Stacey A. Rutherford

Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts

The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in approximately sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in approximately 22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.

Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex

Significance The toxin-producing bacterium Clostridium difficile is the leading cause of antibiotic-associated infection in hospitals worldwide. An estimated 500,000 cases of C. difficile infection (CDI) occur annually in the US, with a cost approaching 3 billion dollars. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of entering host cells to cause fluid secretion, inflammation, and necrosis of the colonic mucosa. Given that numerous investigators are currently targeting these toxins for the development of novel CDI therapies, understanding the mechanism of toxicity is a significant priority. Clostridium difficile infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI.

Structural determinants of Clostridium difficile toxin A glucosyltransferase activity.

Background: C. difficile TcdA and TcdB glucosylate small GTPases. Results: Structural and functional studies reveal comparable activities with Rho substrates, enhanced activities following autoprocessing, and TcdA-specific modification of Rap2A. Conclusion: TcdA is a potent enzyme and modifies a broader array of GTPase substrates than TcdB. Significance: These findings highlight the importance of autoprocessing for activity and reveal differences in target specificity between the toxins. The principle virulence factors in Clostridium difficile pathogenesis are TcdA and TcdB, homologous glucosyltransferases capable of inactivating small GTPases within the host cell. We present crystal structures of the TcdA glucosyltransferase domain in the presence and absence of the co-substrate UDP-glucose. Although the enzymatic core is similar to that of TcdB, the proposed GTPase-binding surface differs significantly. We show that TcdA is comparable with TcdB in its modification of Rho family substrates and that, unlike TcdB, TcdA is also capable of modifying Rap family GTPases both in vitro and in cells. The glucosyltransferase activities of both toxins are reduced in the context of the holotoxin but can be restored with autoproteolytic activation and glucosyltransferase domain release. These studies highlight the importance of cellular activation in determining the array of substrates available to the toxins once delivered into the cell.

Inactivation of Src Family Tyrosine Kinases by Reactive Oxygen Species in Vivo

Reactive oxygen species, including H2 O2, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}\mathrm{H}^{{\cdot}}\) \end{document} are constantly produced in the human body and are involved in the development of cardiovascular diseases. Emerging evidence suggests that reactive oxygen species, besides their deleterious effects at high concentrations, may be protective. However, the mechanism underlying the protective effects of reactive oxygen species is not clear. Here, we reported a novel finding that H2O2 at low to moderate concentrations (50-250 μm) markedly inactivated Src family tyrosine kinases temporally and spatially in vivo but not in vitro. We further showed that Src family kinases localized to focal adhesions and the plasma membrane were rapidly and permanently inactivated by H2O2, which resulted from a profound reduction in phosphorylation of the conserved tyrosine residue at the activation loop. Interestingly, the cytoplasmic Src family kinases were activated gradually by H2O2, which partially compensated for the loss of total activities of Src family kinases but not their functions. Finally, H2O2 rendered endothelial cells resistant to growth factors and cytokines and protected the cells from inflammatory activation. Because Src family kinases play key roles in cell signaling, the rapid inactivation of Src family kinases by H2 O2 may represent a novel mechanism for the protective effects of reactive oxygen species.

Crystal structure of Clostridium difficile toxin A

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Suppression of the Phosphorylation of Receptor Tyrosine Phosphatase-α on the Src-Independent Site Tyrosine 789 by Reactive Oxygen Species

Oxidation of receptor protein tyrosine phosphatase-α (RPTPα) is emerging as an important yet poorly characterized regulatory mechanism for RPTPα signaling in cells. RPTPα has been shown to be reversibly oxidized and inhibited by reactive oxygen species. However, it is not known whether oxidative stress could regulate the phosphorylation of Tyr789, a critical tyrosine residue for RPTPα signaling that modulates the function of Grb2 and the activation of Src family kinases. In the present study, we have taken advantage of a phosphospecific antibody against Tyr789-phosphorylated RPTPα and characterized the phosphorylation of RPTPα Tyr789 in various cultured cells, including SYF cells lacking all three ubiquitously expressed members (Src, Yes, and Fyn) of Src family kinases. We have obtained substantial evidence indicating that the phosphorylation of RPTPα Tyr789 is regulated predominantly by an Src kinase inhibitor, protein phosphatase 1 (PP1)-sensitive but Src/Yes/Fyn-independent tyrosine kinase, in cells. We further reported a novel finding that, besides the inhibition of RPTPαs activity, H2O2 at low to moderate concentrations (50-250 μM) markedly suppressed the phosphorylation of RPTPα Tyr789 and the association of RPTPα with Grb2 in cultured cells, which may result from inhibition of such a PP1-sensitive but Src/Yes/Fyn-independent tyrosine kinase. Because Tyr789 plays an important role in RPTPα signaling, our findings may provide new insights into the functional regulation of RPTPα by oxidative stress in cells.

Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.

Integrated, High-Throughput, Multiomics Platform Enables Data-Driven Construction of Cellular Responses and Reveals Global Drug Mechanisms of Action

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10 000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

ABSTRACT Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly β-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the β-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the β-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly.