Toxigenic Clostridium perfringens Isolated from At-Risk Paediatric Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Potent Killing of Pseudomonas aeruginosa by an Antibody-Antibiotic Conjugate.

Pseudomonas aeruginosa causes life-threatening infections that are associated with antibiotic failure. Previously, we identified the antibiotic G2637, an analog of arylomycin, targeting bacterial type I signal peptidase, which has moderate potency against P. aeruginosa. We hypothesized that an antibody-antibiotic conjugate (AAC) could increase its activity by colocalizing P. aeruginosa bacteria with high local concentrations of G2637 antibiotic in the intracellular environment of phagocytes. Using a novel technology of screening for hybridomas recognizing intact bacteria, we identified monoclonal antibody 26F8, which binds to lipopolysaccharide O antigen on the surface of P. aeruginosa bacteria. This antibody was engineered to contain 6 cysteines and was conjugated to the G2637 antibiotic via a lysosomal cathepsin-cleavable linker, yielding a drug-to-antibody ratio of approximately 6. The resulting AAC delivered a high intracellular concentration of free G2637 upon phagocytosis of AAC-bound P. aeruginosa by macrophages, and potently cleared viable P. aeruginosa bacteria intracellularly. The molar concentration of AAC-associated G2637 antibiotic that resulted in elimination of bacteria inside macrophages was approximately 2 orders of magnitude lower than the concentration of free G2637 required to eliminate extracellular bacteria. This study demonstrates that an anti-P. aeruginosa AAC can locally concentrate antibiotic and kill P. aeruginosa inside phagocytes, providing additional therapeutic options for antibiotics that are moderately active or have an unfavorable pharmacokinetics or toxicity profile. IMPORTANCE Antibiotic treatment of life-threatening P. aeruginosa infections is associated with low clinical success, despite the availability of antibiotics that are active in standard microbiological in vitro assays, affirming the need for new therapeutic approaches. Antibiotics often fail in the preclinical stage due to insufficient efficacy against P. aeruginosa. One potential strategy is to enhance the local concentration of antibiotics with limited inherent anti-P. aeruginosa activity. This study presents proof of concept for an antibody-antibiotic conjugate, which releases a high local antibiotic concentration inside macrophages upon phagocytosis, resulting in potent intracellular killing of phagocytosed P. aeruginosa bacteria. This approach may provide new therapeutic options for antibiotics that are dose limited.

Pseudomonas aeruginosa aggregates in cystic fibrosis sputum produce exopolysaccharides that likely impede current therapies.

In cystic fibrosis (CF) airways, Pseudomonas aeruginosa forms cellular aggregates called biofilms that are thought to contribute to chronic infection. To form aggregates, P. aeruginosa can use different mechanisms, each with its own pathogenic implications. However, how they form in vivo is controversial and unclear. One mechanism involves a bacterially produced extracellular matrix that holds the aggregates together. Pel and Psl exopolysaccharides are structural and protective components of this matrix. We develop an immunohistochemical method to visualize Pel and Psl in CF sputum. We demonstrate that both exopolysaccharides are expressed in the CF airways and that the morphology of aggregates is consistent with an exopolysaccharide-dependent aggregation mechanism. We reason that the cationic exopolysaccharide Pel may interact with some of the abundant anionic host polymers in sputum. We show that Pel binds extracellular DNA (eDNA) and that this interaction likely impacts current therapies by increasing antimicrobial tolerance and protecting eDNA from digestion.

cGAS and Ifi204 cooperate to produce type I IFNs in response to Francisella infection.

Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264.7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS-STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response.