Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.

Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation.

Actin filament assembly at the cell surface of the pathogenic bacterium Listeria monocytogenes requires the bacterial ActA surface protein and the host cell Arp2/3 complex. Purified Arp2/3 complex accelerated the nucleation of actin polymerization in vitro, but pure ActA had no effect. However, when combined, the Arp2/3 complex and ActA synergistically stimulated the nucleation of actin filaments. This mechanism of activating the host Arp2/3 complex at the L. monocytogenes surface may be similar to the strategy used by cells to control Arp2/3 complex activity and hence the spatial and temporal distribution of actin polymerization.

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.

Heterogeneity of the vanA gene cluster in clinical isolates of enterococci from the northeastern United States.

In several strains of Enterococcus faecium isolated in Europe, the cluster of genes encoding high-level resistance to vancomycin (VanA phenotype) resides on a 10.85-kb transposon, Tn1546, or closely related elements. To determine whether Tn1546 was conserved in recent enterococcal isolates from the northeastern United States, seven strains were compared by restriction mapping and DNA hybridization with probes from within the van cluster. Two of the seven strains contained intact Tn1546-like sequences; however, in five of the strains, the organization of the van cluster differed from that of Tn1546. Three of the five strains with variations harbored a novel DNA segment within the van gene cluster. This 1,496-bp segment was similar to IS1165 of Leuconostoc mesenteroides and IS1181 of Staphylococcus aureus and was flanked by 24- and 23-bp imperfect inverted repeats and 8-bp direct repeats. On the basis of these findings, we propose that this element comprises a novel insertion-like sequence, IS1251. Multiple copies of IS1251 were also present at other sites in both resistant and susceptible clinical isolates. Our findings suggest that the van cluster in recent isolates from the northeastern United States differs from that present in the early European VanA phenotype strains.

Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.

Direct observation of microtubule-f-actin interaction in cell free lysates.

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.