Dataset Information

Investigating the effect of an identified mutation within a critical site of PAS domain of WalK protein in a vancomycin-intermediate resistant Staphylococcus aureus by computational approaches.

ABSTRACT:

Background

Vancomycin-intermediate resistant Staphylococcus aureus (VISA) is becoming a common cause of nosocomial infections worldwide. VISA isolates are developed by unclear molecular mechanisms via mutations in several genes, including walKR. Although studies have verified some of these mutations, there are a few studies that pay attention to the importance of molecular modelling of mutations.

Method

For genomic and transcriptomic comparisons in a laboratory-derived VISA strain and its parental strain, Sanger sequencing and reverse transcriptase quantitative PCR (RT-qPCR) methods were used, respectively. After structural protein mapping of the detected mutation, mutation effects were analyzed using molecular computational approaches and crystal structures of related proteins.

Results

A mutation WalK-H364R was occurred in a functional zinc ion coordinating residue within the PAS domain in the VISA strain. WalK-H364R was predicted to destabilize protein and decrease WalK interactions with proteins and nucleic acids. The RT-qPCR method showed downregulation of walKR, WalKR-regulated autolysins, and agr locus.

Conclusion

Overall, WalK-H364R mutation within a critical metal-coordinating site was presumably related to the VISA development. We assume that the WalK-H364R mutation resulted in deleterious effects on protein, which was verified by walKR gene expression changes.. Therefore, molecular modelling provides detailed insight into the molecular mechanism of VISA development, in particular, where allelic replacement experiments are not readily available.

SUBMITTER: Baseri N

PROVIDER: S-EPMC8414773 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) isolates are considered to have emerged from VAN-susceptible S. aureus (VSSA) by spontaneous mutation during VAN exposure. We previously reported that laboratory mutant H14, obtained from VSSA strain Delta IP by exposure to imipenem (IPM), showed overexpression of the vraSR two-component system and a typical hVISA phenotype. In the present study, to elucidate the mechanism of VSSA conversion to hVISA, we further characterized strain H14 by determining its whole-genome sequence, morphology, cell wall synthetic activity, and gene expression. Genome sequencing revealed that H14 harbored a mutated vraS (designated vraS(H14)) that caused an amino acid substitution (S(329)-->L). This mutation is different from the VraS mutation (N(5)-->I) identified in representative clinical hVISA strain Mu3. However, H14 exhibited a phenotype similar to that of Mu3, including heterogeneous resistance to VAN, enhanced cell wall synthetic activity, and vraSR overexpression. Replacement of the vraS gene of DeltaIP with the mutated vraS(H14) gene confirmed that the S(329)-->L substitution was responsible for both the upregulation of vraSR and conversion to the hVISA phenotype. This conversion was also achieved by using the vraS gene of Mu3, which carries a mutation (N(5)-->I), but not with the native vraS gene of strain N315. Finally, we carried out a study to analyze the appearance of hVISA from VSSA by exposure of Delta IP to selective concentrations of VAN and beta-lactam antibiotics. A total of 8 and 5 hVISA isolates were detected among 50 isolates selected with VAN and IPM, respectively. Among the 13 hVISA mutants, mutation in vraSR was detected only in mutant strain H14, suggesting that additional mutational mechanisms can be responsible for evolution to the hVISA phenotype. We conclude that exposure not only to VAN but also to beta-lactams may select for reduced glycopeptide susceptibility in S. aureus.