Campylobacter jejuni, a zoonotic pathogen that frequently colonizes poultry, possesses two Microbial Surface Components Recognizing Adhesive Matrix Molecule(s) (MSCRAMMs) termed CadF and FlpA that bind to the glycoprotein fibronectin (FN). Previous to this study, it was not known whether the CadF and FlpA proteins were functionally redundant or if both were required to potentiate host cell binding and signaling processes. We addressed these questions by generating a complete repertoire of cadF and flpA mutants and complemented isolates, and performing multiple phenotypic assays. Both CadF and FlpA were found to be necessary for the maximal binding of C. jejuni to FN and to host cells. In addition, both CadF and FlpA are required for the delivery of the C. jejuni Cia effector proteins into the cytosol of host target cells, which in turn activates the MAPK signaling pathway (Erk 1/2) that is required for the C. jejuni invasion of host cells. These data demonstrate the non-redundant and bi-functional nature of these two C. jejuni FN-binding proteins. Taken together, the C. jejuni CadF and FlpA adhesins facilitate the binding of C. jejuni to the host cells, permit delivery of effector proteins into the cytosol of a host target cell, and aid in the rewiring of host cell signaling pathways to alter host cell behavior.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the on state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficient. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the on state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Puroindolines are small, amphipathic, wheat proteins that determine the hardness of the wheat kernel and protect crops from different pathogens. Puroindoline A (PinA) and puroindoline B (PinB) are two major isoforms of puroindolines. These proteins have antibacterial and antifungal properties mainly attributed to their characteristic tryptophan-rich domains (TRDs). In this in vitro study, we investigated the antimicrobial effect of PinA and PinB synthetic peptides against the growth and biofilm formation of Campylobacter jejuni. C. jejuni is an important microaerobic, foodborne pathogen that causes gastrointestinal and neurological diseases in humans. Our results showed that: (1) PinA, but not PinB, has strong antimicrobial activity against C. jejuni clinical strains 81-176 and F38011, Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes; (2) The substitution of two tryptophan residues to glycine (W→G) in the TRD of PinA abolishes its antimicrobial activity against these microorganisms; (3) PinA functions additively with two common antibiotics (ciprofloxacin and erythromycin) to inhibit or inactivate C. jejuni strains; (4) PinA damages the C. jejuni cellular membrane, (5) PinA is cytotoxic to human INT 407 cells at high concentrations; and (6) PinA inhibits C. jejuni biofilm formation. In summary, this study demonstrates the antimicrobial activity of PinA against C. jejuni growth and biofilm formation and further confirms the potential use of PinA as a therapeutic agent in health care or as preservatives in the agri-food industry.

Oxidative damage to DNA is a significant source of mutations in living organisms. While DNA damage must be repaired to maintain the integrity of the genome and cell survival, errors made during DNA repair may contribute to evolution. Previous work has revealed that Campylobacter jejuni growth in the presence of bile salt deoxycholate (DOC) causes an increase in reactive oxygen species and the occurrence of 8-oxo-deoxyguanosine (8-oxo-dG) DNA lesions. The fundamental goal of this project was to determine if C. jejuni growth in a medium containing DOC contributes to DNA mutations that provide a fitness advantage to the bacterium. Co-culture experiments revealed that C. jejuni growth in a DOC-supplemented medium increases the total number of ciprofloxacin-resistant isolates compared to C. jejuni grown in the absence of DOC. We recovered two individual isolates grown in a medium with DOC that had a point mutation in the gene encoding the EptC phosphoethanolamine transferase. Transformants harboring the EptC variant protein showed enhanced resistance to the antimicrobial agent polymyxin B and DOC when compared to an eptC deletion mutant or the isolate complemented with a wild-type copy of the gene. Finally, we found that the base excision repair (BER), homologous recombination repair (HRR), and nucleotide excision repair (NER) are involved in general oxidative damage repair in C. jejuni but that the BER pathway plays the primary role in the repair of the 8-oxo-dG lesion. We postulate that bile salts drive C. jejuni mutations (adaptations) and enhance bacterial fitness in animals.