Comparing the performance of conventional PCR, RTQ-PCR, and droplet digital PCR assays in detection of Shigella.

The incidence of foodborne infections caused by Shigella spp. is still very high in every year, which poses a great potential threat to public health. Conventional quantification methods based on culture techniques, biochemical, and serological identification are time-consuming and labor-intensive. To develop a more rapid and efficient detection method of Shigella spp., we compared the sensitivity and specificity of three different polymerase chain reaction (PCR) methods, including conventional PCR, quantitative real-time PCR (RTQ-PCR), and droplet digital PCR (ddPCR). Our results indicated that ddPCR method exhibited higher sensitivity, and the limit of detection was 10-5 ng/µl for genomic DNA templates, 10-1 cfu/ml for Shigella bacteria culture. In addition, we found that ddPCR was a time-saving method, which required a shorter pre-culturing time. Collectively, ddPCR assay was a reliable method for rapid and effective detection of Shigella spp.

Hijacking of death receptor signaling by bacterial pathogen effectors.

Death receptors such as Tumor necrosis factor receptor 1, FAS and TNF-associated apoptosis-inducing ligand-R1/2 play a major role in counteracting with bacterial pathogen infection through regulation of inflammation and programmed cell death. The highly regulated death receptor signaling is frequently targeted by gram-negative bacterial pathogens such as Salmonella, Shigella, enteropathogenic Escherichia coli and enterohamorrhagic Escherichia coli, which harbor a conserved type III secretion system that delivers a repertoire of effector proteins to manipulate host signal transductions for their own benefit. This review focuses on how bacterial gut pathogens hijack death receptor signaling to inhibit host NF-κB and programmed cell death pathways.

Arginine GlcNAcylation and Activity Regulation of PhoP by a Type III Secretion System Effector in Salmonella.

Salmonella type III secretion system (T3SS) effector SseK3 is a glycosyltransferase delivered directly into the host cells to modify host protein substrates, thus manipulating host cellular signal transduction. Here, we identify and characterize the Arg-GlcNAcylation activity of SseK3 inside bacterial cells. Combining Arg-GlcNAc protein immunoprecipitation and mass spectrometry, we found that 60 bacterial proteins were GlcNAcylated during Salmonella infection, especially the two-component signal transduction system regulatory protein PhoP. Moreover, the Arg-GlcNAcylation of PhoP by SseK3 was detected in vivo and in vitro, and four arginine residues, Arg65, Arg66, Arg118, and Arg215 were identified as the GlcNAcylation sites. Site-directed mutagenesis showed that the PhoP R215A change significantly reduced the DNA-binding ability and arginine to alanine change at all four sites (PhoP 4RA) completely eliminated the DNA-binding ability, suggesting that Arg215 is essential for the DNA-binding activity of PhoP and GlcNAcylation of PhoP affects this activity. Additionally, GlcNAcylation of PhoP negatively regulated the activity of PhoP and decreased the expression of its downstream genes. Overall, our work provides an example of the intra-bacterial activities of the T3SS effectors and increases our understanding of endogenous Arg-GlcNAcylation.