Proteomic and Systematic Functional Profiling Unveils Citral Targeting Antibiotic Resistance, Antioxidant Defense, and Biofilm-Associated Two-Component Systems of Acinetobacter baumannii To Encumber Biofilm and Virulence Traits

Abstract

Acinetobacter baumannii has been reported as a multidrug-resistant bacterium due to biofilms and antimicrobial resistance mechanisms. Hence, novel therapeutic strategies are necessary to overcome A. baumannii infections. This study revealed that citral at 200 μg/ml attenuated A. baumannii biofilms by up to 90% without affecting viability. Furthermore, microscopic analyses and in vitro assays confirmed the antibiofilm efficacy of citral. The global effect of citral on A. baumannii was evaluated by proteomic, transcriptional, and in silico approaches. Two-dimensional (2D) gel electrophoresis and matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) analyses were used to assess the effect of citral on the A. baumannii cellular proteome. Quantitative real-time PCR (qPCR) analysis was done to validate the proteomic data and identify the differentially expressed A. baumannii genes. Protein-protein interactions, gene enrichment, and comparative gene network analyses were performed to explore the interactions and functional attributes of differentially expressed proteins of A. baumannii. Global omics-based analyses revealed that citral targeted various mechanisms such as biofilm formation, antibiotic resistance, antioxidant defense, iron acquisition, and type II and type IV secretion systems. The results of antioxidant analyses and antibiotic sensitivity, blood survival, lipase, and hemolysis assays validated the proteomic results. Cytotoxicity analysis showed a nontoxic effect of citral on peripheral blood mononuclear cells (PBMCs). Overall, the current study unveiled that citral has multitarget efficacy to inhibit the biofilm formation and virulence of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial-infection-causing bacterium and also possesses multidrug resistance to a wide range of conventional antibiotics. The biofilm-forming ability of A. baumannii plays a major role in its resistance and persistence. There is an alarming need for novel treatment strategies to control A. baumannii biofilm-associated issues. The present study demonstrated the strong antibiofilm and antivirulence efficacy of citral against A. baumannii. In addition, proteomic analysis revealed the multitarget potential of citral against A. baumannii. Furthermore, citral treatment enhances the susceptibility of A. baumannii to the host innate immune system and reactive oxygen species (ROS). Cytotoxicity analysis revealed the nonfatal effect of citral on human PBMCs. Therefore, citral could be the safest therapeutic compound and can be taken for further clinical evaluation for the treatment of biofilm-associated infections by A. baumannii.

Polyphosphate Kinase Is Required for the Processes of Virulence and Persistence in Acinetobacter baumannii

“…In recent years, A. baumannii has aroused widespread concern due to its virulence and persistence ( 1 ). The pathogenicity of A. baumannii is mediated mainly by several vital virulence factors that are associated with motility ( 2 ) and adhesion to and invasion of host cells ( 3 ) that collectively enable A. baumannii to successfully infect its host. Regarding drug resistance, A. baumannii is also considered an emerging health threat due to its multidrug-resistant characteristics ( 4 ).…”

Anti-Virulence Properties of Plant Species: Correlation between In Vitro Activity and Efficacy in a Murine Model of Bacterial Infection

“…In this regard, it was reported that various derivatives of phenolic compounds control ExoS expression via the interference of a TCS called GacS-GacA [25]. Additionally, in a global analysis based on omics, it was found that citral (essential oil) acts through several mechanisms to reduce virulence (QS-regulated factors, type II and type IV secretion systems) of A. baumannii [28].…”

“…In most studies evaluating anti-virulence activity with substances of plant origin, inhibition of pigment biosynthesis regulated by QS, such as violacein in Chromobacterium violaceum and pyocyanin in P. aeruginosa, has been commonly used [17][18][19]. To a lesser extent, other relevant targets such as type III secretion systems (T3SSs) [20,21], key enzymes [22], toxins [23], or two-component systems, among others, have also been evaluated [24][25][26][27][28].…”

Non-antibiotic prevention and treatment against Acinetobacter baumannii infection: Are vaccines and adjuvants effective strategies?

“…Citral, a mixture of monoterpene aldehydes from plants, has no bactericidal effect but a strong detoxification effect with naturally safe annon-toxic, which can reduce the toxicity of A. baumannii biofilm by 90%. Citral is targeted to various mechanisms, such as biofilm formation, antibiotic resistance, antioxidant defense, iron acquisition, and type II and type IV secretion systems enhance the sensitivity of A. baumannii to the host innate immune system and reactive oxygen species (ROS) (Selvaraj et al, 2020 ).…”