Actin-based motility is sufficient for bacterial membrane protrusion formation and host cell uptake

Abstract

SummaryShigella flexneri replicates in the cytoplasm of host cells, where it nucleates host cell actin filaments at one pole of the bacterial cell to form a`comet tail' that propels the bacterium through the host's cytoplasm. To determine whether the ability to move by actinbased motility is sufficient for subsequent formation of membrane-bound protrusions and intercellular spread, we conferred the ability to nucleate actin on a heterologous bacterium, Escherichia coli. Previous work has shown that IcsA (VirG), the molecule that is necessary and sufficient for actin nucleation and actin-based motility, is distributed in a unipolar fashion on the surface of S. flexneri. Maintenance of the unipolar distribution of IcsA depends on both the S. flexneri outer membrane protease IcsP (SopA) and the structure of the lipopolysaccharide (LPS) in the outer membrane. We co-expressed IcsA and IcsP in two strains of E. coli that differed in their LPS structures. The E. coli were engineered to invade host cells by expression of invasin from Yersinia pseudotuberculosis and to escape the phagosome by incubation in purified listeriolysin O (LLO) from Listeria monocytogenes. All E. coli strains expressing IcsA replicated in host cell cytoplasm and moved by actin-based motility. Actin-based motility alone was sufficient for the formation of membrane protrusions and uptake by recipient host cells. The presence of IcsP and an elaborate LPS structure combined to enhance the ability of E. coli to form protrusions at the same frequency as S. flexneri, quantitatively reconstituting this step in pathogen intercellular spread in a heterologous organism. The frequency of membrane protrusion formation across all strains tested correlates with the efficiency of unidirectional actin-based movement, but not with bacterial speed.

Abl Kinases Regulate Actin Comet Tail Elongation via an N-WASP-Dependent Pathway

“…The protocol for time-lapse microscopy and quantification of bacterial velocity was designed based on previous reports (27,28). Cells were plated on 35-mm cover glass bottom dishes (Mat-Tek), infected with GFP-expressing Shigella as previously described (24,42), and maintained on the microscope in a 37°C chamber with humidified 5% CO 2 .…”

Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell Phagosomes

“…Cells were 60 -80% confluent on the day of infection. Infections were performed with L. monocytogenes strains 10403S (wild-type) (Bishop and Hinrichs, 1987), DP-L973 (Sun et al, 1990), DP-L3078 ⌬actA (Skoble et al, 2000), DP-L2319 ⌬hly ⌬plcA ⌬plcB (Gedde et al, 2000;O'Riordan et al, 2002), and DP-L4032 GGG actA allele (Skoble et al, 2001); as well as E. coli (ATM379) expressing invasin from Y. pseudotuberculosis (Isberg and Falkow, 1985;Monack and Theriot, 2001). L. monocytogenes strains were provided by Dr. D. A. Portnoy (University of California, Berkeley, CA) and E. coli (invasin) was provided by Dr. D. M. Monack (Stanford University, Stanford, CA).…”

“…Cells were infected with L. monocytogenes and E. coli (invasin) as described previously (Robbins et al, 1999;Monack and Theriot, 2001). After 45-60 min at 37°C, 5% CO 2 , infected cells were washed three times with DMEM supplemented with 10% fetal bovine serum (FBS) (DMEM/FBS) to remove unattached bacteria, and fresh DMEM/FBS was added.…”

Listeria monocytogenesFlagella Are Used for Motility, Not as Adhesins, To Increase Host Cell Invasion

“…It would be advantageous for bacteria to interact head on with host cell mem-branes as internalin A, a major cell surface adhesin of L. monocytogenes, accumulates at the bacterial poles (19). In addition, the physical force generated by unidirectional movement may enhance host cell uptake, as was shown for intercellular spread of bacteria that use an actin-based mechanism of motility (15).…”