Time-resolved proteomic adaptation of multidrug-resistant <i>Acinetobacter baumannii</i> to antimicrobial stress induced by partially purified fraction from <i>Caesalpinia pulcherrima</i> flower using DEqMS

ABSTRACT The global prevalence of multidrug-resistant (MDR) bacteria represents an urgent public health challenge, emphasizing the critical need for novel antimicrobial agents. MDR Acinetobacter baumannii , a nosocomial pathogen of critical global concern owing to its capacity to acquire and disseminate antimicrobial resistance, was employed as a bacterial model to investigate the antimicrobial potential of natural products derived from Caesalpinia pulcherrima (L.) Sw. (Fabaceae). This medicinal plant represents a promising reservoir of novel bioactive compounds; however, its molecular effects on the A. baumannii proteome had not previously been characterized. The partially purified ethyl acetate fraction of C. pulcherrima flowers (CPF4) exhibited potent bactericidal activity against susceptible A. baumannii (minimum inhibitory concentration and minimum bactericidal concentration = 31.25 µg/mL), and time-resolved label-free quantitative LC-MS/MS proteomics was subsequently performed on MDR A. baumannii cultures treated with CPF4 at 24 h, 48 h, and 72 h post-treatment alongside untreated controls in biological triplicate, with differential protein expression assessed using differential expression of quantified mass spectrometry data. No significantly differentially expressed proteins were detected at 24 h or 48 h relative to the control, indicating that the proteomic effects of CPF4 manifest predominantly at the late treatment stage. In contrast, a robust late-phase response was identified at 72 h, comprising the coordinated induction of proteins associated with DNA damage repair, transcriptional regulation, and cell surface glycosylation remodeling. The sensor histidine kinase PmrB was significantly upregulated at 72 h vs 48 h (adjusted P = 0.029), implicating the PmrA/PmrB two-component system in late-phase colistin tolerance acquisition under sustained CPF4 exposure. IMPORTANCE These findings provide mechanistic insight into the adaptive survival strategies employed by multidrug-resistant Acinetobacter baumannii in response to plant-derived antimicrobial challenge and support the further development of Caesalpinia pulcherrima -derived natural products as candidate antimicrobial agents.