Advancements in studies on the evolutionary mechanisms underlying bacterial antibiotic resistance are unclear. This study aimed to investigate the evolutionary mechanism underlying bacterial antibiotic resistance using isobaric tags for relative and absolute quantitationbased quantitative proteomics along with functional validation. Quantitative analysis revealed 101, 325, and 428 differentially expressed proteins (DEPs) at three drug resistance levels (low-R, 0.2 μg/mL; medium-R, 5 μg/mL; high-R, 15 μg/mL). Continuous adjustment of metabolic patterns to enhance nucleotide synthesis and energy generation may underlie evolution. Indeed, nucleotide levels were elevated and strengthened ciprofloxacin resistance. Quorum sensing (QS) genes were upregulated in the early growth phase, thus potentially improving survival. Further, a thicker cell wall potentially serves as a stronger barrier and reduces drug permeation. The aforementioned three drug resistance levels displayed continuity and differences; the low-resistant level displayed no prominent mechanism; medium, a more focused change in nucleotide metabolism; and high, a thorough evolution to a complete systematic mechanism with higher adenosine 5′-triphosphate levels, serving as a defense mechanism for reducing drug-induced stress. Thus, gradual increments in nucleotide synthesis, energy synthesis, cell wall synthesis, QS, and biofilm formation may direct the evolution of bacterial resistance.

Streptococcus pneumoniae, a Gram-positive human pathogen, causes a series of serious diseases in humans. SPD_1495 from S. pneumoniae is annotated as a hypothetical ABC sugar-binding protein in the NCBI database, but there are few reports on detailed biological functions of SPD_1495. To fully study the influence of SPD_1495 on bacterial virulence in S. pneumoniae, we constructed a deletion mutant (D39Δspd1495) and an overexpressing strain (D39spd1495+). Comparative analysis of iTRAQ-based quantitative proteomic data of the wild-type D39 strain (D39-WT) and D39Δspd1495 showed that several differentially expressed proteins that participate in capsular polysaccharide synthesis, such as Cps2M, Cps2C, Cps2L, Cps2T, Cps2E, and Cps2D, were markedly upregulated in D39Δspd1495. Subsequent transmission electron microscopy and uronic acid detection assay confirmed that capsular polysaccharide synthesis was enhanced in D39Δspd1495 compared to that in D39-WT. Moreover, knockout of spd1495 resulted in increased capsular polysaccharide synthesis, as well as increased bacterial virulence, as confirmed by the animal study. Through a coimmunoprecipitation assay, surface plasmon resonance, and electrophoretic mobility shift assay, we found that SPD_1495 negatively regulated cps promoter expression by interacting with phosphorylated ComE, a negative transcriptional regulator for capsular polysaccharide formation. Overall, this study suggested that SPD_1495 negatively regulates capsular polysaccharide formation and thereby enhances bacterial virulence in the host. These findings also provide valuable insights into understanding the biology of this clinically important bacterium. IMPORTANCE Capsular polysaccharide is a key factor underlying the virulence of Streptococcus pneumoniae in human diseases. Thus, a deep understanding of capsular polysaccharide synthesis is essential for uncovering the pathogenesis of S. pneumoniae infection. In this study, we show that protein SPD_1495 interacts with phosphorylated ComE to negatively regulate the formation of capsular polysaccharide. Deletion of spd1495 increased capsular polysaccharide synthesis and thereby enhanced bacterial virulence. These findings further reveal the synthesis mechanism of capsular polysaccharide and provide new insight into the biology of this clinically important bacterium.

Infections caused by drug-resistant bacteria are a serious problem worldwide. Therefore, there is an urgent need to find novel drugs with good antibacterial activity against multidrug-resistant bacteria.

The unreasonable misuse of antibiotics has led to the emergence of large-scale drug-resistant bacteria, seriously threatening human health. Compared with drug-sensitive bacteria, resistant bacteria are difficult to clear by host immunity. To fully explore the adaptive mechanism of resistant bacteria to the iron-restricted environment, we performed data-independent acquisition-based quantitative proteomics on ciprofloxacin (CIP)-resistant (CIP-R) Staphylococcus aureus in the presence or absence of iron. On bioinformatics analysis, CIP-R bacteria showed stronger amino acid synthesis and energy storage ability. Notably, CIP-R bacteria increased virulence by upregulating the expression of many virulence-related proteins and enhancing the synthesis of virulence-related amino acids under iron-restricted stress. This study will help us to further explain the adaptive mechanisms that lead to bacterial resistance to antibiotics depending on the host environment and provide insights into the development of novel drugs for the treatment of drug-resistant bacterial infections.

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