Cellular Activity of \u3ci\u3eSalmonella\u3c/i\u3e Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonella enterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and D-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella

Cystic Fibrosis

Cystic fibrosis was first recognized as a new disease in 1938 when autopsies of children who died from malnourishment revealed similar findings. Currently, there are approximately 30,000 people living in the U.S. who are affected by this rare genetic disease. Cystic fibrosis is a congenital progressive disorder caused by a mutation in the gene encoding cystic fibrosis transmembrane conductance regulator (CTFR). Mutations in the CTFR gene cause the mucus in the body to become thick and glue-like, blocking ducts and tubes distributed within the body. Buildup in mucus can cause a multitude of problems, eventually leading to death. Treatment of an individual with cystic fibrosis depends on the severity of symptoms. Antibiotics can prevent and treat chest infections. Other medications can also treat thick mucus in the lungs by thinning and hydrating the mucus, allowing it to clear and also widening the airways to make breathing easier. Lung transplant may be needed in the case of lung damage. The FDA recently approved Trikafta, the first triple combination therapy available to treat CF. Here, we explore the manner in which this trifecta of drugs helps the protein encoded by the CTFR gene mutation function more effectively

Cloning and Purification of the ArtB Binding Subunit From a Novel Toxin in Salmonella enterica Typhimurium

According to the CDC, Salmonella is responsible for about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the United States every year. Food is the source for most of these illnesses. Salmonella enterica is common in cattle and has a number of serovars that are pathogenic in humans. Many Salmonella serovars encode for the AB5 toxin, or ArtAB, which is similar in structure to pertussis toxin. The pentameric B subunit is non-covalently linked to the A subunit, thus allowing separation of the receptor-binding B subunit from the holotoxin. We hypothesize that being able to extract and use ArtAB or the non-toxic ArtB subunit for vaccines could decrease prevalence of Salmonella infections in cows and potentially prevent transmission from cattle and agriculture to humans. The B subunit can be transformed into E. coli using a vector plasmid and then purified and extracted using D-galactose and fetuin affinity chromatography methods. Our results show that we have successfully cloned the B subunit into a plasmid, transformed the plasmid into E. coli, and had ArtB protein expression. Further research will examine the toxicity of ArtAB and ArtB to determine whether or not they are suitable for use in vaccines

Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonellaenterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella