Overexpression of BIT33_RS14560 Enhances the Biofilm Formation and Virulence of Acinetobacter baumannii

Ruifu Yang; Bipeng Lai; Kang Liao; Baomo Liu; Lixia Huang; Shaoli Li; Jincui Gu; Ziying Lin; Yili Chen; Shuaishuai Wang; Yanli Qiu; Jiating Deng; Simin Chen; Chao Zhuo; Yanbin Zhou

doi:10.3389/fmicb.2022.867770

Overexpression of BIT33_RS14560 Enhances the Biofilm Formation and Virulence of Acinetobacter baumannii

Front Microbiol. 2022 Apr 25:13:867770. doi: 10.3389/fmicb.2022.867770. eCollection 2022.

Authors

Ruifu Yang¹, Bipeng Lai¹, Kang Liao², Baomo Liu¹, Lixia Huang¹, Shaoli Li¹, Jincui Gu¹, Ziying Lin¹, Yili Chen², Shuaishuai Wang¹, Yanli Qiu¹, Jiating Deng¹, Simin Chen¹, Chao Zhuo³, Yanbin Zhou¹

Affiliations

¹ Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
² Department of Clinical Laboratory, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
³ State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Abstract

Acinetobacter baumannii, a strictly aerobic, non-lactose fermented Gram-negative bacteria, is one of the important pathogens of nosocomial infection. Major facilitator superfamily (MFS) transporter membrane proteins are a class of proteins that widely exists in microbial genomes and have been revealed to be related to biofilm formation in a variety of microorganisms. However, as one of the MFS transporter membrane proteins, little is known about the role of BIT33_RS14560 in A. baumannii. To explore the effects of BIT33_RS14560 on biofilm formation of A. baumannii, the biofilm formation abilities of 62 isolates were firstly investigated and compared with their transcript levels of BIT33_RS14560. Then, this specific gene was over-expressed in a standard A. baumannii strain (ATCC 19606) and two isolates of extensively drug-resistant A. baumannii (XDR-Ab). Bacterial virulence was observed using a Galleria mellonella infection model. High-throughput transcriptome sequencing (RNA seq) was performed on ATCC 19606 over-expressed strain and its corresponding empty plasmid control strain. Spearman's correlation analysis indicated a significant negative correlation (R = -0.569, p = 0.000) between the △CT levels of BIT33_RS1456 and biofilm grading of A. baumannii isolates. The amount of A. baumannii biofilm was relatively high within 12-48 h. Regardless of standard or clinical strains; the biofilm biomass in the BIT33_RS14560 overexpression group was significantly higher than that in the control group ( p < 0.0001). Kaplan-Meier survival curve analysis showed that the mortality of G. mellonella was significantly higher when infected with the BIT33_RS14560 overexpression strain (χ² = 8.462, p = 0.004). RNA-Seq showed that the mRNA expression levels of three genes annotated as OprD family outer membrane porin, glycosyltransferase family 39 protein, and glycosyltransferase family 2 protein, which were related to bacterial adhesion, biofilm formation, and virulence, were significantly upregulated when BIT33_RS14560 was over-expressed. Our findings provided new insights in identifying potential drug targets for the inhibition of biofilm formation. We also developed a practical method to construct an over-expressed vector that can stably replicate in XDR-Ab isolates.

Keywords: Acinetobacter baumannii; BIT33_RS14560; RNA sequencing; biofilm; extensively drug-resistant; overexpression.