Lambda Red Recombineering in Shigella flexneri.

Shigellosis remains a major cause of severe diarrheal disease and death throughout the world. Vaccine development against shigellosis has been hampered by an incomplete understanding of the molecular mechanisms by which Shigella spp. causes disease and difficulties in manipulating Shigella spp. genomes. While homologous recombination protocols for the construction of precise gene deletions exist, construction of mutants in S. flexneri has not become commonplace. We describe the steps for construction of gene deletions using λ-red recombination using tools that we have developed in our laboratory.

Photodynamic Inactivation of Human Coronaviruses.

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

Genomic and phenotypic analysis of SspH1 identifies a new Salmonella effector, SspH3.

Salmonella is a major foodborne pathogen and is responsible for a range of diseases. Not all Salmonella contributes to severe health outcomes as there is a large degree of genetic heterogeneity among the 2,600 serovars within the genus. This variability across Salmonella serovars is linked to numerous genetic elements that dictate virulence. While several genetic elements encode virulence factors with well-documented contributions to pathogenesis, many genetic elements implicated in Salmonella virulence remain uncharacterized. Many pathogens encode a family of E3 ubiquitin ligases that are delivered into the cells that they infect using a Type 3 Secretion System (T3SS). These effectors, known as NEL-domain E3s, were first characterized in Salmonella. Most Salmonella encodes the NEL-effectors sspH2 and slrP, whereas only a subset of Salmonella encodes sspH1. SspH1 has been shown to ubiquitinate the mammalian protein kinase PKN1, which has been reported to negatively regulate the pro-survival program Akt. We discovered that SspH1 mediates the degradation of PKN1 during infection of a macrophage cell line but that this degradation does not impact Akt signaling. Genomic analysis of a large collection of Salmonella genomes identified a putative new gene, sspH3, with homology to sspH1. SspH3 is a novel NEL-domain effector.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.

Recalls of Foods due to Microbial Contamination Classified by the Canadian Food Inspection Agency, 2000 to 2017.

Recall of microbial-contaminated food products is an important intervention in preventing the transmission of foodborne illness. Here, we summarize the number and nature of foods recalled as a result of microbial contamination, classified by the Canadian Food Inspection Agency, for the period 1 January 2000 through 31 December 2017. A total of 10,432 food products were recalled from 2,094 recall events in Canada because of microbial contamination during this period. The meat, meat products and poultry category, followed by fishery and seafood products and nuts and edible seeds, contained the food products most commonly associated with microbial contamination. Most microbial-contaminated food products reported were recalled because of the presence bacterial pathogens. Salmonella contamination was responsible for the largest number of recall events, whereas Listeria monocytogenes contamination accounted for the greatest number of food products recalled because of microbial contamination. L. monocytogenes contamination was also most commonly associated with major food recall events, although records may be inflated because of an invested effort to prevent future L. monocytogenes outbreaks following a 2008 deli meat recall. The findings and data we present in this study will support future surveillance and analysis of microbial-contaminated food recalls in Canada.