Biomedical subjects

Daoguo Zhou

Publications and source records attributed to Daoguo Zhou.

10 recordsLinked to original sources

PubMed2006

Exo70 interacts with the Arp2/3 complex and regulates cell migration.

The exocyst is a multiprotein complex essential for tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. Here, we report that the exocyst component Exo70 interacts with the Arp2/3 complex, a key regulator of actin polymerization. We further show that the exocyst-Arp2/3 interaction is regulated by epidermal growth factor (EGF) signalling. Inhibition of Exo70 by RNA interference (RNAi) or antibody microinjection blocks the formation of actin-based membrane protrusions and affects various aspects of cell motility. We propose that Exo70, in addition to functioning in exocytosis, also regulates actin at the leading edges of migrating cells, therefore coordinating cytoskeleton and membrane traffic during cell migration.

Actin-Related Protein 2-3 Complex↗

PubMed2006

The inflammation-associated Salmonella SopA is a HECT-like E3 ubiquitin ligase.

Salmonella translocate a group of type III effectors into the host cells to induce entry, promote survival and cause intestinal inflammation. Although the biochemical and cellular mechanisms of how bacterial effectors function inside host cells remain largely unknown, studies have indicated that a likely strategy is to exploit host cellular pathways through functional mimicry. We report here that SopA, a Salmonella type III effector, mimics the mammalian HECT E3 ubiquitin ligase. SopA preferentially uses the host UbcH5a, UbcH5c and UbcH7 as E2s, which are involved in inflammation. Both the wild-type SopA and the mutant SopAC753S were expressed and translocated at similar levels during the infection of HeLa cells. A Salmonella strain expressing a catalytically incompetent SopAC753S mutant had reduced Salmonella-induced polymorphonuclear leukocytes transepithelial migration. We speculate that SopA ubiquitinate bacterial/host proteins involved in Salmonella-induced intestinal inflammation.

Amino Acid Sequence↗

PubMed2006

Inactivation of Bacillus anthracis spores in murine primary macrophages.

The current model for pathogenesis of inhalation anthrax indicates that the uptake and fate of Bacillus anthracis spores in alveolar macrophages are critical to the infection process. We have employed primary macrophages, which are more efficient for spore uptake than the macrophage-like cell line RAW264.7, to investigate spore uptake and survival. We found that at a multiplicity of infection (moi) of 5, greater than 80% of the spores of the Sterne strain containing only the pXO1 plasmid were internalized within 1 h. Within 4 h post infection, viability of internalized Sterne spores decreased to approximately 40%. Intracellular vegetative bacteria represented less than 1% of the total spore inoculum throughout the course of infection suggesting effective killing of germinated spores and/or vegetative bacteria. The Sterne spores trafficked quickly to phagolysosomes as indicated by colocalization with lysosome-associated membrane protein 1 (LAMP1). Expression of a dominant-negative Rab7 that blocked lysosome fusion enhanced Sterne spore survival. Addition of d-alanine to the infection resulted in 75% inhibition of spore germination and increased survival of internalized spores of the Sterne strain and a pathogenic strain containing both the pXO1 and pXO2 plasmids. Inhibition was reversed by the addition of l-alanine, which resumed spore germination and subsequent spore killing. Our data indicate that B. anthracis spores germinate in and are subsequently killed by primary macrophages.

Animals↗

PubMed2006

The first 45 amino acids of SopA are necessary for InvB binding and SPI-1 secretion.

Salmonella enterica serovar Typhimurium encodes two type III secretion systems (TTSSs) within pathogenicity island 1 (SPI-1) and island 2 (SPI-2). These type III protein secretion and translocation systems transport a panel of bacterial effector proteins across both the bacterial and the host cell membranes to promote bacterial entry and subsequent survival inside host cells. Effector proteins contain secretion and translocation signals that are often located at their N termini. We have developed a ruffling-based translocation reporter system that uses the secretion- and translocation-deficient catalytic domain of SopE, SopE78-240, as a reporter. Using this assay, we determined that the N-terminal 45 amino acid residues of Salmonella SopA are necessary and sufficient for directing its secretion and translocation through the SPI-1 TTSS. SopA1-45, but not SopA1-44, is also able to bind to its chaperone, InvB, indicating that SPI-1 type III secretion and translocation of SopA require its chaperone.

Amino Acid Sequence↗

PubMed2005

Recognition and ubiquitination of Salmonella type III effector SopA by a ubiquitin E3 ligase, HsRMA1.

Salmonella translocate bacterial effectors into host cells to confer bacterial entry and survival. It is not known how the host cells cope with the influx of these effectors. We report here that the Salmonella effector, SopA, interacts with host HsRMA1, a ubiquitin E3 ligase with a previously unknown function. SopA is ubiquitinated and degraded by the HsRMA1-mediated ubiquitination pathway. A sopA mutant escapes out of the Salmonella-containing vacuoles less frequently to the cytosol than wild type Salmonella in HeLa cells in a HsRMA1-dependent manner. Our data suggest that efficient bacterial escape into the cytosol of epithelial cells requires HsRMA1-mediated SopA ubiquitination and contributes to Salmonella-induced enteropathogenicity.

Bacterial Proteins↗

PubMed2005

Delineation and characterization of the actin nucleation and effector translocation activities of Salmonella SipC.

Salmonella type III secreted SipC possesses dual functions: translocation of effectors and actin modulation. The biological significance of SipC's actin nucleation activity in Salmonella-induced actin cytoskeleton rearrangements has not been studied. We report here the delineation of the actin nucleation activity from the effector translocation activity of SipC. Our data show that the central amino acid region (residues: 201-220) is essential for its actin nucleation activity and the C-terminal amino acid region (321-409) is required for translocation of effectors. A SipC nucleation-deficient mutant, which maintained its effector translocation activity, was obtained. This nucleation-deficient mutant had significantly reduced ability to induce actin cytoskeleton rearrangements, resulting in lower bacterial invasion into HeLa cells. Contrary to a previous report, we found that the purified recombinant wild-type SipC(199-409) protein is monomeric in solution by size exclusion chromatography coupled with multiangle laser light scattering assays (SEC-LS). Our data established that the actin nucleation activity of SipC plays a vital role in Salmonella-induced membrane ruffles and subsequent bacteria invasion.

Actins↗

PubMed2004

Efficient Salmonella entry requires activity cycles of host ADF and cofilin.

Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.

Actin Depolymerizing Factors↗

PubMed2004

Secretion and function of Salmonella SPI-2 effector SseF require its chaperone, SscB.

Salmonella strains utilize a type III secretion system for their successful survival and replications inside host cells. SseF is one of the several effector proteins that are required for conferring this survival ability by altering the trafficking of the Salmonella-containing vacuoles. These effector proteins often require appropriate chaperones to maintain their stabilities inside the bacteria. These chaperones are also known to assist the subsequent secretion and translocation of their substrates. We report here that SscB acts as the chaperone for SseF, an effector for the Salmonella pathogenicity island 2 (SPI-2). We found that the sscB gene is required for the formation of Salmonella sp.-induced continuous filaments in epithelial cells. Efficient Salmonella replication in macrophages requires SscB function. Intracellular and secretion levels of SseF are greatly reduced in an sscB mutant strain compared to the wild-type strain. A protein stability assay demonstrated that the half-life of SseF is significantly shortened in the absence of SscB. Transcriptional analysis of the sseF gene showed that the effect of SscB on the SseF level is not at the transcriptional level. A coprecipitation experiment indicated that SscB interacts with SseF. In summary, our results indicate that SscB is a chaperone for SPI-2 effector SseF to facilitate its secretion and function inside the host cells.

Animals↗

PubMed2002

The bacterial protein SipA polymerizes G-actin and mimics muscle nebulin.

SipA is a Salmonella protein delivered into host cells to promote efficient bacterial entry, which is essential for pathogenicity. SipA exerts its function by binding F-actin, resulting in the stabilization of F-actin and the stimulation of the bundling activity of fimbrin. Here we show that under low salt conditions where spontaneous nucleation and polymerization of actin do not occur, SipA induces extensive polymerization. We have used electron microscopy and a method for helical image analysis to visualize the complex of actin with the actin-binding fragment of SipA. The SipA fragment binds to actin as a tubular molecule extending approximately 95 A. The main sites of SipA binding on actin involve sequence insertions that are not present in the bacterial homolog of actin, MreB, suggesting a mechanism for preventing SipA from interacting with bacterial MreB filaments. Remarkably, the pattern of SipA binding, which connects subunits on opposite actin strands and explains the stabilization of F-actin, is similar to that shown for a fragment of the giant muscle protein nebulin. We suggest that SipA is a bacterial structural mimic of muscle nebulin and nebulin-like proteins in non-muscle cells that are involved in the regulation of the actin-based cytoskeleton.

Actins↗

PubMed2002

Involvement of SipA in modulating actin dynamics during Salmonella invasion into cultured epithelial cells.

Salmonella entry into epithelial host cells results from the host actin cytoskeleton reorganization that is induced by a group of bacterial proteins delivered to the host cells by the Salmonella type III secretion system. SopE, SopE2 and SopB activate CDC42 and Rac1 to intercept the signal transduction pathways involved in actin cytoskeleton rearrangements. SipA and SipC directly bind actin to modulate the actin dynamics facilitating bacterial entry. Biochemical studies have indicated that SipA decreases the critical concentration for actin polymerization and may be involved in promoting the initial actin polymerization in Salmonella-induced actin reorganization. In this report, we conducted experiments to analyze the in vivo function(s) of SipA during Salmonella invasion. SipA was found to be preferentially associated with peripheral cortical actin filaments but not stress fibres using permeabilized epithelial cells. When polarized Caco-2 cells were infected with Salmonella, actin cytoskeleton rearrangements induced by the wild-type strain had many filopodia structures that were intimately associated with the bacteria. In contrast, ruffles induced by the sipA null mutant were smoother and distant from the bacteria. We also found that the F-actin content in cells infected with the sipA mutant decreased nearly 80% as compared to uninfected cells or those infected with the wild-type Salmonella strain. Furthermore, expression of either the full-length or the SipA(459-684) actin-binding fragment induced prominent punctuate actin assembly in the cortical region of COS-1 cells. These results indicate that SipA is involved in modulating actin dynamics in cultured epithelial cells during Salmonella invasion.

Actin Cytoskeleton↗