colistin or CSA-13. However, as I read further and only after going back to and reading the method, I realized that this experiment was to compare gene expression profiles between the wild-type strain and isolates (either resistant to colistin or CSA-13).
-In general, the authors' objective for comparing gene expression profiles between the wildtype and a resistant isolate is unclear. Any mutations (i.e., deletion, point mutation, insertion, or combination of mutations in multiple chromosomal loci) in resistant strains would be expected to affect expression of certain genes as compared with the wildtype strain. If the authors clarify the purpose of this experiment, it would be helpful for readers to understand the significance of this study.
Ln 139: "In comparison to colistin" This is not "colistin" and, again, it is misleading. This is a colistin resistant strain.
Ln 139-140: "CSA-13 exerted more extensive dysregulation of the E. hormaechei transcriptome" In general, statements like above need to be revised: for example, in the CSA-13 resistant strain, more extensive transcriptional changes were observed.

Reviewer #2 (Comments for the Author):
This manuscript focuse well on experiments and methodological part is precise

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Here, we examined the genomic and transcriptome responses by Enterobacter hormaechei after 26 prolonged exposure to either CSA-13 or colistin. Resistance of E. hormaechei 4236 strain 27 (ST89) to colistin and CSA-13 was induced in vitro during serial passages with sub-lethal doses 28 of tested agents. The genomic and metabolic profiles of the tested isolates were characterized 29 using a combination of the whole genome (WGS) and transcriptome (RNA-seq) sequencing, 30 followed by metabolic mapping of differentially expressed genes using Pathway Tools  Colistin and ceragenins, like CSA-13, are cationic antimicrobials that disrupt the bacterial cell 48 envelope through different mechanisms. Here, we examined the genomic and transcriptome 49 changes in Enterobacter hormaechei ST89, an emerging hospital pathogen, after prolonged 50 exposure to these agents to identify potential resistance mechanisms. Interestingly, we observed 51 down-regulation of genes associated with acid stress response as well as distinct dysregulation 52 of genes involved in carbon metabolism, resulting in a switch from pyruvate fermentation to 53 acetoin (colistin) and the glyoxylate pathway (CSA-13). Therefore, we hypothesize that 54 repression of the acid stress response, which alkalinizes cytoplasmic pH and, in turn, suppresses 55 resistance to cationic antimicrobials, could be interpreted as an adaptation that prevents 56 alkalinization of cytoplasmic pH in emergencies induced by colistin and CSA-13.

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Colistin is one of the first antibiotics discovered and is nearly as old as antimicrobial 62 therapy. However, its recent re-emergence as the 'last resort' drug against carbapenem-resistant 63 bacteria revealed our misunderstanding of its properties, even at the level of antibiotic 64 susceptibility testing (AST) (1-3).

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Colistin is problematic in testing due to the variety and constantly evolving (hetero-66 )resistance mechanisms and paradoxical effects, such as 'skipped well' or species-dependent 67 increasing/decreasing MICs in the presence of divalent cations (2, 4). Therefore, recent findings 68 connecting resistance to colistin with globally regulated physiological processes, such as ion 69 homeostasis, membrane potential, respiration, and carbon metabolism, regardless of LPS 70 modification, e.g., by L-Ara4N, are very engaging (5-7). Notably, a study by Panta et al. (2001) 71 demonstrated a universal relationship between colistin activity and cytoplasmic pH 72 homeostasis, likely mediated by proton and non-proton pumping systems (5). Although the 73 molecular basis of this phenomenon remains to be elucidated, metabolic processes preventing 74 intracellular alkalization, such as fermentation, are certainly implicated (5, 7). Remarkably, in 75 this scenario, the proton motive force may directly impact interactions between colistin and 76 LPS due to remodeled metabolism (8).

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In light of this, the purpose of the present research was to determine whether another    variety of DEGs as well as their vitality for the cell. Overall, 6.1 % and 2.4 % of the total gene content, 165 representing multiple categories, from ribosomal proteins to flagella components ( Fig. 1 and 2), were 166 affected in Eh4236Csa13R and Eh4236ColR, respectively. In addition, the ratio between up-and  Eh4236ColR and Eh4236Csa13R (Fig. 3). Nevertheless, a periplasmic protein VirK/YbjX (318 in colistin and CSA-13 resistant isolates, respectively (Fig. 4).

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The putrescine-ornithine antiporter PotE, encoded in one operon, with ornithine 248 decarboxylase SpeF, seems to be the central point in this acid response suppression network. Accordingly, the patterns of abnormally expressed genes involved in pyruvate 288 metabolism appear to reflect distinct stress response strategies induced by colistin and CSA-13 289 (Fig. 4). Specifically, in Eh4236ColR, a tendency to reduce the formation of acidic products, 290 i.e., formate, acetate, and propionate was observed, which may explain the attenuation of the  Alteration of LPS by L-Ara4N residues, as a mechanism mediated by proton motive 338 force (PMF)-dependent transporters, should be perceived in light of the aforementioned 339 observations. In fact, L-Ara4N incorporation into LPS seems to be another physiological 340 response to acidic stress coordinated by DedA family transporters (5, 6, 23, 45). Indeed, the 341 DedA transporter was overexpressed in both isolates at comparable levels (Fig. 3). In addition,  Moreover, another VirK/YbjX variant (326 aa) was also overexpressed in both isolates (Fig. 3). 346 Initially, these proteins were identified with Shigella flexneri virulence factor VirK (46).

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However, VirK/YbjX should be rather perceived as membrane stress chaperons, preventing