Wza gene knockout decreases Acinetobacter baumannii virulence and affects Wzy-dependent capsular polysaccharide
Tianshui Niu1,2, Lihua Guo1, Qixia Luo1
1Collaborative Initiative Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis & Treatment of Infectious Diseases, the First Affiliated Hospital, college of Medicine, Zhejiang University, Hangzhou, China.
Abstract:
To investigate the virulence of capsular polysaccharide export protein (Wza) in carbapenem-resistant Acinetobacter baumannii and its effect on capsule formation.wza gene knockout and complementation strains were constructed, and changes in bacterial virulence were observed using in vitro adhesion, antiserum complement killing, anti-oxidation experiments, and infections in Galleria mellonella and mice. The effect of wza knockout on the genes wzb and wzc and wzi were assessed by RT-PCR.We successfully constructed wza knockout and complementation strains. Compared with wild-type (WT) strains, wza knockout strains displayed lower adhesion to A549 cells (p = 0.044), lower antiserum complement killing ability (p = 0.001), and lower mortality of G. mellonella (p = 0.010) and mice (p = 0.033). Expression levels of wzb, wzc and wzi were decreased in wza knockout strains. The antioxidant capacity of Wza knockout bacteria was only slightly decreased. Complementation of the wza gene returned the adhesion ability, antiserum complement killing ability, and mortality of G. mellonella and mice to WT levels. Expression of wzb, wzc and wzi was also returned to WT levels following wza complementation.The results clearly demonstrate that Wza is toxic. Wza affects the expression of other proteins of the Wzy capsule polysaccharide synthesis pathway, which affects the assembly, export, and extracellular fixation of capsular polysaccharide, resulting in synergistic effects that decrease bacterial virulence.
Insights
The capsular polysaccharide export protein (Wza) in carbapenem-resistant Acinetobacter baumannii significantly impacts bacterial virulence. Knocking out the wza gene reduces virulence, affecting capsule formation and gene expression, with complementation restoring these traits.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Carbapenem-resistant *Acinetobacter baumannii* (CRAB) poses a significant threat due to its multidrug resistance.
- Capsular polysaccharides play a crucial role in bacterial virulence and immune evasion.
- The specific role of the capsular polysaccharide export protein (Wza) in CRAB virulence remains to be fully elucidated.
Purpose of the Study:
- To investigate the virulence of the capsular polysaccharide export protein (Wza) in CRAB.
- To determine the effect of Wza on capsule formation and its contribution to CRAB pathogenicity.
- To analyze the impact of Wza on the expression of other genes involved in the Wzy capsule polysaccharide synthesis pathway.
Main Methods:
- Construction of *wza* gene knockout and complementation strains of CRAB.
- Assessment of bacterial virulence through *in vitro* adhesion assays (A549 cells), antiserum complement killing, and anti-oxidation experiments.
- Evaluation of *in vivo* pathogenicity using *Galleria mellonella* and mouse infection models.
- Analysis of *wzb*, *wzc*, and *wzi* gene expression levels using RT-PCR.
Main Results:
- *wza* knockout strains exhibited significantly reduced adhesion to A549 cells, lower antiserum complement killing ability, and decreased mortality in *G. mellonella* and mouse models compared to wild-type (WT) strains.
- Expression of *wzb*, *wzc*, and *wzi* genes was significantly downregulated in *wza* knockout strains.
- Complementation of the *wza* gene restored virulence, adhesion, complement killing ability, and gene expression to WT levels.
- Antioxidant capacity was only slightly decreased in *wza* knockout bacteria.
Conclusions:
- Wza is a critical determinant of CRAB virulence, contributing to adhesion, immune evasion, and overall pathogenicity.
- Wza influences the expression of other genes within the Wzy capsule polysaccharide synthesis pathway, affecting capsule assembly, export, and extracellular fixation.
- Targeting Wza presents a potential strategy for combating CRAB infections by attenuating its virulence.
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