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.

Sound the (Smaller) Alarm: The Triphosphate Magic Spot Nucleotide pGpp.

It has recently become evident that the bacterial stringent response is regulated by a triphosphate alarmone (pGpp) as well as the canonical tetra- and pentaphosphate alarmones ppGpp and pppGpp [together, (p)ppGpp]. Often dismissed in the past as an artifact or degradation product, pGpp has been confirmed as a deliberate endpoint of multiple synthetic pathways utilizing GMP, (p)ppGpp, or GDP/GTP as precursors. Some early studies concluded that pGpp functionally mimics (p)ppGpp and that its biological role is to make alarmone metabolism less dependent on the guanine energy charge of the cell by allowing GMP-dependent synthesis to continue when GDP/GTP has been depleted. However, recent reports that pGpp binds unique potential protein receptors and is the only alarmone synthesized by the intestinal pathogen Clostridioides difficile indicate that pGpp is more than a stand-in for the longer alarmones and plays a distinct biological role beyond its functional overlap (p)ppGpp.

Unique Features of Alarmone Metabolism in Clostridioides difficile.

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. IMPORTANCE Despite the role of the stringent response in antibiotic survival and recurrent infections, it has been a challenging target for antibacterial therapies because it is so ubiquitous. This is an especially relevant consideration for the treatment of Clostridioides difficile infection (CDI), as exposure to broad-spectrum antibiotics that harm commensal microbes is a major risk factor for CDI. Here, we report that both of the alarmone synthetase enzymes that mediate the stringent response in this organism employ a unique mechanism that requires the hydrolysis of two phosphate bonds and synthesize the triphosphate alarmone pGpp exclusively. Inhibitors targeted against these noncanonical synthetases have the potential to be highly specific and minimize detrimental effects to stringent response pathways in commensal microbes.

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCEClostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

Single cell analysis of nutrient regulation of Clostridioides (Clostridium) difficile motility.

Regulation of bacterial motility to maximize nutrient acquisition or minimize exposure to harmful substances plays an important role in microbial proliferation and host colonization. The technical difficulties of performing high-resolution live microscopy on anaerobes have hindered mechanistic studies of motility in Clostridioides (formerly Clostridium) difficile. Here, we present a widely applicable protocol for live cell imaging of anaerobic bacteria that has allowed us to characterize C. difficile swimming at the single-cell level. This accessible method for anaerobic live cell microscopy enables inquiry into previously inaccessible aspects of C. difficile physiology and behavior. We present the first report that vegetative C. difficile are capable of regulated motility in the presence of different nutrients. We demonstrate that the epidemic C. difficile strain R20291 exhibits regulated motility in the presence of multiple nutrient sources by modulating its swimming velocity. This is a powerful illustration of the ability of single-cell studies to explain population-wide phenomena such as dispersal through the environment.

How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3.

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide-copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue.

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase.

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability.

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile.

UNLABELLED: The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCE: How Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

Cyclic di-GMP riboswitch-regulated type IV pili contribute to aggregation of Clostridium difficile.

Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile.

Nanosecond pulsed electric fields increase antibiotic susceptibility in methicillin-resistant Staphylococcus aureus.

IMPORTANCE: We have found that treatment with short electric pulses potentiates the effects of multiple antibiotics against methicillin-resistant Staphylococcus aureus. By reducing the dose of antibiotic necessary to be effective, co-treatment with electric pulses could amplify the effects of standard antibiotic dosing to treat S. aureus infections such as skin and soft-tissue infections (SSTIs). SSTIs are accessible to physical intervention and are good candidates for electric pulse co-treatment, which could be adopted as a step-in wound and abscess debridement.

RelQ-mediated alarmone signaling regulates growth, sporulation, and stress-induced biofilm formation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signaling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors, and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sub-lethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and sporulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

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.

The second messenger cyclic Di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD.

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile.

Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection.

Second messenger signaling in Clostridioides difficile.

Small, diffusible second messenger molecules transmit information about extracellular conditions to intracellular machinery in order to influence transcription, translation, and metabolism. The enteropathogenic bacterium Clostridioides difficile coordinates its response to a dynamic and hostile environment via nucleotide second messengers. While riboswitch-mediated cyclic diguanylate regulation has been extensively characterized in C. difficile, signaling by cyclic diadenylate and by guanosine alarmones has only recently been confirmed in this organism. This review summarizes the current knowledge of how nucleotide second messenger signaling regulates physiological processes in C. difficile.

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.

Growth in a biofilm sensitizes Cutibacterium acnes to nanosecond pulsed electric fields.

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells.

An analysis of the solution structure and signaling mechanism of LovK, a sensor histidine kinase integrating light and redox signals.

Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria. These approximately 100-residue photosensory modules are generally encoded within larger, multidomain proteins that control a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes a soluble LOV-histidine kinase, LovK, that regulates the adhesive properties of the cell. Full-length LovK is dimeric as are a series of systematically truncated LovK constructs containing only the N-terminal LOV sensory domain. Nonconserved sequence flanking the LOV domain functions to tune the signaling lifetime of the protein. Size exclusion chromatography and small-angle X-ray scattering (SAXS) demonstrate that the LOV sensor domain does not undergo a large conformational change in response to photon absorption. However, limited proteolysis identifies a sequence flanking the C-terminus of the LOV domain as a site of light-induced change in protein conformation and dynamics. On the basis of SAXS envelope reconstruction and bioinformatic prediction, we propose this dynamic region of structure is an extended C-terminal coiled coil that links the LOV domain to the histidine kinase domain. To test the hypothesis that LOV domain signaling is affected by cellular redox state in addition to light, we measured the reduction potential of the LovK FMN cofactor. The measured potential of -258 mV is congruent with the redox potential of Gram-negative cytoplasm during logarithmic growth (-260 to -280 mV). Thus, a fraction of LovK in the cytosol may be in the reduced state under typical growth conditions. Chemical reduction of the FMN cofactor of LovK attenuates the light-dependent ATPase activity of the protein in vitro, demonstrating that LovK can function as a conditional photosensor that is regulated by the oxidative state of the cellular environment.

Electronic and protein structural dynamics of a photosensory histidine kinase.

The bacterium Caulobacter crescentus encodes a two-component signaling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.