Food-Borne Bacterial Toxins

Food poisoning caused by the ingestion of preformed bacterial toxins is considered in relation to comparative symptoms, procedures for extraction and purification of the causal toxins, their chemistry, serology, assay procedures and pharmacology, in so far as these are known.The bacteria discussed in this context are Clostridium botulinum, C. perfringens, Staphylococcus aureus, Bacillus cereus, and Vibrio parahemolyticus. The possible roles of the enterococci, Proteus, E. coli and of unknown species, in relation to production of non-antigenic toxic substances, are discussed briefly.Requirements for prevention of the various forms of bacterial food poisoning are outlined.     

Indigenous Bacteria and Fungi Drive Traditional Kimoto Sake Fermentations

Sake (Japanese rice wine) production is a complex, multistage process in which fermentation is performed by a succession of mixed fungi and bacteria. This study employed high-throughput rRNA marker gene sequencing, quantitative PCR, and terminal restriction fragment length polymorphism to characterize the bacterial and fungal communities of spontaneous sake production from koji to product as well as brewery equipment surfaces. Results demonstrate a dynamic microbial succession, with koji and early moto fermentations dominated by Bacillus, Staphylococcus, and Aspergillus flavus var. oryzae, succeeded by Lactobacillus spp. and Saccharomyces cerevisiae later in the fermentations. The microbiota driving these fermentations were also prevalent in the production environment, illustrating the reservoirs and routes for microbial contact in this traditional food fermentation. Interrogating the microbial consortia of production environments in parallel with food products is a valuable approach for understanding the complete ecology of food production systems and can be applied to any food system, leading to enlightened perspectives for process control and food safety.     

Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state.     

Modulation of gut physiology through enteric toxins

Diarrheal diseases caused by microorganisms and their toxins are a major cause of mortality and morbidity throughout the world. Acute diarrhea is mainly caused due to increased intestinal secretion, commonly as a result of infection with enterotoxin producing organisms (enterotoxigenic Escherichia coli, Vibrio cholera) or due to decreased intestinal absorption from infection with organisms that damage the intestinal epithelium (enteropathogenic E. coli sp., Shigella sp., Salmonella sp.) The studies of the impact of enteric pathogens and their virulence factors exert their effect by producing toxins, called bacterial toxins. The protein toxins are produced by diverse group of bacteria. Most of the bacterial toxins exert their effect through involvement of ADP-ribosylation proteins; otherwise essential for several cellular functions while other toxins involve guanylate cyclase systems or calcium and protein kinases for their ultimate action.     

Small Changes in pH Have Direct Effects on Marine Bacterial Community Composition: A Microcosm Approach

As the atmospheric CO₂ concentration rises, more CO₂ will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15–8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.     

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol,Nivalenol, and HT-2 Toxin in Cereal Matrices

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K_m of 5.4 mM and a V_max of 16 μmol min⁻¹ mg⁻¹ were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.     

Potentialities of fluorescent carbon nanomaterials as sensor for food analysis

Food safety and quality are among the most significant and prevalent research areas worldwide. The fabrication of appropriate technical procedures or devices for the recognition of hazardous features in foods is essential to safeguard food materials. In the recent era, developing high-performance sensors based on carbon nanomaterial for food safety investigation has made noteworthy progress. Hence this review briefly highlights the different detection approaches (colorimetric sensor, fluorescence sensor, surface-enhanced Raman scattering, surface plasmon resonance, chemiluminescence, and electroluminescence), functional carbon nanomaterials with various dimensions (quantum dots, graphene quantum dots) and detection mechanisms. Further, this review emphasizes the assimilation of carbon nanomaterials with optical sensors to identify multiple contaminants in food products. The insights of carbon-based nanomaterials optical sensors for pesticides and insecticides, toxic metals, antibiotics, microorganisms, and mycotoxins detection are described in detail. Finally, the opportunities and future perspectives of nanomaterials-based optical analytical approaches for detecting various food contaminants are discussed.     

Glutathione transferase activity and oocyte development in copepods exposed to toxic phytoplankton

Organisms present a series of cellular mechanisms to avoid the effects of toxic compounds. Such mechanisms include the increase in activity of detoxification enzymes [e.g., 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST)], which could explain the low retention of ingested toxins generally observed in copepods. In addition, decreasing gross growth efficiency (GGE) of copepods with increasing concentration of toxic diets could be caused either by a high expenditure coping with toxins (e.g., increase in the activity of detoxification enzymes) or by a deterioration of reproductive tissues. To assess the effect of toxic phytoplankton on the activity of detoxification enzymes and on oocyte maturation of Acartia tonsa and Temora longicornis, feeding and egg production experiments were carried out with a variety of toxic diets and an adequate non-toxic food control (Rhodomonas spp.) all provided as single species diets. Toxic diets included the nodularin-producing cyanobacterium Nodularia spumigena, the dinoflagellates Alexandrium minutum, and A. tamarense, which contained Paralytic Shellfish Poisoning (PSP) toxins, the dinoflagellate Prorocentrum lima with Diarrhetic Shellfish Poisoning (DSP) toxins and the haptophyte Prymnesium parvum, which produces ichtyotoxins with haemolytic activity. Feeding on toxic diets was lower than on Rhodomonas spp., except for A. minutum and A. tamarense. In addition, toxic diets negatively affected reproduction in both copepod species with the production of oocytes and oocyte development impaired with A. minutum and N. spumigena. While the negative effect of N. spumigena seemed to be connected to gonad atresia likely caused by severe food limitation (starvation), the negative effect of A. minutum could have been either caused by a direct effect of saxitoxins or nutritional inadequacy on oocyte production. We could not detect EROD activity in the copepods, while the activity of GST was generally higher with the non-toxic food control and positively related to the feeding and egestion rates, suggesting relation to feeding conditions rather than to exposure to toxic diets. No relationship was found between GGE and GST activity. Our results refute the hypothesis that toxic diets, provided at ecologically relevant levels, would induce cellular mechanisms in copepods regarding GST activity. GST activity thus seems to play no role in detoxification of copepods confronted with toxic phytoplankton. Toxin detoxification and its cost for copepods still remain an open question.     

Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.     

Isolation of deoxynivalenol-transforming bacteria from the chicken intestines using the approach of PCR-DGGE guided microbial selection

Background  

Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Bacterial diseases of crabs: a review

Bacterial diseases of crabs are manifested as bacteremias caused by organisms such as Vibrio, Aeromonas, and a Rhodobacteriales-like organism or tissue and organ tropic organisms such as chitinoclastic bacteria, Rickettsia intracellular organisms, Chlamydia-like organism, and Spiroplasma. This paper provides general information about bacterial diseases of both marine and freshwater crabs. Some bacteria pathogens such as Vibrio cholerae and Vibrio vulnificus occur commonly in blue crab haemolymph and should be paid much attention to because they may represent potential health hazards to human beings because they can cause serious diseases when the crab is consumed as raw sea food. With the development of aquaculture, new diseases associated with novel pathogens such as spiroplasmas and Rhodobacteriales-like organisms have appeared in commercially exploited crab species in recent years. Many potential approaches to control bacterial diseases of crab will be helpful and practicable in aquaculture.     

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.     

Bacterial toxin and effector glycosyltransferases

Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.     

Predator-Prey Chemical Warfare Determines the Expression of Biocontrol Genes by Rhizosphere-Associated Pseudomonas fluorescens

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.Bacterial communities are heavily consumed by protozoan predators (30), and predation is a major force shaping the structure of microbial communities in both aquatic and terrestrial ecosystems (34, 35). The competitiveness of bacteria strongly depends on their ability to avoid predation (9, 22), and many species have developed defense mechanisms such as the production of toxins, which reduces mortality by repelling or killing predators (21, 24). Toxin production, however, is energetically costly, and defense theory predicts that prey species should optimize the investment in defense according to the resources available and the predation risk (40), for example, in response to predator-associated chemical cues (4, 15). In bacteria the production of defense traits is tightly regulated by various sensor cascades (11), and defense mechanisms, such as the formation of inedible morphotypes or microcolonies, can be elicited in the presence of predators (45).Toxin production is one of the most powerful defense strategies, and in the present study we tested whether bacteria can also modulate the production of toxic secondary metabolites in response to protozoan predators. We investigated the chemical communication between the soil bacterium Pseudomonas fluorescens CHA0 and the bacterivorous amoeba Acanthamoeba castellanii, a ubiquitous soil protist feeding on a wide range of bacterial species (33). P. fluorescens CHA0 effectively colonizes the rhizosphere of plants and produces various extracellular toxins including pyrrolnitrin (PRN), 2,4-diacetylphloroglucinol (DAPG), hydrogen cyanide (HCN), and pyoluteorin (PLT) (18). These toxins reduce predation pressure and enhance the competitiveness of the bacteria in the rhizosphere (22) but also act antagonistically against plant pathogens, thereby promoting plant growth (11).The production of secondary metabolites by P. fluorescens is a dynamic process that depends on environmental factors, such as nutrient availability (12), cell density (18), or the presence of phytopathogens (27). We hypothesized that P. fluorescens is also able to sense predators and responds by increasing the expression of toxin genes.Predators or competitors susceptible to toxins can adopt counterstrategies to repress their production. For example, the fungal pathogen Fusarium can inhibit the production of DAPG by pseudomonads (26), and we hypothesized that A. castellanii can counteract prey defense by inhibiting bacterial toxin production.We investigated the effects of predator-prey interactions on the regulation of the production of the extracellular toxins DAPG, PLT, PRN, and HCN using a set of autofluorescent green fluorescent protein (GFP)- and mCherry-based reporter fusions (2, 32). Autofluorescent reporter fusions allow in situ measurement of gene expression patterns and have been applied to monitor the expression of antifungal genes in the rhizosphere (10, 32). The response of the bacteria to predators or associated signal molecules was investigated in batch experiments and on barley roots.     

Abundance of type I toxin–antitoxin systems in bacteria: searches for new candidates and discovery of novel families

Small, hydrophobic proteins whose synthesis is repressed by small RNAs (sRNAs), denoted type I toxin–antitoxin modules, were first discovered on plasmids where they regulate plasmid stability, but were subsequently found on a few bacterial chromosomes. We used exhaustive PSI-BLAST and TBLASTN searches across 774 bacterial genomes to identify homologs of known type I toxins. These searches substantially expanded the collection of predicted type I toxins, revealed homology of the Ldr and Fst toxins, and suggested that type I toxin–antitoxin loci are not spread by horizontal gene transfer. To discover novel type I toxin–antitoxin systems, we developed a set of search parameters based on characteristics of known loci including the presence of tandem repeats and clusters of charged and bulky amino acids at the C-termini of short proteins containing predicted transmembrane regions. We detected sRNAs for three predicted toxins from enterohemorrhagic Escherichia coli and Bacillus subtilis, and showed that two of the respective proteins indeed are toxic when overexpressed. We also demonstrated that the local free-energy minima of RNA folding can be used to detect the positions of the sRNA genes. Our results suggest that type I toxin–antitoxin modules are much more widely distributed among bacteria than previously appreciated.     

Identification of Household Bacterial Community and Analysis of Species Shared with Human Microbiome

Microbial populations in indoor environments, where we live and eat, are important for public health. Various bacterial species reside in the kitchen, and refrigerators, the major means of food storage within kitchens, can be a direct source of food borne illness. Therefore, the monitoring of microbiota in the refrigerator is important for food safety. We investigated and compared bacterial communities that reside in the vegetable compartment of the refrigerator and on the seat of the toilet, which is recognized as highly colonized by microorganisms, in ten houses using high-throughput sequencing. Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were predominant in refrigerator and toilet samples. However, Proteobacteria was more abundant in the refrigerator, and Firmicutes was more abundant in the toilet. These household bacterial communities were compared with those of human skin and gut to identify potential sources of household bacteria. Bacterial communities from refrigerators and toilets shared more species in common with human skin than gut. Opportunistic pathogens, including Propionibacterium acnes, Bacteroides vulgatus, and Staphylococcus epidermidis, were identified as species shared with human skin and gut microbiota. This approach can provide a general background of the household microbiota and a potential method of source-tracking for public health purposes.     

Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.