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Back to Journals » Infection and Drug Resistance » Volume 15
Authors Chen M, Li Y, Li S, Cui W, Zhou Y, Qu Q, Che R, Li L, Yuan S, Liu X
Received 20 June 2022
Accepted for publication 16 September 2022
Published 26 October 2022 Volume 2022:15 Pages 6165—6176
DOI https://doi.org/10.2147/IDR.S379264
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 3
Editor who approved publication: Professor Suresh Antony
Mo Chen,1 Yanhua Li,1 Shu Li,2 Wenqiang Cui,2 Yonghui Zhou,3 Qianwei Qu,1 Ruixiang Che,4 Lu Li,5 Shuguang Yuan,2 Xin Liu3
1College of Veterinary Medicine, Northeast Agricultural University, Harbin, People’s Republic of China; 2Research Center for Computer-Aided Drug Discovery, Shenzhen Institutes of Advanced Technology, Shenzhen, People’s Republic of China; 3College of Basic Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang, People’s Republic of China; 4College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, People’s Republic of China; 5College of Life Sciences, Northeast Agricultural University, Harbin, People’s Republic of China
Correspondence: Shuguang Yuan, Research Center for Computer-Aided Drug Discovery, Shenzhen Institutes of Advanced Technology, Shenzhen, People’s Republic of China, Tel +86-150-0209-0670, Email [email protected] Xin Liu, College of Basic Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang, People’s Republic of China, Tel +86-188-8605-6643, Email [email protected]
Purpose: Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs. Strains that are resistant to multiple drugs pose severe clinical problems and cost lives. However, systematic studies on cross-resistance of Staphylococcus xylosus have been missing.
Methods: Here, we investigated various mutations in the sequence of ribosomal proteins involved in cross-resistance. To understand this effect on a molecular basis and to further elucidate the role of cross-resistance, we computationally constructed the 3D model of the large ribosomal subunit from S. xylosus as well as its complexes with both tylosin and florfenicol. Meanwhile, all-atom molecular dynamics simulations was used. In addition, the regulation of protein networks also played an essential role in the development of cross-resistance in S. xylosus.
Results: We discovered that the minimum inhibitory concentration against both tylosin and florfenicol of the mutant strain containing the insertion L22 97KRTSAIN98 changed dramatically. Further, we found that unique structural changes in the β-hairpin of L22 played a central role in this variant in the development of antibiotic resistance in S. xylosus. The regulation of protein networks also played an essential role in the development of cross-resistance in S. xylosus.
Conclusion: Our work provides insightful views into the mechanism of S. xylosus resistance that could be useful for the development of the next generation of antibiotics.
Keywords: antimicrobial resistance, Staphylococcus xylosus, cross-resistance evolution, ribosomal protein L22
Antibiotic resistance occurs when bacteria or fungi develop the ability to defeat the drugs designed to kill them.1 Worldwide, about 700,000 people die each year due to drug-resistant diseases, according to a report from the World Health Organization.2,3 No one can completely avoid the risk of being affected by resistant infections. If antibiotics lose effectiveness, we lose the ability to treat serious infections and control public health.
Staphylococcus xylosus is a coagulase-negative, Gram-positive coccus that is widely distributed in the environment, as well as in the skin and mucosal surfaces of mammals.4,5 S. xylosus is also naturally present in raw meat and milk, and in addition is used as a starter culture for manufacturing sausage, fermented cheese, and meat.6 Although this strain is defined as an apparent nonpathogenic Staphylococcus, some strains unexpectedly were involved in bacterial infections in human and animal.7,8 Therefore, ever-increasing attention is paid to the role S. xylosus plays in infectious diseases.
During the last few years, an increasing number of human infections have been associated with this species, such as urinary tract, pancreatic, cardiac, ophthalmologic, dental, bloodstream, erythema chest infections, and nodosum, as well as otogenic brain abscess.8–11 Currently, S. xylosus has become the prevalent cause of mastitis in dairy herds in many countries, including the US, Finland, Canada, Sweden, Switzerland, Norway, and China.12–15 The average proportion of mastitis due to S. xylosus increases year by year according to a nationwide survey.13,16 S. xylosus can also lead to other diseases in animals, including dermatitis in gerbils and immunodeficient mice, pulmonary and lymph-node lesions in immunodeficient mice, or transmural necrotizing cystitis and secondary peritonitis in a 4-month-old male Holstein calf.7,17
Nowadays, macrolides (tylosin), florfenicol, aminoglycosides (gentamicin and neomycin), lincomycin, and β-lactam antimicrobials (including penicillin and cephalosporins) are commonly used to treat mastitis. In general terms, treatment with antimicrobials is regarded as the main driver of antimicrobial resistance. As a result, resistance to antibiotics used in mastitis is increasing.18,19 CNS(coagulase-negative Staphylococcus) resistance to erythromycin in the milk of cows with mastitis of 14.3% has been reported.20 Another study observed the highest percentage of isolates resistant to erythromycin among S. xylosus strains from the milk of cows with mastitis.21 Strains resistant to lincomycin (53%) have also been observed among isolates of S. xylosus.22 Some studies have reported an increase in central nervous system resistance to β-lactam antibiotics, particularly penicillin and ampicillin, which is a worldwide trend.23,24
In several studies addressing evolution of resistance, one fearsome fact was observed: bacteria develop cross-resistance against antimicrobial drugs to which they never have been exposed before.25 Cross-resistance is a severe obstacle for designing effective drug therapies, as it limits possible antibiotic options following unsuccessful drug treatment. Surprisingly, evolution of cross-resistance in S. xylosus did not receive much attention until very recently. The ribosomal peptidyl transferase center (PTC) is critical for protein synthesis.26 Peptidyl transferase reaction involves aminolysis by the α-amino group of the A-site aminoacyl-tRNA of the ester bond that links the nascent peptide to the 3 hydroxyl of the 3 terminal ribose of the P-site tRNA.27 Many antibiotics that inhibit growth of microorganisms do so by binding to the PTC and inhibiting its activity.28
The structural mode of macrolides binding to the entrance of the peptide exit tunnel of the large (50S) ribosomal subunit has been characterized at atomic resolution with X-ray crystallography.29 Although macrolides bind in the vicinity of the PTC, they do not inhibit peptide bond formation. On the contrary, macrolides block entrance of the nascent chain to the peptide exit tunnel. The study suggested that mutations in ribosomal proteins L4 and L22 in the 50S subunit reduced macrolide susceptibility. Similarly, chloramphenicol, the first broad-spectrum antibiotic, inhibits protein synthesis by targeting the PTC of the bacterial ribosome.30 Accordingly, macrolides and chloramphenicol share the common mechanism of inhibition of PTC activity in bacteria, and the cross-resistance mechanism of macrolides and chloramphenicol might be related to the PTC.
To better explore mechanism of antibiotic cross-resistance, we introduced site-selective mutations in ribosomal proteins and computational modeling. We propose that in addition to the relationship of ribosomal proteins of cross-resistance, regulation of the protein network may be involved in the mechanism of cross-resistance. In our previous study on tylosin-resistant mechanisms, we suggested that some resistant proteins played an important role.31 To elucidate the mechanism of resistance evolution of S. xylosus against tylosin and florfenicol under selection pressure, we here further explored the aforementioned resistant proteins. We investigated whether distinct mutations in ribosomal proteins were responsible for cross-resistance establishing mutant strains using protein sequencing and computer simulations. Ultimately, quantitative PCR (qPCR) and computer simulations were applied to demonstrate that the resistant proteins were regulated by ribosomal proteins. Our present results demonstrated that mutations in ribosomal proteins were of central importance for the evolution of tylosin and florfenicol cross-resistance.
Plasmids used in this study are listed in Supplementary Table 1. S. xylosus ATCC 700404 was purchased from the American Type Culture Collection (ATCC) and preserved in our lab. All tylosin-resistant S. xylosus strains were induced under tylosin pressure in vitro and kept in our lab. All mutants of S. xylosus were constructed in this study. The strains in (Supplementary Table 2) were cultured in tryptic soy broth (TSB) at 37°C.
Based on the genomes of S. xylosus and tylosin-resistant S. xylosus (tylosin 128 µg/mL), the rplC, rplD, and rplV fragments were amplified. All primers are listed in Supplementary Table 3. According to a previous describedly method in our lab, the genomes of S. xylosus and tylosin-resistant S. xylosus were extracted. We chosen the PrimeStar GXL DNA polymerase to amplify the fragments of rplC, rplD, and rplV. Then, the PCR products were visualized by agarose gel electrophoresis. DNA sequences of PCR products were tested at Comate Bioscience. Subsequently, the Dnaman software was used to compare amino acid (nucleotide) sequences of S. xylosus with tylosin-resistant S. xylosus.
The recombinant shuttle plasmids (PBT2-rplC, PBT2-rplD, and PBT2-rplV) were used according to a protocol in our lab with some modifications.32 Upstream and downstream fragments of rplC, rplD, and rplV genes were amplified from the genomic DNA of S. xylosus. The gfp gene and mutant fragments of rplC, rplD and rplV were obtained from plasmids, including gfp-T, mutant rplC-T, rplD-T, and rplV-T. Then, in determinate order, the above PCR products were linked by overlap PCR and cloned to pClone007 blunt simple vector. The constructed plasmids were named based on mutant sites as pClone007-rplC, pClone007-rplD, and pClone007-rplV. Next, the constructed plasmids were digested and cloned using the restriction sites of the E. coli–Staphylococcus shuttle vector pBT2, and thus the recombinant shuttle plasmids PBT2-rplC, PBT2-rplD, and PBT2-rplV were obtained. The primers and restriction sites used in this section are listed in Supplementary Table 4
The mutant strains were created using a previously described protocol with some modifications.33 The recombinant shuttle plasmids were introduced into S. xylosus by electroporation. The strains with recombinant shuttle plasmids were grown in tryptic soy broth at 37°C for 24 hours, then transferred to fresh medium at 42°C for another 24 hours, which was identified as one passage. Based on the gfp gene, flow cytometry (FC) was used to detect the mutant strains of seven, nine, and eleven passages. The blank strains without recombinant shuttle plasmids served as controls. The mutant strains screened by FC were identified by PCR amplification and sequences of mutant fragments (Supplementary Figure 1). Finally, the mutant strains were named L3 (rplC) N135G, A137G, S142A and R162K mutant S. xylosus, L4 (rplD) S158N and A164E mutant S. xylosus, and L22 (rplV) 97KRTSAIN98 insertion mutant S. xylosus.
Minimum inhibitory concentration (MIC) assays of tylosin and florfenicol to S. xylosus and mutant S. xylosus were done as previously reported.34 Briefly, S. xylosus and mutant strains were grown overnight at 37°C. The overnight cultures were diluted in sterile physiological saline, which corresponded to 108 colony-forming units/mL. Then, the cultures were diluted again with TSB to 106 colony-forming units/mL. Finally, 100 mL samples were added to the 96-well plate containing serial dilutions of tylosin in culture medium. Control bacterial and medium were cultivated in the absence of tylosin. The MIC was defined as the lowest concentration of inhibitor to visually inhibit growth. The assays were repeated three times.
We employed MD simulations for S. xylosus 23S rRNA, L22, and L22 97KRTSAIN98 insertion mutants to obtain corresponding three-dimensional structures and studied the functional changes in the structure. All MD simulations were performed using Gromacs 2018.35 All systems were solvated in a cubic water box with 12 Å buffer distance between the solvent box wall and the nearest solute atoms and neutralized by addition of 150 mM Na+ or Cl– ions. The Amber99SB-ILDN force field was assigned to the RNA, protein, and ions.36 Water was modeled using the TIP3P water model. All structure models were first minimized to relax the solvent and optimize the system using steepest descent. Then, a short 100 ps NVT preequilibration simulation was performed whereby the heavy atoms of RNA or protein were positionally restrained using a force constant of 200 kJ/(mol•nm). Finally, the production run 23S rRNA and L22 were positionally restrained to the backbone of ribose (C1’ atom), phosphate (P atom), and Cα atom and a positional force constant of 200 kJ/(mol•nm) was applied. L22 and L22 97KRTSAIN98 insertion mutant production runs were performed without any positional constraints. The temperature of simulations was controlled using the Nosé–Hoover thermostat and the pressure was held at 1 bar using an isotropic coupling with the Parrinello–Rahman barostat. Long-range electrostatic interactions were calculated using the particle mesh Ewald method with a real-space cutoff value of 1.2 nm. Unbonded interactions were cut off at 1.2 nm.
The docking of antibiotics to the receptor was performed using Glide.37 Tylosin and florfenicol were produced in Schrödinger Maestro software. The LigPrep module in the software was introduced for geometric optimization using the OPLS_2005 force field. The receptor models were prepared in the software under the OPLS_2005 force field. Hydrogen atoms were added according to the physiological pH (7.5) with the Propka tool in Maestro Protein Preparation Wizard to optimize the hydrogen-bond network. Constrained energy minimization was conducted on the fully atomic models. Cubic boxes were centered on the tylosin and florfenicol mass center with a radius of 12 Å for the peptide exit tunnel and PTC of receptor.
Here, five genes related to resistance were investigated (16s rRNA was used as an internal control). The primers of target genes are listed in Supplementary Table 5. Total RNA of different levels of tylosin-resistant S. xylosus was extracted as specified by manufacturer, including tylosin (8 µg/mL, 32 µg/mL, and 128 µg/mL). Next, an equivalent amount of total DNA-free RNA from the last sample was reverse-transcribed using a PrimEScript RT reagent kit with gDNA Eraser. Finally, real-time PCR was run for 40 cycles (95°C for 15 s, 60°C for 35 s) after initial 30 s incubation at 95°C. The assays were repeated three times.
Five genes related to resistance were investigated at mRNA level, and 16S rRNA was used as an internal control. The list of primers is shown in Supplementary Table 5. Total RNA of S. xylosus and mutant S. xylosus was extracted. Then, the equivalent amount of total DNA-free RNA from the last sample was reverse-transcribed using PrimeScript kit with gDNA Eraser. Real-time PCR reactions were run for 40 cycles (95°C for 15 s, 60°C for 35 s) after initial 30 s incubation at 95°C. The assays were repeated three times.
All MIC values were determined in triplicate to ensure reliability. Data on real-time PCR were analyzed (and figures drawn) using GraphPad Prism. Dnaman and BLAST were used to identify DNA sequences. Values are expressed as means ± SD. Statistical differences among the groups were compared by one-way ANOVA, and significant means were separated using Tukey’s honestly significant difference at p<0.05.
To probe the molecular evolution of tylosin-resistant S. xylosus, we sequenced PCR-amplified DNA of ribosomal proteins L3, L4, and L22 from S. xylosus and tylosin-resistant S. xylosus. Sequencing revealed mutations N135G, A137G, S142A and R162K in rplC (ribosomal protein L3), S158N and A164E in rplD (ribosomal protein L4), and 97KRTSAIN98 insertion in rplV (ribosomal protein L22.) The L3 loop F127–P170 locates in the vicinity of PTC, and deletions or mutations in the loop may reshape the drug-binding pocket, hindering antibiotic binding of S. aureus.38 The mutations of L3 found in our present study were consistent with results of the previous study. Hence, we concluded that mutations N135G, A137G, S142A and R162K in L3 were involved in the molecular evolution of cross-resistance. In addition, the L4 W65–Q75 loop locates in the vicinity of the phosphate of ribosomal RNA A2059, which belongs to the multidrug-binding pocket, and mutations in the W65–Q75 loop might affect the conformation of A2059 leading to multidrug resistance [34]. However, the mutations of L4 found in our present study were not in line with the region mentioned before. In addition, the mutations, deletions, and insertions in the L22 hairpin loop, which were part of the exit tunnel R80-S108, would confer resistance to erythromycin, synercid and telithromycin in multidrug-resistant S. aureus.39,40 Hence, it is reasonable that 97KRTSAIN98 insertion in L22 was involved in the molecular evolution of cross-resistance.
Our protein sequence analysis point out that mutations of ribosomal proteins L3 (rplC), L4 (rplD) and L22 (rplV) may relate to tylosin resistance of S. xylosus. As such, a ribosomal mutant S. xylosus was constructed, and the gfp gene instead of resistance genes as screening flags was used to find mutant strains, since our aim was to clarify the drug-resistant mechanism, and resistance genes would disturb the results. Then, FC was used to detect mutant strains on the basis of the fluorescence of the expressed gfp gene. Compared with the wild-type strain, the screened strains showed strong fluorescence, and fluorescence intensity continually increased along with screening generations (Figure 1). The screened strains were further identified by PCR amplification and sequencing (Supplementary Figure 2). As expected, the sequencing results of the amplified fragments were in accord with the introduced mutant sites.
 |
Figure 1 In order to screen the mutant strains, we used flow cytometry to measure the fluorescence of GFP in mutated S. xylosus, after seven, nine, and eleven generations. |
Figure 1 In order to screen the mutant strains, we used flow cytometry to measure the fluorescence of GFP in mutated S. xylosus, after seven, nine, and eleven generations.
Next, we carried out MIC assays to seek mutant sites thatwere related to the molecular evolution mechanism of tylosin resistance. The resistance to tylosin increased from 0.5 µg/mL in wild-type S. xylosus to 128 µg/mL in the L22 (rplV) mutant. An additional interesting result was that the MIC against florfenicol increased from 0.5 µg/mL in wild-type S. xylosus to 2 µg/mL in L22 (rplV) mutant. In contrast, the resistance to tylosin and florfenicol did not change for L3 (rplC) or L4 (rplD) mutants compared to wild-type S. xylosus.
Crystal structures of the ribosomal subunit from S. xylosus in the absence or presence of bound antibiotic and S. xylosus–antibiotic complex have not been reported yet, making it difficult to understand the structural relationship between S. xylosus and the two antibiotics (tylosin and florfenicol). Fortunately, co-crystal structure of tylosin bound to the related 50S ribosomal subunit of Haloarcula marismortui has been resolved. This study revealed tylosin bound in a narrow part of the ribosomal peptide exit tunnel at a site that lies between the PTC and the constriction in the tunnel near protein L22.41 The crystal structure of the 50S ribosomal subunit complexed with the clinically relevant antibiotic chloramphenicol, with a closely related chemical structure to florfenicol, showed that the antibiotic-binding site was composed exclusively of segments of 23S ribosomal RNA at the entrance to the peptide exit tunnel or the peptidyl transferase cavity.42 Based on these studies, we suggested that tylosin and florfenicol contact primarily the peptide exit tunnel or PTC of 23S rRNA of S. xylosus and the mycinose moiety of tylosin binds L22.
We used computational methods to construct a three-dimensional structure of 23S rRNA and L22 of S. xylosus and then docked antibiotics to the structure to elucidate the structural basis of ribosome–antibiotic interactions. We used a fully automatic procedure with Swiss-Model43 to model L22 of S. xylosus based on a related homologous L22 from S. aureus (PDB ID: 4WCE)38 that shared 96% sequence identity. The homology model of S. xylosus L22 shared a common overall structure of L22 from other species.38,41 It consisted of a globular domain with three α-helices and an extended loop with two- or three-stranded antiparallel β-sheets forming a β-hairpin, which extended approximately 30 Å from the globular domain (Figure 2A and B). Because homology modeling of RNA remains a challenge,44 a more effective method to build S. xylosus 23S rRNA was implemented. We found that the sequence alignment of the 23S rRNA of 50S nucleotides showed 93% identity with only 97 different bases between S. xylosus and S. aureus (Supplementary Figure 3). For high sequence conservation, we directly mutated these local bases in the crystal structure of S. aureus 23S rRNA to build the initial three-dimensional structure for S. xylosus 23S rRNA. Then, we combined the homology model of L22 and the mutant 23S rRNA into a new S. xylosus model. The interactions between bases and sidechains of amino acids, and those between side chains of amino acids in this new model were recalibrated by performing MD simulations which provided a more reasonable S. xylosus structure (Figure 2C). The MD trajectory showed that RMSD of 23S rRNA and L22 within 1.4 Å during 5 ns simulation (Supplementary Figure 4), suggesting that the molecular interactions in this new S. xylosus mode were stable.
 |
Figure 2 (A) 3D model of S. xylosus L22. The overall structure consists of three α-helices and three β-strands. The encircled box indicates the location of β-hairpin. (B) The amino acid sequence of L22 together with distribution of particular secondary structures. (C) The overall structure of S. xylosus ribosomal subunit. The 23S rRNA is shown in gray, the L22 is shown in orange, the antibiotic-binding site is shown in cyan, and the PTC location is marked as a purple dot. |
Figure 2 (A) 3D model of S. xylosus L22. The overall structure consists of three α-helices and three β-strands. The encircled box indicates the location of β-hairpin. (B) The amino acid sequence of L22 together with distribution of particular secondary structures. (C) The overall structure of S. xylosus ribosomal subunit. The 23S rRNA is shown in gray, the L22 is shown in orange, the antibiotic-binding site is shown in cyan, and the PTC location is marked as a purple dot.
Then, we extracted the last configuration from the simulation trajectory as a starter to obtain a S. xylosus–antibiotic complex model. As indicated in Figure 3, S. xylosus ribosomal subunit complexed with tylosin shows that the saccharide branch linked to lactone ring C5 contacts with the PTC and the mycinose linked to C14 that points to the exit tunnel consisting of L22 and domain II of 23S rRNA. It was similar to the binding orientation in other ribosome–tylosin complexes elsewhere.41 The tylosin-binding sites include nucleotides A798 (A748, E. coli), A2086 (A2041, E. coli), A2090 (A2045, E. coli), G2533 (G2508, E. coli), U2637 (U2575, E. coli) and L22 Gln90, which establish either hydrophobic or hydrogen bond interactions with ligands. In the model of S. xylosus-florfenicol, florfenicol was bound at the PTC with the OH– and the NH groups forming hydrogen bonds with A2479 (A2454, E. coli) and A2533 (A2508, E. coli). These results clearly illustrate that tylosin is bound at the polypeptide exit tunnel, which may inhibit translation by blocking the egress of nascent polypeptides. In contrast, the florfenicol-binding site is located at the A-site and may block PTC activity by hampering the binding of transfer RNA to the A-site.
 |
Figure 3 A model of antibiotics in complex with the S. xylosus ribosome. (A) Superposition of the binding locations of tylosin (red) and florfenicol (green). Surface representation of the structure of 23S rRNA (cyan) and L22 (orange). Tylosin binds to the peptide exit tunnel, whereas florfenicol is above the exit tunnel of PTC peptide (B). The hydrogen bond interactions (purple dash) of florfenicol (C) and tylosin (D) with 23S rRNA in the ribosome. |
Figure 3 A model of antibiotics in complex with the S. xylosus ribosome. (A) Superposition of the binding locations of tylosin (red) and florfenicol (green). Surface representation of the structure of 23S rRNA (cyan) and L22 (orange). Tylosin binds to the peptide exit tunnel, whereas florfenicol is above the exit tunnel of PTC peptide (B). The hydrogen bond interactions (purple dash) of florfenicol (C) and tylosin (D) with 23S rRNA in the ribosome.
The resistance to tylosin increased from 0.5 µg/mL in wild-type S. xylosus to 128 µg/mL in L22 (rplV) mutant (97KRTSAIN98 insertion). Previous research showed that the point mutations, deletions, and insertions in the uL22 hairpin loop that is part of the exit tunnel wall in residues R80–S108 would confer resistance to erythromycin, synercid, and telithromycin in multidrug-resistant S. aureus.39,40 Along similar lines, resistance mutations were also observed in additional bacterial species, such as Legionella pneumophila.45 Hence, it is reasonable to infer that 97KRTSAIN98 insertion in L22(rplV) was involved in the molecular evolution mechanism of cross-resistance. In order to illustrate the above result, the effect of the mutations identified on the L22 structure was investigated by both molecular modeling and MD simulations. According to the modeled structures, the mutated site of L22 (rplV) was located in the β-hairpin of the wild-type L22 (Supplementary Figure 5). Therefore, it could potentially destroy the hairpin secondary structure. Considering the flexibility of protein that ruled function and controlled activity, we further investigated the structure and motion of wild-type as well as mutant L22 using MD simulations. As shown in Figure 4A, the L22 protein extended β-hairpin remained stable in a folded-sheet structure in the wild type. In contrast, the mutant protein in this region became highly flexible and disordered, supporting mutant-induced β-hairpin disruption in L22 (rplV). We used principal component analysis (PCA)46 to calculate the protein backbone motion in the MD trajectory. This analysis revealed that the mutant insertion residues were unstructured and underwent large changes, breaking the β-hairpin formation (Figure 4B). The docking study indicated that the tylosin-binding site interacted with the mycinose moiety with the L22 β-hairpin. An apparent disruption of the conformation of the extended β-hairpin might directly influence tylosin binding. In addition, this hairpin contributed to the formation of part of the ribosomal tunnel wall for the exit of the nascent peptide (Figure 4C and D) along with 23S rRNA and ribosomal protein L22. The L22 mutant could change the surface properties or perturb the three-dimensional structure of the tunnel wall. Therefore, it might influence translation by regulating the exit of nascent peptides. Taken together, the L22 (rplV) 97KRTSAIN98 insertion mutant has double effects on the tylosin resistance. (1) Breaking the interaction of the tylosin mycinose moiety with the L22 β-hairpin. (2) Altering the structure of the exit tunnel wall. In contrast, as no direct contact was found between L22 and florfenicol, the resistance most likely occurs indirectly via perturbation of the exit tunnel wall. Therefore, the MIC of mutant strains against florfenicol did not return to the level of resistant strains (0.5 µg/mL to 2 µg/mLin L22 (rplV) mutant).
 |
Figure 4 L22 resistance mechanism. (A) Secondary structure changes of the L22 β-hairpin during MD simulations in the wild type (upper panel) and 97KRTSAIN98 insertion mutant (lower panel). (B) Intrinsic mobility of the L22 mutant calculated from PCA analysis. The lengths of the green vectors scale with the domain motions. (C and D) Structure of the nascent polypeptide in the exit tunnel. The model of the nascent polypeptide chain is shown in red. The structure of the ribosome shown comprises the 23S rRNA and the homology model of L4 and L22 mutants. The 23S rRNA is shown in gray, the antibiotic-binding site in cyan, L4 in green, and L22 in orange. Wild-type L22 is represented as a white cartoon (D). |
Figure 4 L22 resistance mechanism. (A) Secondary structure changes of the L22 β-hairpin during MD simulations in the wild type (upper panel) and 97KRTSAIN98 insertion mutant (lower panel). (B) Intrinsic mobility of the L22 mutant calculated from PCA analysis. The lengths of the green vectors scale with the domain motions. (C and D) Structure of the nascent polypeptide in the exit tunnel. The model of the nascent polypeptide chain is shown in red. The structure of the ribosome shown comprises the 23S rRNA and the homology model of L4 and L22 mutants. The 23S rRNA is shown in gray, the antibiotic-binding site in cyan, L4 in green, and L22 in orange. Wild-type L22 is represented as a white cartoon (D).
To further investigate the role of mutations in L3 and L4, we constructed three-dimensional structures of L3 and L3 (rplC) mutant, and L4 and L4 (rplD) mutant based on homologous structure from S. aureus (PDB ID: 4WCE), which shares 90% sequence identity. Comparing these modeled structures, no structural difference between wild-type and mutant was evident (Supplementary Figure 6A and B). In particular, none of the mutated amino acids in the modeled structures was adjacent to the tylosin- and florfenicol-binding pockets or to the exit tunnel of nascent peptide (Supplementary Figure 6C), indicating that these mutations do not alter the binding of these two antibiotics and disrupt the progression of the nascent peptide.
Antibiotic resistance might not only depend on mutations in ribosomal proteins but might also be influenced by the interaction protein network.47 Our previous study showed that proteins related to stress-response and transcription in the tylosin resistant S. xylosus markedly changed at mRNA level. We concluded that during the evolution of tylosin and florfenicol resistance, expression of the above resistant proteins may change at mRNA level. In order to identify the relationship of the resistant proteins to the evolution of tylosin-resistance in S. xylosus, the involved proteins have been probed at the mRNA level. As shown in Figure 5, the mRNA level of rplW (50S ribosomal protein L23), aldA (aldehyde dehydrogenase), trxA (thioredoxin), gros (chaperonin) gradually increased during the evolution of tylosin-resistance in S. xylosus while the mRNA level of ldh (lactate dehydrogenase) was reduced. These results confirmed the influence of the corresponding proteins on the evolution of tylosin resistance in S. xylosus.
 |
Figure 5 Changes in resistant proteins at mRNA level of different tylosin-resistance levels in S. xylosus, including thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh), and chloramphenicol-resistant protein (cl). |
Figure 5 Changes in resistant proteins at mRNA level of different tylosin-resistance levels in S. xylosus, including thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh), and chloramphenicol-resistant protein (cl).
To explore the relationship of mutations in the ribosomal proteins for the development of tylosin-resistance in S. xylosus further, we determined the mRNA level of resistant proteins in the L22 mutant of S. xylosus. The results (Figure 6) showed that the mRNA level of the resistant proteins, including thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh) and chloramphenicol resistant protein (cl) in the L22 mutant strain changed significantly, demonstrating that the resistant proteins were regulated by ribosomal protein L22.
 |
Figure 6 Changes in resistant proteins at mRNA level of L22 (rplV) mutant S. xylosus, including thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh), and chloramphenicol-resistant protein (cl). |
Figure 6 Changes in resistant proteins at mRNA level of L22 (rplV) mutant S. xylosus, including thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh), and chloramphenicol-resistant protein (cl).
In summary, our study strongly suggested that tylosin-resistant S. xylosus cross-resistance to florfenicol was related to ribosomal protein L22. Computational analysis fostered this proposition, revealing atomic details for the action of the two antibiotics (Figure 7): Tylosin was bound to the ribosomal polypeptide exit tunnel adjacent to the PTC, whereas florfenicol was bound to the PTC blocking A-site. The evolution of increasing resistance of S. xylosus against tylosin and florfenicol was related to the interaction protein network, which included thioredoxin (trxA), 50S ribosomal protein L23 (rplW), aldehyde dehydrgenase (aldA-1), chaperonin (gros), L-lactic dehydrogenase (ldh) and chloramphenicol-resistant protein (cl). Our work provides insightful views into the molecular mechanism of S. xylosus resistance that could be useful for the development of next-generation antibiotics.
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Figure 7 The drug-resistance mechanism of tylosin and florfenicol. Florfenicol (gray and red spheres) binds to the deep narrow pocket of the nascent peptide exit tunnel, which is far away from the β-hairpin of L22. A loop (in cyan) inserted into the β-hairpin of L22 has limited influence on the drug resistance of florfenicol. Tylosin (blue and red spheres), which is much larger than florfenicol, binds to the entrance of the nascent peptide exit tunnel. It closes into the β-hairpin of L22. The insertion of additional amino acid (in cyan) shows great impact on the drug resistance of tylosin. |
Figure 7 The drug-resistance mechanism of tylosin and florfenicol. Florfenicol (gray and red spheres) binds to the deep narrow pocket of the nascent peptide exit tunnel, which is far away from the β-hairpin of L22. A loop (in cyan) inserted into the β-hairpin of L22 has limited influence on the drug resistance of florfenicol. Tylosin (blue and red spheres), which is much larger than florfenicol, binds to the entrance of the nascent peptide exit tunnel. It closes into the β-hairpin of L22. The insertion of additional amino acid (in cyan) shows great impact on the drug resistance of tylosin.
We appreciate financial support from the Science and Technology Foundation of Guizhou Province through the Qianke He Foundation (ZK[2021]General 083). The work of Yanhua Li was supported by China Agriculture Research System and National Key Research and Development Program of China (2018YFD0500300) and National Nature Science Foundation of China (31772787). The work of Shuguang Yuan was supported by funding from Chinese Academy of Sciences Shenzhen Institutes of Advanced Technology (JCYJ20200109114818703) and Guangdong Province (2019QN01Y306).
The authors declare no competing interests.
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