Cationic Homopolymers Inhibit Spore and Vegetative Cell Growth of Clostridioides difficile.

A wide range of synthetic polymers have been explored for antimicrobial activity. These materials usually contain both cationic and hydrophobic subunits because these two characteristics are prominent among host-defense peptides. Here, we describe a series of nylon-3 polymers containing only cationic subunits and their evaluation against the gastrointestinal, spore-forming pathogen Clostridioides difficile. Despite their highly hydrophilic nature, these homopolymers showed efficacy against both the vegetative and spore forms of the bacterium, including an impact on C. difficile spore germination. The polymer designated P34 demonstrated the greatest efficacy against C. difficile strains, along with low propensities to lyse human red blood cells or intestinal epithelial cells. To gain insight into the mechanism of P34 action, we evaluated several cell-surface mutant strains of C. difficile to determine the impacts on growth, viability, and cell morphology. The results suggest that P34 interacts with the cell wall, resulting in severe cell bending and death in a concentration-dependent manner. The unexpected finding that nylon-3 polymers composed entirely of cationic subunits display significant activities toward C. difficile should expand the range of other polymers considered for antibacterial applications.

Regulation and Anaerobic Function of the Clostridioides difficile ß-Lactamase.

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to ß-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to ß-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of ß-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a ß-lactamase, which is a common ß-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a ß-lactamase that was greatly induced in response to several classes of ß-lactam antibiotics. An in-frame deletion of the operon abolished ß-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the ß-lactam ampicillin. We found that the activity of this ß-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to ß-lactams. These results provide support for the function of a ß-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile ß-lactamase activity.

.Clostridioides difficile.

Clostridioides difficile is a spore-forming, anaerobic, intestinal pathogen that causes severe diarrhea that can lead to death. In 2011, C. difficile infected ∼500000 people in the USA and killed ∼29000 people. C. difficile infection (CDI) is the most common healthcare-related infection in the USA, leading to increased healthcare costs of $4.8 billion. This pathogen transmits via the oral-fecal route as a highly contagious and resilient spore. Upon exposure to primary bile acids in the intestine, C. difficile germinates, and in the absence of colonization resistance from the normal microbiota, the bacterium colonizes the colon and produces toxins. These toxins inhibit actin polymerization in host cells, leading to cell death. C. difficile cells can then sporulate in the intestine and exit the body via diarrheal shedding. In culture, sporulation is induced at stationary phase in a nutrient-limiting environment, but the intestinal triggers of sporulation are still unknown.

EGF promotes the shedding of soluble E-cadherin in an ADAM10-dependent manner in prostate epithelial cells.

During the progression of prostate cancer, the epithelial adhesion molecule E-cadherin is cleaved from the cell surface by ADAM15 proteolytic processing, generating an extracellular 80kDa fragment referred to as soluble E-cadherin (sE-cad). Contrary to observations in cancer, the generation of sE-cad appears to correlate with ADAM10 activity in benign prostatic epithelium. The ADAM10-specific inhibitor INCB8765 and the ADAM10 prodomain inhibit the generation of sE-cad, as well as downstream signaling and cell proliferation. Addition of EGF or amphiregulin (AREG) to these untransformed cell lines increases the amount of sE-cad shed into the conditioned media, as well as sE-cad bound to EGFR. EGF-associated shedding appears to be mediated by ADAM10 as shRNA knockdown of ADAM10 results in reduced shedding of sE-cad. To examine the physiologic role of sE-cad on benign prostatic epithelium, we treated BPH-1 and large T immortalized prostate epithelial cells (PrEC) with an sE-cad chimera comprised of the human Fc domain of IgG(1), fused to the extracellular domains of E-cadherin (Fc-Ecad). The treatment of untransformed prostate epithelial cells with Fc-Ecad resulted in phosphorylation of EGFR and downstream signaling through ERK and increased cell proliferation. Pre-treating BPH-1 and PrEC cells with cetuximab, a therapeutic monoclonal antibody against EGFR, decreased the ability of Fc-Ecad to induce EGFR phosphorylation, downstream signaling, and proliferation. These data suggest that ADAM10-generated sE-cad may have a role in EGFR signaling independent of traditional EGFR ligands.

Microsphere-based scaffolds for cartilage tissue engineering: using subcritical CO(2) as a sintering agent.

Shape-specific, macroporous tissue engineering scaffolds were fabricated and homogeneously seeded with cells in a single step. This method brings together CO(2) polymer processing and microparticle-based scaffolds in a manner that allows each to solve the key limitation of the other. Specifically, microparticle-based scaffolds have suffered from the limitation that conventional microsphere sintering methods (e.g., heat, solvents) are not cytocompatible, yet we have shown that cell viability was sustained with subcritical (i.e., gaseous) CO(2) sintering of microspheres in the presence of cells at near-ambient temperatures. On the other hand, the fused microspheres provided the pore interconnectivity that has eluded supercritical CO(2) foaming approaches. Here, fused poly(lactide-co-glycolide) microsphere scaffolds were seeded with human umbilical cord mesenchymal stromal cells to demonstrate the feasibility of utilizing these matrices for cartilage regeneration. We also demonstrated that the approach may be modified to produce thin cell-loaded patches as a promising alternative for skin tissue engineering applications.