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Role of Type VI Secretion System Core Proteins Hcp and VgrG in Antibiotic Resistance of Acinetobacter baumannii

ABSTRACT: The antimicrobial resistance of Acinetobacter baumannii (A. baumannii) is becoming increasingly severe. This study aimed to investigate the role and mechanism of the type VI secretion system (T6SS) core protein hemolysin coregulated protein (Hcp) and valine-glycine repeat protein G (VgrG) in antibiotic resistance regulation in Acinetobacter baumannii. Using ATCC 17978 as the parental strain, we constructed Δhcp, ΔvgrG3, and ΔhcpΔvgrG3 knockout mutants. Transcriptomic and proteomic analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation, and antibiotic susceptibility testing (AST) in the presence or absence of the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) were performed. The results showed that the Δhcp mutant primarily downregulated pathways related to amino acid metabolism, secretion systems, and biofilm formation, with no significant changes in the minimum inhibitory concentrations (MICs) against all tested antibiotics. In contrast, the ΔvgrG3 mutant significantly upregulated the resistance-nodulation-division (RND) efflux pump AdeIJK, as well as pathways associated with resistance to β-lactam antibiotics and cationic antimicrobial peptides, resulting in broadly elevated MICs. The double knockout mutant ΔhcpΔvgrG3 further downregulated adeN, leading to enhanced AdeIJK expression. It also synergistically regulated major facilitator superfamily (MFS) efflux pumps, and lipopolysaccharide biosynthesis, causing further increases in resistance to piperacillin/tazobactam, ceftazidime, cefepime, and meropenem. Treatment with the efflux pump inhibitor PAβN partially reversed the resistance phenotypes of the mutants. In conclusion, Hcp and VgrG3 exhibit distinct functional differentiation in the regulation of antibiotic resistance in A. baumannii. Hcp has a limited impact on resistance, whereas VgrG3 critically contributes to multidrug resistance by negatively regulating the expression of the AdeIJK efflux pump system.

ORGANISM(S): Acinetobacter Baumannii

SUBMITTER: Daojun Yu

PROVIDER: PXD078173 | iProX | Fri May 08 00:00:00 GMT+01:00 2026

REPOSITORIES: iProX

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Project description:Acinetobacter baumannii has emerged as one of the most problematic opportunist bacterial pathogen responsible for hospital-acquired and community infections worldwide. Besides its high capacities to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development, a lifestyle conferring an additional protection against various treatments, and allowing it to persist for long periods in various hospital niches. Due to their increasing resilience to antimicrobial treatments, A. baumannii biofilms are difficult to control and ultimately eradicate. Overcoming the significant challenges pose by A. baumannii require fundamental insights into mechanisms involved in the development of mature biofilm and thus, may help to develop novel strategies for biofilm prevention and control. To unravel critical determinants of this sessile lifestyle, we compared the proteome profiles of bacteria grown in planktonic stationary phase with those of two A. baumannii strains (ATCC 17978 and SDF), harboring specific features regarding biofilm formation, grown in mature solid-liquid (S-L) biofilm using a proteomic quantitative study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump appears to play a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm and contributes to maintain WT membrane rigidity as well as tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen facing and adapting to biofilm architecture which is heterogeneous in space and time, especially in mature biofilm. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

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Role of Type VI Secretion System Core Proteins Hcp and VgrG in Antibiotic Resistance of Acinetobacter baumannii

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