Unique Features of Alarmone Metabolism in \u3ci\u3eClostridioides difficile\u3c/i\u3e

The “magic spot” alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by the RelA-SpoT homolog (RSH) of C. difficile (RSHCd) and RelQCd. These enzymes are transcriptionally activated by diverse environmental stresses. RSHCd has previously been reported to synthesize ppGpp, but in this study, we found that both clostridial enzymes exclusively synthesize pGpp. While direct synthesis of pGpp from a GMP substrate, and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases, have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5′ phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes alarmone signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms

Characterizing the Activity of Antimicrobial Peptides Against the Pathogenic Bacterium \u3ci\u3eClostridium difficile\u3c/i\u3e in an Anaerobic Environment

In our lab we study oxidative stress response of Clostridioides difficile in relation to biofilm formation. Oxidative stress is the imbalance between oxidation and anti-oxidation in the bacterial system. Biofilm are extracellular matrices produced by the bacteria. C. difficile forms biofilm one of the phenotypic expression response to oxidative stress. We use peptides and metals as the major source of oxidative stress. Metals like copper and silver which are already known for their antimicrobial effect, showed stimulation of biofilm at sub-inhibitory concentrations while we notice no difference in biofilm formation when exposed to magnesium. Piscidin one of the host defense peptides (HDPs) produced by the innate immune system combats pathogens entering the host body by inducing oxidative stress. The peptide did not show significant biofilm stimulation suggesting that the response of the bacteria to different sources of oxidative stress may vary and this is worthy of more investigations

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C. difficile to clinically relevant antibiotics tested at sublethal concentrations. Both peptides bind to Cu2+ using an amino terminal copper and nickel binding motif. Here, we investigate the two peptides in the apo and holo states as antibiotic adjuvants against an epidemic strain of C. difficile. We find that the presence of the peptides leads to lower doses of metronidazole, vancomycin, and fidaxomicin to kill C. difficile. The activity of metronidazole, which targets DNA, is enhanced by a factor of 32 when combined with p3, previously shown to bind and condense DNA. Conversely, the activity of vancomycin, which acts at bacterial cell walls, is enhanced 64-fold when combined with membrane-active p1-Cu2+. As shown through microscopy monitoring the permeabilization of membranes of C. difficile cells and vesicle mimics of their membranes, the adjuvant effect of p1 and p3 in the apo and holo states is consistent with a mechanism of action where the peptides enable greater antibiotic penetration through the cell membrane to increase their bioavailability. The variations in effects obtained with the different forms of the peptides reveal that while all piscidins generally sensitize C. difficile to antibiotics, co-treatments can be optimized in accordance with the underlying mechanism of action of the peptides and antibiotics. Overall, this study highlights the potential of antimicrobial peptides as antibiotic adjuvants to increase the lethality of currently approved antibiotic dosages, reducing the risk of incomplete treatments and ensuing drug resistance