Adoption of an in-silico analysis approach to assess the functional and structural impacts of rpoB-encoded protein mutations on Chlamydia pneumoniae sensitivity to antibiotics

Abstract

Background: Antibiotics are frequently used to treat infections caused by Chlamydia pneumoniae; an obligate intracellular gram-negative bacterium commonly associated with respiratory diseases. However, improper or overuse of these drugs has raised concerns about the development of antibiotic resistance, which poses a significant global health challenge. Previous studies have revealed a link between mutations in the rpoB-encoded protein of C. pneumoniae and antibiotic resistance. This study assessed these mutations via various bioinformatics tools to predict their impact on function, structural stability, antibiotic binding, and, ultimately, their effect on bacterial sensitivity to antibiotics.

Results: Eight mutations in the rpoB-encoded protein (R421S, F450S, L456I, S454F, D461E, S476F, L478S, and S519Y) are associated with resistance to rifampin and rifalazil. These mutations occur in conserved regions of the protein, leading to decreased stability and affecting essential functional sites of RNA polymerase, the target of these antibiotics. Although the structural differences between the native and mutant proteins are minimal, notable changes in local hydrogen bonding have been observed. Despite similar binding energies, variations in hydrogen bonds and hydrophobic interactions in certain mutants (for instance, D461E for rifalazil and S476F for rifampin) indicate that these changes may diminish ligand affinity and specificity. Furthermore, protein-protein network analysis demonstrated a strong correlation between wild-type rpoB and ten C. pneumoniae proteins, each fulfilling specific functional roles. Consequently, some of these mutations can reduce the bacterium's sensitivity to rifampin and rifalazil, thereby contributing to antibiotic resistance.

Conclusion: The findings of this study indicate that mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase, are pivotal in the resistance of C. pneumoniae to rifampin and rifalazil. Some of these mutations may result in reduced protein stability and changes in the structure, function, and antibiotic binding. As a consequence, the efficacy of these drugs in inhibiting RNA polymerase is compromised, allowing the bacteria to persist in transcription and replication even in the presence of antibiotics. Overall, these insights enhance our understanding of the resistance mechanisms in C. pneumoniae and could guide the development of strategies to address this challenge.

Clinical trial number: Not applicable.

Keywords: Chlamydia pneumoniae; RpoB gene; Antibiotic; In Silico analysis; Mutations; Resistance.

Fig. 1

Study steps fowchart

Fig. 2

Prediction of changes in protein stability via the SAAFECSEQ and Dynamut2 tools

Fig. 3

[a] Comparison of rpoB-encoded protein hydrogen bonding, from the native to the mutant structure. a Wild type R and mutant S residues at 421th position (R421S). b Wild type F and mutant S residues at 450th position (F450S). c Wild type S and mutant F residues at 454th position (S454F). d Wild type L and mutant I residues at 456th position (L456I). [b] Comparison of Hydrogen Bonding in the rpoB-Encoded Protein Between the Native and Mutant Structures. e Wild type D and mutant E residues at 461th position (D461E). f Wild type S and mutant F residues at 476th position (S4767). g Wild type L and mutant S residues at 478th position (L478S). h Wild type S and mutant Y residues at 519th position (S519Y)

Fig. 4

Three-dimensional structures of the predicted mutant proteins, along with Ramachandran favorable regions and corresponding plots generated by MolProbity

Fig. 5

The superimposed structures of the mutant and wild-type amino acids of the rpoB-encoded protein at different positions. The native amino acid is red, and the mutant amino acid is green

Fig. 6

Binding energy (Kcal /Mol) trends for wild-type and mutant proteins

Fig. 7

2D representations of interactions between mutant models of rpoB-encoded protein and rifalazil and rifampin

Fig. 8

2D docking interaction representations of the wild-type rpoB -encoded protein with rifampin (a) and rifalazil (b)

Fig. 9

Overview of rpoB-encoded protein network construction via the STRING server. The evidence view and confidence view are given