Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology

Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing Escherichia coli O104:H4

The outbreak of diarrhea and hemolytic uremic syndrome that occurred in Germany in 2011 was caused by a Shiga toxin-producing enteroaggregative Escherichia coli (EAEC) strain. The strain was classified as EAEC due to the presence of a plasmid (pAA) that mediates a characteristic pattern of aggregative adherence on cultured cells, the defining feature of EAEC that has classically been associated with virulence. Here, we describe an infant rabbit-based model of intestinal colonization and diarrhea caused by the outbreak strain, which we use to decipher the factors that mediate the pathogen’s virulence. Shiga toxin is the key factor required for diarrhea. Unexpectedly, we observe that pAA is dispensable for intestinal colonization and development of intestinal pathology. Instead, chromosome-encoded autotransporters are critical for robust colonization and diarrheal disease in this model. Our findings suggest that conventional wisdom linking aggregative adherence to EAEC intestinal colonization is false for at least a subset of strains

A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala

Background: Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients. Method Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing. Results: The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage. Conclusion: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1121-7) contains supplementary material, which is available to authorized users

The Regulation of Sulfur Metabolism in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention

Human alkaloid biosynthesis : chemical inducers of Parkinson's disease?

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.Includes bibliographical references (leaves 26-29).The occurrence of certain alkaloids in the human brain appears to be associated with the onset of Parkinson's disease (PD). Recently, a human protein bearing homology to an alkaloid synthase in plants was identified. This protein, termed BSCv, may catalyze alkaloid formation in humans. If such activity is confirmed, regulation of BSCv through the use of small molecule inhibitors could provide novel drug therapies for PD patients. This paper describes the first heterologous expression and purification of this transmembrane protein and examines its biological function through a series of enzyme assays. The assays used to evaluate enzyme activity were modeled after the Pictet-Spengler condensation catalyzed by the plant enzyme. Substrates were selected based on their potential to form alkaloids previously identified in central nervous system tissue. Product formation was monitored via high-performance liquid chromatography. Preliminary data suggest that BSCv does not function as an alkaloid synthase. However, further studies are needed to ascertain such conclusions. Alternative detergents should be evaluated to assess their influence on enzyme activity. The use of an expanded substrate pool may also provide insight into protein function since substrate specificity may have restricted product formation in the performed assays. Finally, incubation of BSCv with rat brain extract, which contains another species homologue of the protein, could provide insight into its natural substrates. If these studies are unsuccessful, consideration should be given to the possibility that BSCv may function as a receptor. Once the mechanistic and structural properties of the plant enzyme are elucidated, it may be possible to take a more direct approach to the characterization of its human homologue.by Stavroula K. Hatzios.S.B

Sulfur metabolism genes from <i>M. tuberculosis</i> induced by various conditions of environmental stress.

a<p>The <i>cysN</i> and <i>cysC</i> genes of <i>Mtb</i> are fused into a single, bifunctional <i>cysNC</i> gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002036#ppat.1002036-Pinto1" target="_blank">[22]</a>. However, the transcript is often reported as <i>cysN</i> in microarray data.</p><p>SDS, sodium dodecyl sulfate.</p

Venn diagram illustrating the convergent transcriptional regulation of <i>Mtb</i> sulfur metabolism genes by various conditions of environmental stress.

<p>Venn diagram illustrating the convergent transcriptional regulation of <i>Mtb</i> sulfur metabolism genes by various conditions of environmental stress.</p