LcrV mutants that abolish Yersinia type III injectisome function.

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Pathogenesis

Background:

• LcrV is the type III needle cap protein in pathogenic Yersinia.
• It is proposed to link needle protein YscF with YopB-YopD to form the injectisome.
• The injectisome facilitates effector protein translocation into host cells.

Purpose of the Study:

• To investigate the role of the LcrV needle cap in responding to low-calcium signals.
• To test the hypothesis that LcrV promotes injectisome assembly upon sensing low calcium.
• To identify specific sites within LcrV critical for its function.

Main Methods:

• Genetic analysis of Yersinia pestis.
• Isolation of dominant-negative lcrV alleles (lcrV(*327)) using calcium-depleted growth conditions.
• Introduction of C-terminal tags (amino acids or Strep tag) to LcrV.
• Analysis of LcrV variants' effect on injectisome assembly and type III secretion.

Main Results:

• Modifying the C terminus of LcrV, such as adding a Strep tag, resulted in a low-calcium response-negative (LCR-) phenotype.
• These LcrV variants capped type III needles but failed to assemble the YopD component of the injectisome.
• The C-terminal Strep tag blocked effector transport, even in calcium-blind YscF variants.
• Mutants like LcrV(*327) were locked in a state unresponsive to low-calcium signals, halting injectisome assembly and type III secretion.
• Tag insertion at other LcrV positions yielded different phenotypes, pinpointing essential sites for secretion, capping, and assembly.

Conclusions:

• LcrV's C terminus is crucial for its response to low-calcium signals.
• LcrV acts as a critical sensor and regulator of injectisome assembly and type III secretion.
• Specific regions of LcrV dictate its function as a secretion substrate, needle cap, and assembly factor.