YgiV promoter mutations cause resistance to cystobactamids and reduced virulence factor expression in Escherichia coli

Abstract

Antimicrobial resistance is one of the major health threats of the modern world. Thus, new structural classes of antimicrobial compounds are needed in order to overcome existing resistance. Cystobactamids represent one such new compound class that inhibit the well-established target bacterial type II topoisomerases while exhibiting superior antibacterial and resistance-breaking properties. Understanding potential mechanisms of emerging resistances is crucial in the development of novel antibiotics as they directly impact the future therapeutic application and market success. Therefore, the frequency and molecular basis of cystobactamid resistance in Escherichia coli was analyzed. High-level resistant E. coli mutants were selected and found to harbor single nucleotide polymorphisms in the promotor region of the ygiV gene, causing an upregulation of the respective protein. These stable mutations are contrary to what was observed as a resistance genotype in the structurally related albicidins, where ygiV gene amplifications were identified as causing resistance. Overexpression of YgiV in the mutants was additionally amplified upon cystobactamid exposition, showing further adaptation to this compound class under treatment. YgiV binds cystobactamids with high binding affinity, thereby preventing their interaction with the antimicrobial targets topoisomerase IV and DNA gyrase. In addition, we observed a substantial impact of YgiV on in vitro gyrase activity by leading to increased DNA cleavage and concurrent reduction in the efficacy of cystobactamids in inhibiting gyrase supercoiling activity. Furthermore, we identified co-upregulation of membrane-modifying proteins, such as EptC, and the transcriptional regulator QseB. This presumably contributes to the observed reduced motility and fimbrial protein expression in resistant mutants, resulting in a reduced expression of virulence factors and potentially pathogenicity, associated with ygiV promotor mutations.

Fig. 1. Chemical structures of tested cystobactamid derivatives CN-861-2 [1], CNDM-861 [2] and Cysto-180 [3] in comparison to the structurally related albicidin [4].

Structural differences to cystobactamid CN-861-2 [1] (reference in this study) are marked in red.

Fig. 2. Cystobactamid induced mutations in Escherichia coli are located in direct genetic neighborhood.

Single nucleotide polymorphisms were found in the promotor region of ygiV of all cystobactamid-resistant mutants, with some carrying additional mutations in qseC (cp. Table 2) (created with BioRender.com).

Fig. 3. Full-proteome analysis revealed high consensus in upregulated proteins of tested mutants and indicate an important role of YgiV in resistance.

a Comparison of upregulated proteins in mutants M3, M6 and M8 showed an intersection of 18 out of 32 proteins. (created using BioVenn). b Two main functional clusters were identified, including phage-shock proteins and membrane modifying proteins (created using STRING database). c Volcano plot shows that YgiV was the most upregulated protein in highly resistant strain M6 and major downregulated proteins in all tested mutants were found to be OmpF and FimA. d YgiV expression levels correlated to the level of resistance (M6 > M3 > M8). Data shown are mean ± SD of n = 4 (**** p-value < 0.0001). Significant up-/downregulation was analyzed by two-tailed student’s t-test with p-value [–log₁₀] > 1.3 and abundance difference [log₂] > 1.5 as cut-offs (n = 4).

Fig. 4. Cystobactamids lose their inhibitory activity on the bacterial gyrase in the presence of YgiV due to high-affinity binding and potentially by the DNA interaction of YgiV.

a Gyrase supercoiling assay showed neutralization of CN-861-2 [1] inhibitory activity (mean ± SD of n = 3) by addition of YgiV. b Native MS measurements showed binding of CN-861-2 [1] to YgiV with at least one but potentially multiple binding sites. c Microscale thermophoresis revealed low nanomolar binding affinity (K_D = 36.41 ± 16.86 nM, mean ± SD of n = 3) of CN-861-2 [1] to YgiV. d YgiV induced linearization of plasmid DNA in a concentration dependent manner. Intensity after background subtraction of the linear plasmid band at 20 µM YgiV was set as 100%. Negative control was set as 0%. (n = 3).

Fig. 5. YgiV overexpression in cystobactamid-resistant mutants was significantly induced by treatment with cystobactamid.

a Volcano plot of differerencial protein abundance observed for CYS^R M6 treated vs. untreated, and c string protein network analysis of overexpressed proteins after CYS treatment (created using STRING database), revealed mainly DNA repair proteins as part of SOS response commonly upregulated. b YgiV overexpression in cystobactamid-resistant mutants was induced via cystobactamid treatment, whereas only minor effects were observed in WT cells (*p-value = 0.0344, ****p-value < 0.0001). d YgiV upregulation could not be induced via CIP treatment (* p-value = 0.0142). Data shown are mean ± SD of n = 4. Significant up-/downregulation was analyzed by two-tailed unpaired student’s t-test (cut-offs: p-value [–log₁₀] > 1.3 and abundance difference [log₂] > 1.5) (n = 4).

Fig. 6. Affinity-based protein profiling (AfBPP) in M6 revealed intracellular binding of cystobactamid to YgiV and other proteins.

a AfBPP cross-linked the photo-affinity probe Cysto-33 [5] to target proteins after UV-irradiation with following click-reaction to biotin used for avidin-bead enrichment of bound proteins. After tryptic digest, the samples were measured using nanoElute-timsTOF (Bruker) and analyzed using DIA-NN and Perseus (created with BioRender.com). b YgiV was significantly enriched (p-value [-log₁₀] > 1.3, difference [log₂] > 1.0), thereby confirming binding to the cystobactamid probe inside the whole cell environment (*** p-value = 0.0001, two-tailed unpaired t-test, mean ± SD of n = 4). c Known target proteins, gyrase and topoisomerase IV, could be identified, as well as AcrB, SohB and YdgA as potential cystobactamid binding proteins. However, of these only the efflux pump protein AcrB seemed to have an effect on cystobactamid´s bioactivity (Supplementary Table 3).