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Profilin is required for sustaining efficient intra- and intercellular spreading of Shigella flexneri
H Mimuro1, T Suzuki, S Suetsugu
1Division of Bacterial Infection, Department of Microbiology and Immunology, Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Abstract:
The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-l-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Deltap) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Deltap but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Deltap reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.
Insights
Profilin I and neural Wiskott-Aldrich syndrome protein (N-WASP) are crucial for Shigella's actin-based motility and spread within host cells. Disrupting their interaction significantly impairs bacterial movement and infection progression.
Area of Science:
- Microbiology
- Cell Biology
- Infectious Diseases
Background:
- Shigella causes bacillary dysentery through actin-based motility within host cells.
- This motility relies on the interaction between VirG and neural Wiskott-Aldrich syndrome protein (N-WASP), which recruits the Arp2/3 complex.
- Host cell factors are critical for pathogen dissemination.
Purpose of the Study:
- To investigate the role of profilin I in Shigella's actin-based motility.
- To determine the critical interactions between profilin I, N-WASP, and G-actin in bacterial pathogenesis.
- To elucidate the contribution of profilin I-N-WASP complex to Shigella's intra- and intercellular spread.
Main Methods:
- Utilized COS-7 cells overexpressing wild-type and mutant profilin I (H119E, H133S) and N-WASP (Deltap).
- Performed Xenopus egg extract assays with depleted profilin and N-WASP.
- Conducted plaque formation assays in Madin-Darby canine kidney cell monolayers.
Main Results:
- Profilin I's interaction with G-actin and N-WASP is essential for Shigella motility.
- Mutants of profilin I (H119E, H133S) and N-WASP (Deltap) significantly inhibited bacterial motility and actin tail formation.
- Depletion and rescue experiments confirmed profilin I's necessity, while N-WASP's proline-rich domain is vital for profilin binding.
- Impaired host cell factors reduced Shigella's cell-to-cell spreading.
Conclusions:
- Profilin I, in association with N-WASP, is an essential host factor for Shigella pathogenesis.
- The interaction between profilin I and N-WASP is critical for efficient intra- and intercellular spread of Shigella.
- Targeting this host-pathogen interaction could offer therapeutic strategies against Shigella infections.