Pathogen Clostridioides Difficile Research Articles

Penicillin-binding proteins exhibit catalytic redundancy during asymmetric cell division in Clostridioides difficile.

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of SEDS family glycosyltransferases that polymerize glycan strands and class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While most bacteria encode multiple bPBPs that perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVD's catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by the catalytic activity of another sporulation-specific bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Proteolytic activity of surface-exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile.

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

Inhibition of Clostridioides difficile toxins TcdA and TcdB by the amiodarone derivative dronedarone.

The dreaded nosocomial pathogen Clostridioides difficile causes diarrhea and severe inflammation of the colon, especially after the use of certain antibiotics. The bacterium releases two deleterious toxins, TcdA and TcdB, into the gut, which are mainly responsible for the symptoms of C.difficile-associated diseases (CDADs). Both toxins are capable of entering independently into various host cells, e.g., intestinal epithelial cells, where they mono-O-glucosylate and inactivate Rho and/or Ras GTPases, important molecular switches for various cellular functions. We have shown recently that the cellular uptake of the Clostridioides difficile toxins TcdA and TcdB (TcdA/B) is inhibited by the licensed class III antiarrhythmic drug amiodarone (Schumacher et al. in Gut Microbes 15(2):2256695, 2023). Mechanistically, amiodarone delays the cellular uptake of both toxins into target cells most likely by lowering membrane cholesterol levels and by interfering with membrane insertion and/or pore formation of TcdA/B. However, serious side effects, such as thyroid dysfunction and severe pulmonary fibrosis, limit the clinical use of amiodarone in patients with C. difficile infection (CDI). For that reason, we aimed to test whether dronedarone, an amiodarone derivative with a more favorable side effect profile, is also capable of inhibiting TcdA/B. To this end, we tested in vitro with various methods the impact of dronedarone on the intoxication of Vero and CaCo-2 cells with TcdA/B. Importantly, preincubation of both cell lines with dronedarone for 1h at concentrations in the low micromolar range rendered the cells less sensitive toward TcdA/B-induced Rac1 glucosylation, collapse of the actin cytoskeleton, cell rounding, and cytopathic effects, respectively. Our study points toward the possibility of repurposing the already approved drug dronedarone as the preferable safer-to-use alternative to amiodarone for inhibiting TcdA/B in the (supportive) therapy of CDADs.

An organometallic hybrid antibiotic of metronidazole with a Gold(I) N-Heterocyclic Carbene overcomes metronidazole resistance in Clostridioides difficile

Antimicrobial resistance (AMR) has been emerging as a major global health threat and calls for the development of novel drug candidates. Metal complexes have been demonstrating high efficiency as antibacterial agents that differ substantially from the established types of antibiotics in their chemical structures and their mechanism of action. One strategy to exploit this potential is the design of metal-based hybrid organometallics that consist of an established antibiotic and a metal-based warhead that contributes an additional mechanism of action different from that of the parent antibiotic. In this communication, we describe the organometallic hybrid antibiotic 2c, in which the drug metronidazole is connected to a gold(I) N-heterocyclic carbene warhead that inhibits bacterial thioredoxin reductase (TrxR). Metronidazole can be used for the treatment with the obligatory anaerobic pathogen Clostridioides difficile (C. difficile), however, resistance to the drug hampers its clinical success. The gold organometallic conjugate 2c was an efficient inhibitor of TrxR and it was inactive or showed only minor effects against eucaryotic cells and bacteria grown under aerobic conditions. In contrast, a strong antibacterial effect was observed against both metronidazole-sensitive and -resistant strains of C. difficile. This report presents a proof-of-concept that the design of metal-based hybrid antibiotics can be a viable approach to efficiently tackle AMR.Graphical abstractA metronidazole-gold hybrid metalloantibiotic with high efficacy against resistant C. difficile

Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

Novel insights into phage biology of the pathogen Clostridioides difficile based on the active virome.

The global pathogen Clostridioides difficile is a well-studied organism, and researchers work on unraveling its fundamental virulence mechanisms and biology. Prophages have been demonstrated to influence C. difficile toxin expression and contribute to the distribution of advantageous genes. All these underline the importance of prophages in C. difficile virulence. Although several C. difficile prophages were sequenced and characterized, investigations on the entire active virome of a strain are still missing. Phages were mainly isolated after mitomycin C-induction, which does not resemble a natural stressor for C. difficile. We examined active prophages from different C. difficile strains after cultivation in the absence of mitomycin C by sequencing and characterization of particle-protected DNA. Phage particles were collected after standard cultivation, or after cultivation in the presence of the secondary bile salt deoxycholate (DCA). DCA is a natural stressor for C. difficile and a potential prophage-inducing agent. We also investigated differences in prophage activity between clinical and non-clinical C. difficile strains. Our experiments demonstrated that spontaneous prophage release is common in C. difficile and that DCA presence induces prophages. Fourteen different, active phages were identified by this experimental procedure. We could not identify a definitive connection between clinical background and phage activity. However, one phage exhibited distinctively higher activity upon DCA induction in the clinical strain than in the corresponding non-clinical strain, although the phage is identical in both strains. We recorded that enveloped DNA mapped to genome regions with characteristics of mobile genetic elements other than prophages. This pointed to mechanisms of DNA mobility that are not well-studied in C. difficile so far. We also detected phage-mediated lateral transduction of bacterial DNA, which is the first described case in C. difficile. This study significantly contributes to our knowledge of prophage activity in C. difficile and reveals novel aspects of C. difficile (phage) biology.

Exogenous butyrate inhibits butyrogenic metabolism and alters virulence phenotypes in Clostridioides difficile.

The gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood. A prominent class of gut microbiome-produced metabolites important for colonization resistance against C. difficile is short-chain fatty acids (SCFAs). In particular, one SCFA (butyrate) decreases the fitness of C. difficile in vitro and is correlated with C. difficile-inhospitable gut environments, both in mice and in humans. Here, we demonstrate that butyrate-dependent growth inhibition in C. difficile occurs under conditions where C. difficile also produces butyrate as a metabolic end product. Furthermore, we show that exogenous butyrate is internalized into C. difficile cells and is incorporated into intracellular CoA pools where it is metabolized in a reverse (energetically unfavorable) direction to crotonyl-CoA and (S)-3-hydroxybutyryl-CoA and/or 4-hydroxybutyryl-CoA. This internalization of butyrate and reverse metabolic flow of a butyrogenic pathway(s) in C. difficile coincides with alterations in toxin release and sporulation. Together, this work highlights butyrate as a marker of a C. difficile-inhospitable environment to which C. difficile responds by releasing its diarrheagenic toxins and producing environmentally resistant spores necessary for transmission between hosts. These findings provide foundational data for understanding the molecular and genetic basis of how C. difficile growth is inhibited by butyrate and how butyrate alters C. difficile virulence in the face of a highly competitive and dynamic gut environment.IMPORTANCEThe gut microbiome engenders colonization resistance against the diarrheal pathogen Clostridioides difficile, but the molecular basis of this colonization resistance is incompletely understood, which hinders the development of novel therapeutic interventions for C. difficile infection (CDI). We investigated how C. difficile responds to butyrate, an end-product of gut microbiome community metabolism which inhibits C. difficile growth. We show that exogenously produced butyrate is internalized into C. difficile, which inhibits C. difficile growth by interfering with its own butyrate production. This growth inhibition coincides with increased toxin release from C. difficile cells and the production of environmentally resistant spores necessary for transmission between hosts. Future work to disentangle the molecular mechanisms underlying these growth and virulence phenotypes will likely lead to new strategies to restrict C. difficile growth in the gut and minimize its pathogenesis during CDI.

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.

An Updated View on the Cellular Uptake and Mode-of-Action of Clostridioides difficile Toxins.

Research on the human gut pathogen Clostridioides (C.) difficile and its toxins continues to attract much attention as a consequence of the threat to human health posed by hypervirulent strains. Toxin A (TcdA) and Toxin B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (C. difficile transferase). C. difficile toxins are the causative agents of C. difficile-associated diseases (CDADs), such as antibiotics-associated diarrhea and pseudomembranous colitis. For that reason, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Many of these studies have been conducted in European laboratories. Here, we provide an update on our previous review (Papatheodorou et al. Adv Exp Med Biol, 2018) on important advances in C. difficile toxins research.

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile

The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions.

Exploring the inhibitory potential of the antiarrhythmic drug amiodarone against Clostridioides difficile toxins TcdA and TcdB

ABSTRACT The intestinal pathogen Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans. The symptoms of C. difficile-associated diseases (CDADs) are directly associated with the pathogen’s toxins TcdA and TcdB, which enter host cells and inactivate Rho and/or Ras GTPases by glucosylation. Membrane cholesterol is crucial during the intoxication process of TcdA and TcdB, and likely involved during pore formation of both toxins in endosomal membranes, a key step after cellular uptake for the translocation of the glucosyltransferase domain of both toxins from endosomes into the host cell cytosol. The licensed drug amiodarone, a multichannel blocker commonly used in the treatment of cardiac dysrhythmias, is also capable of inhibiting endosomal acidification and, as shown recently, cholesterol biosynthesis. Thus, we were keen to investigate in vitro with cultured cells and human intestinal organoids, whether amiodarone preincubation protects from TcdA and/or TcdB intoxication. Amiodarone conferred protection against both toxins independently and in combination as well as against toxin variants from the clinically relevant, epidemic C. difficile strain NAP1/027. Further mechanistic studies suggested that amiodarone’s mode-of-inhibition involves also interference with the translocation pore of both toxins. Our study opens the possibility of repurposing the licensed drug amiodarone as a novel pan-variant antitoxin therapeutic in the context of CDADs.

Expanding our grasp of two-component signaling in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile encodes roughly 50 TCS, but very few have been characterized in terms of their activating signals or their regulatory roles. A. G. Pannullo, B. R. Zbylicki, and C. D. Ellermeier (J Bacteriol 205:e00164-23, 2023, https://doi.org/10.1128/jb.00164-23) have identified both for the novel C. difficile TCD DraRS. DraRS responds to antibiotics that target lipid-II molecules in the bacterial cell envelope, and regulates the production of a novel glycolipid necessary for bacitracin and daptomycin resistance in C. difficile.

Dual RNA-seq identifies genes and pathways modulated during Clostridioides difficile colonization.

The gastrointestinal pathogen Clostridioides difficile is the most common cause of hospital-acquired diarrhea. Bacterial interactions with the gut mucosa are crucial for the establishment of C. difficile infection; however, key infection events like bacterial attachment and gut penetration are still poorly defined. To better understand the initial events that occur when this anaerobe interacts with the human gut epithelium, we employed a dual RNA-sequencing approach to study the bacterial and host transcriptomic profiles during C. difficile infection in a dual environment in vitro human gut model. Temporal changes in gene expression during infection were studied in bacterial and epithelial cells over 3-24 hours. While there were several common differentially expressed bacterial genes across different timepoints after infection, mammalian transcriptional profiles were quite distinct, with little overlap. Interestingly, an induction of colonic receptors for C. difficile toxins was observed, along with the downregulation of genes encoding immune response markers. Several cell wall-associated proteins were downregulated in C. difficile when associated with host cells, including slpA, which encodes the main S-layer protein. Gene function and pathway enrichment analyses revealed a potential modulation of the purine/pyrimidine synthesis pathways both in the mammalian and bacterial cells. We observed that proline-proline endopeptidase, a secreted metalloprotease responsible for cell surface protein cleavage, is downregulated during infection, and a mutant lacking this enzyme demonstrated enhanced adhesion to epithelial cells during infection. This study provides new insight into the host and bacterial pathways based on gene expression modulation during the initial contact of C. difficile with gut cells. IMPORTANCE The initial interactions between the colonic epithelium and the bacterium are likely critical in the establishment of Clostridioides difficile infection, one of the major causes of hospital-acquired diarrhea worldwide. Molecular interactions between C. difficile and human gut cells have not been well defined mainly due to the technical challenges of studying cellular host-pathogen interactions with this anaerobe. Here we have examined transcriptional changes occurring in the pathogen and host cells during the initial 24 hours of infection. Our data indicate several changes in metabolic pathways and virulence-associated factors during the initial bacterium-host cell contact and early stages of infection. We describe canonical pathways enriched based on the expression profiles of a dual RNA sequencing in the host and bacterium, and functions of bacterial factors that are modulated during infection. This study thus provides fresh insight into the early C. difficile infection process.

Gut microbiota interspecies interactions shape the response of Clostridioides difficile to clinically relevant antibiotics.

In the human gut, the growth of the pathogen Clostridioides difficile is impacted by a complex web of interspecies interactions with members of human gut microbiota. We investigate the contribution of interspecies interactions on the antibiotic response of C. difficile to clinically relevant antibiotics using bottom-up assembly of human gut communities. We identify 2 classes of microbial interactions that alter C. difficile's antibiotic susceptibility: interactions resulting in increased ability of C. difficile to grow at high antibiotic concentrations (rare) and interactions resulting in C. difficile growth enhancement at low antibiotic concentrations (common). Based on genome-wide transcriptional profiling data, we demonstrate that metal sequestration due to hydrogen sulfide production by the prevalent gut species Desulfovibrio piger increases the minimum inhibitory concentration (MIC) of metronidazole for C. difficile. Competition with species that display higher sensitivity to the antibiotic than C. difficile leads to enhanced growth of C. difficile at low antibiotic concentrations due to competitive release. A dynamic computational model identifies the ecological principles driving this effect. Our results provide a deeper understanding of ecological and molecular principles shaping C. difficile's response to antibiotics, which could inform therapeutic interventions.

Regulatory Role of Anti-Sigma Factor RsbW in Clostridioides difficile Stress Response, Persistence, and Infection.

The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σB) is employed to modulate gene transcription, and σB is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW), in which σB is assumed to be "always on," was generated. ΔrsbW did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbW was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbW showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σB-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to ΔrsbW, changes in some σB-controlled stress-associated genes were similar to those reported in the absence of σB. Further analysis of ΔrsbW showed unexpected lower intracellular levels of σB, suggesting an additional post-translational control mechanism for σB in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. IMPORTANCE Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σB) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence, and virulence and suggest alternate σB control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.

Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling

Anaerobic microbial metabolism drives critical functions within global ecosystems, host–microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.

Tomato-fruit-derived extracellular vesicles inhibit Fusobacterium nucleatum via lipid-mediated mechanism.

Nano-sized extracellular vesicles (EV) are essential for cell communication. Studies on EV from natural sources including edible plants are gaining momentum due to the biological implications. In this study, EV from tomato fruit were isolated by ultracentrifugation and their physical and morphological features along with their biocargo profiles were analyzed. We found that tomato EV promote the growth of probiotic Lactobacillus species, while inhibiting growth of the opportunistic intestinal pathogens Clostridioides difficile and Fusobacterium nucleatum. Tomato EV reversed microbiota dysbiosis caused by F. nucleatum in a simulator of the gut microbiota fermentation model. Phospholipid analysis of tomato EV revealed that the anti-bacterial effect of tomato-EV was driven by the presence of specific lipids in the EV, as demonstrated by lipid depletion and reconstitution experiments. The findings suggest the potential of tomato-derived EV for treating gut microbiota dysbiosis and preventing intestinal bacterial infections.

Rounding out cyclic dinucleotide signaling in Clostridioides difficile: A role and a mechanism for c-di-AMP.

The second messenger c-di-AMP contributes to various homeostatic and stress responses in bacteria. In this issue of Science Signaling, Oberkampf et al. have identified it as a mediator of osmotic stress and bile salt resistance in the opportunistic pathogen Clostridioides difficile, with additional roles in cell wall homeostasis and biofilm formation.

Battling Enteropathogenic Clostridia: Phage Therapy for Clostridioides difficile and Clostridium perfringens.

The pathogenic Clostridioides difficile and Clostridium perfringens are responsible for many health care-associated infections as well as systemic and enteric diseases. Therefore, they represent a major health threat to both humans and animals. Concerns regarding increasing antibiotic resistance (related to C. difficile and C. perfringens) have caused a surge in the pursual of novel strategies that effectively combat pathogenic infections, including those caused by both pathogenic species. The ban on antibiotic growth promoters in the poultry industry has added to the urgency of finding novel antimicrobial therapeutics for C. perfringens. These efforts have resulted in various therapeutics, of which bacteriophages (in short, phages) show much promise, as evidenced by the Eliava Phage Therapy Center in Tbilisi, Georgia (https://eptc.ge/). Bacteriophages are a type of virus that infect bacteria. In this review, the (clinical) impact of clostridium infections in intestinal diseases is recapitulated, followed by an analysis of the current knowledge and applicability of bacteriophages and phage-derived endolysins in this disease indication. Limitations of phage and phage endolysin therapy were identified and require considerations. These include phage stability in the gastrointestinal tract, influence on gut microbiota structure/function, phage resistance development, limited host range for specific pathogenic strains, phage involvement in horizontal gene transfer, and—for phage endolysins—endolysin resistance, -safety, and -immunogenicity. Methods to optimize features of these therapeutic modalities, such as mutagenesis and fusion proteins, are also addressed. The future success of phage and endolysin therapies require reliable clinical trial data for phage(-derived) products. Meanwhile, additional research efforts are essential to expand the potential of exploiting phages and their endolysins for mitigating the severe diseases caused by C. difficile and C. perfringens.