Guangzhou Health Center revealed the molecular mechanism of bacterial type VI. secretion effector protein RhsP targeting prey cells

Recently, the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences and the University of Macau jointly published a report entitled Vibrio parahaemolyticus prey targeting requires autoproteolysis-triggered dimerization of the type VI secretion in Cell Reports online Research paper by system effector RhsP.

The study found that the effector protein RhsP of Vibrio enteritidis formed a barrel-like structure, which caused obvious conformational changes in the vir (VgrG2-interacting region) peptide in the barrel through self-hydrolysis, and further proved that this conformational change promoted the formation of RhsP dimer and released nuclease toxins, suggesting that RhsP targets prey cells by self-hydrolysis.
This paper provides a molecular mechanism and theoretical basis
for the development of potential new anti-infective methods.

Type VI secretion system (T6SS) is widely found in gram-negative bacteria, and its structure is similar to the phage tail tube, mainly by mounting and secreting different toxin effector proteins to attack prey cells and gain a competitive advantage
in the flora.
Among them, the Rhs family protein is a large class of effector proteins, which cut the DNA of prey cells through its C-terminal nuclease toxin to achieve the purpose of
killing prey.
At the same time, downstream of the toxin effector protein genome is often accompanied by the expression of immune factors that can neutralize their toxicity, which also allows bacteria carrying Rhs toxin to protect themselves from being affected
.
The molecular mechanism of how T6SS secretes large effector proteins such as Rhs has always been a hot spot and difficult point in the field, and the process of how Rhs family effector proteins release nuclease toxins has not been clearly analyzed
.

In this study, a self-hydrolyzed T6SS effector protein RhsP was first discovered in Vibrio enteritidis, and further biochemical experiments found that RhsP was self-hydrolyzed into three fragments, the N-terminus, the Rhs barrel and the C-terminus with nuclease toxicity (Figure 1A).

To address the effect of this process of autohydrolysis on RhsP secretion and nuclease toxin release, the team used single-particle cryo-EM to obtain high-resolution structures of RhsP after self-hydrolysis (cleaved RhsP) and before RhsP self-hydrolysis (Uncleaved RhsP) (Figure 1B).

After analyzing the high-resolution structure of RhsP before and after hydrolysis, it was found that a closed barrel-like structure
was formed in the Rhs region.
The N-terminal peptide extends from the top gap in the barrel lid, indicating the exit trajectory after N-terminal hydrolysis (Figure 2A).

In addition, the VIR after hydrolysis undergoes a dramatic change in conformation, stretching in a U-shape inside the barrel and sending the VIR-C end containing nuclease toxins out to the gap caused by the upward movement of the barrel lid (Figure 2B).

Finally, through structural analysis, VIR combined with the hydrophobic inner surface of the β-sheet was found, and the F1208A/Y1209A mutant strain at the VIR-C terminal was constructed to verify that RhsP induced VIR conformational changes after hydrolysis to promote the formation
of dimers.

In this study, the instantaneous conformational changes of effector proteins before and after self-hydrolysis were captured by biochemical experiments and cryo-EM, and it was found that the self-hydrolysis process of RhsP triggered conformational changes in the barrel-like structure of Rhs, revealed the molecular mechanism of releasing nuclease toxins, and also found that the formation of RhsP dimers promoted by conformational changes in the VIR region was a key step in targeting prey cells (Figure 3).

Tang Le, a postdoctoral fellow at the University of Macau, Dong Shuqi, a doctoral student at the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Nadia Rasheed, a postdoctoral fellow at the University of Macau, and Hu Qiaoying, a doctoral student, and Zhou Ningkun, a research assistant at Bio Island Laboratory, are the co-first authors
of this paper.
Professor William Chong Hang Chao of the University of Macau, Professor He Jun of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, and Professor Zheng Jun of the University of Macau are co-corresponding authors
of this paper.
The research was supported
by the University of Macau, the Science and Technology Development Fund of the Macao Special Administrative Region, the National Natural Science Foundation of China, and the Natural Science Foundation of Guangdong Province.

Paper link

Figure 1 A: Schematic diagram of the domain of toxin effector protein Rhs P, B: RhsP structure diagram of high-resolution protein before and after hydrolysis

Figure 2 A: N-terminal peptide trajectory map of RhsP before and after hydrolysis, B: Vir conformational change map of RhsP before and after hydrolysis

Figure 3 Step-by-step model of RhsP self-hydrolysis-induced dimer formation and targeting of prey cells