C. difficile exploits a host metabolite produced during toxin-mediated disease.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.

Tc toxin activation requires unfolding and refolding of a ß-propeller.

Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.

Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance.

Within bacterial populations, a small fraction of persister cells is transiently capable of surviving exposure to lethal doses of antibiotics. As a bet-hedging strategy, persistence levels are determined both by stochastic induction and by environmental stimuli called responsive diversification. Little is known about the mechanisms that link the low frequency of persisters to environmental signals. Our results support a central role for the conserved GTPase Obg in determining persistence in Escherichia coli in response to nutrient starvation. Obg-mediated persistence requires the stringent response alarmone (p)ppGpp and proceeds through transcriptional control of the hokB-sokB type I toxin-antitoxin module. In individual cells, increased Obg levels induce HokB expression, which in turn results in a collapse of the membrane potential, leading to dormancy. Obg also controls persistence in Pseudomonas aeruginosa and thus constitutes a conserved regulator of antibiotic tolerance. Combined, our findings signify an important step toward unraveling shared genetic mechanisms underlying persistence.

Remarkable Functional Convergence: Alarmone ppGpp Mediates Persistence by Activating Type I and II Toxin-Antitoxins.

In this issue of Molecular Cell, Verstraeten et al. (2015) demonstrate that the conserved GTPase Obg and the second messenger ppGpp mediate persistence by activation of a type I toxin-antitoxin module (hokB/sokB) in E. coli.

Neobavaisoflavone Inhibits Biofilm Formation and α-Toxin Activity of Staphylococcus aureus.

Neobavaisoflavone had antimicrobial activities against Gram-positive multidrug-resistant (MDR) bacteria, but the effect of neobavaisoflavone on the virulence and biofilm formation of S. aureus has not been explored. The present study aimed to investigate the possible inhibitory effect of neobavaisoflavone on the biofilm formation and α-toxin activity of S. aureus. Neobavaisoflavone presented strong inhibitory effect on the biofilm formation and α-toxin activity of both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains at 25 µM, but did not affect the growth of S. aureus planktonic cells. Genetic mutations were identified in four coding genes, including cell wall metabolism sensor histidine kinase walK, RNA polymerase sigma factor rpoD, tetR family transcriptional regulator, and a hypothetical protein. The mutation of WalK (K570E) protein was identified and verified in all the neobavaisoflavone-induced mutant S. aureus isolates. The ASN501, LYS504, ILE544 and GLY565 of WalK protein act as hydrogen acceptors to form four hydrogen bonds with neobavaisoflavone by molecular docking analysis, and TRY505 of WalK protein contact with neobavaisoflavone to form a pi-H bond. In conclusion, neobavaisoflavone had excellent inhibitory effect on the biofilm formation and α-toxin activity of S. aureus. The WalK protein might be a potential target of neobavaisoflavone against S. aureus.

MazF toxin causes alterations in Staphylococcus aureus transcriptome, translatome and proteome that underlie bacterial dormancy.

Antibiotic resistance is a serious problem which may be caused by bacterial dormancy. It has been suggested that bacterial toxin-antitoxin systems induce dormancy. We analyzed the genome-wide role of Staphylococcus aureus endoribonuclease toxin MazF using RNA-Seq, Ribo-Seq and quantitative proteomics. We characterized changes in transcriptome, translatome and proteome caused by MazF, and proposed that MazF decreases translation directly by cleaving mRNAs, and indirectly, by decreasing translation factors and by promoting ribosome hibernation. Important pathways affected during the early stage of MazF induction were identified: MazF increases cell wall thickness and decreases cell division; MazF activates SsrA-system which rescues stalled ribosomes, appearing as a result of MazF mRNA cleavage. These pathways may be promising targets for new antibacterial drugs that prevent bacteria dormancy. Finally, we described the overall impact of MazF on S. aureus cell physiology, and propose one of the mechanisms by which MazF might regulate cellular changes leading to dormancy.

An RNA thermometer dictates production of a secreted bacterial toxin.

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.

Assembly and Function of the Anthrax Toxin Protein Translocation Complex.

Anthrax toxin is a major virulence factor of Bacillus anthracis, a Gram-positive bacterium which can form highly stable spores that are the causative agents of the disease, anthrax. While chiefly a disease of livestock, spores can be "weaponized" as a bio-terrorist agent, and can be deadly if not recognized and treated early with antibiotics. The intracellular pathways affected by the enzymes are broadly understood and are not discussed here. This chapter focuses on what is known about the assembly of secreted toxins on the host cell surface and how the toxin is delivered into the cytosol. The central component is the "Protective Antigen", which self-oligomerizes and forms complexes with its pay-load, either Lethal Factor or Edema Factor. It binds a host receptor, CMG2, or a close relative, triggering receptor-mediated endocytosis, and forms a remarkably elegant yet powerful machine that delivers toxic enzymes into the cytosol, powered only by the pH gradient across the membrane. We now have atomic structures of most of the starting, intermediate and final assemblies in the infectious process. Together with a major body of biophysical, mutational and biochemical work, these studies reveal a remarkable story of both how toxin assembly is choreographed in time and space.

Mother-to-infant transmission of the carcinogenic colibactin-producing bacteria.

BACKGROUND: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. RESULTS: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. CONCLUSIONS: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

Polycyclic polyprenylated acylphloroglucinol congeners from Garcinia yunnanensis Hu with inhibitory effect on α-hemolysin production in Staphylococcus aureus.

α-Hemolysin (Hla) is an extracellular protein secreted by methicillin-resistant Staphylococcus aureus (MRSA) strains that plays a critical role in the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections, rendering Hla a potential therapeutic target. In this study, 10 unreported polycyclic polyprenylated acylphloroglucinol (PPAP) derivatives, garciyunnanins C-L (1-10), with diverse skeletons, were isolated from Garcinia yunnanensis Hu. The structures of these new compounds were determined by HRMS, NMR, electronic circular dichroism (ECD) calculations, single-crystal X-ray diffraction, and biomimetic transformation. Garciyunnanins C and D (1 and 2) were found to be potent Hla inhibitors in the anti-virulence efficacy evaluation against MRSA strain.

Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Comparative clinical outcomes evaluation of hospitalized patients infected with Clostridioides difficile ribotype 106 vs. other toxigenic strains.

BACKGROUND: Although Clostridioides difficile surveillance often identifies emerging strains, clinical outcome evaluations are rarely performed. Ribotype (RT) 106 is a commonly isolated C. difficile strain worldwide; however, studies investigating RT 106 clinical outcomes are limited. The purpose of this study was to investigate clinical outcomes of RT 106 infections compared with two other endemic strains of varying virulence. METHODS: This multicenter study evaluated adults hospitalized with C. difficile infection (CDI). C. difficile samples underwent PCR ribotyping and patients infected with RT 106 were compared to patients infected with a known hypervirulent strain (RT 027) and a strain associated with less virulence (RT 014-020). Electronic medical records were reviewed by blinded investigators to assess the primary outcome of poor clinical outcome (composite of initial clinical failure, discharge to a higher level of care, 90-day CDI recurrence, and CDI-contributable mortality). RESULTS: A total of 396 patients with CDI were identified (RT 106, 32.3%; RT 027, 29.3%; RT 014-020, 38.3%). Patients infected with RT 014-020 less often experienced a poor clinical outcome (40%) compared with RT 106 (56%) and RT 027 (65%) infection (P < 0.0001). After controlling for covariates and using RT 014-020 as a comparator, patients infected with RT 106 (OR, 2.25; 95% CI, 1.36-3.73) or RT 027 (OR, 2.56; 95% CI, 1.52-4.31) had higher odds of poor clinical outcome. Using RT 027 as the comparator, only RT 014-020 was associated with lower odds of poor clinical outcome (OR, 0.42; 95% CI, 0.27-0.65). CONCLUSION: This study demonstrated that the emergent C. difficile RT 106 was associated with increased rates of poor clinical outcomes compared to RT 014-020 and comparable poor clinical outcomes to RT 027. These findings can help to better understand the clinical significance of this and future emerging ribotypes.

Production of recombinant lethal factor of Bacillus anthracis in Bacillus subtilis.

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.

The 5' NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates.

Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5' ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium's physiology, was found to be 5' NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the -1 position of the P3 promoter. An increase in RNAIII's NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII's secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII's secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria.IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5' NAD cap in specific RNAs. While the presence of the 5' NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5' NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium's virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

Panton-Valentine Leukocidin-Secreting Staphylococcus aureus Pneumonia Complicating COVID-19.

Necrotizing pneumonia induced by Panton-Valentine leukocidin-secreting Staphylococcus aureus is a rare but life-threatening infection that has been described in patients after they had influenza. We report a fatal case of this superinfection in a young adult who had coronavirus disease.

Biochemical determinants of ObgE-mediated persistence.

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence.

The Impact of pH on Clostridioides difficile Sporulation and Physiology.

Clostridioides difficile is a pathogenic bacterium that infects the human colon to cause diarrheal disease. Growth of the bacterium is known to be dependent on certain bile acids, oxygen levels, and nutrient availability in the intestine, but how the environmental pH can influence C. difficile is mostly unknown. Previous studies indicated that C. difficile modulates the intestinal pH, and prospective cohort studies have found a strong association between a more alkaline fecal pH and C. difficile infection. Based on these data, we hypothesized that C. difficile physiology can be affected by various pH conditions. In this study, we investigated the impact of a range of pH conditions on C. difficile to assess potential effects on growth, sporulation, motility, and toxin production in the strains 630Δerm and R20291. We observed pH-dependent differences in sporulation rate, spore morphology, and viability. Sporulation frequency was lowest under acidic conditions, and differences in cell morphology were apparent at low pH. In alkaline environments, C. difficile sporulation was greater for strain 630Δerm, whereas R20291 produced relatively high levels of spores in a broad range of pH conditions. Rapid changes in pH during exponential growth impacted sporulation similarly among the strains. Furthermore, we observed an increase in C. difficile motility with increases in pH, and strain-dependent differences in toxin production under acidic conditions. The data demonstrate that pH is an important parameter that affects C. difficile physiology and may reveal relevant insights into the growth and dissemination of this pathogen.IMPORTANCEClostridioides difficile is an anaerobic bacterium that causes gastrointestinal disease. C. difficile forms dormant spores which can survive harsh environmental conditions, allowing their spread to new hosts. In this study, we determine how intestinally relevant pH conditions impact C. difficile physiology in the two divergent strains, 630Δerm and R20291. Our data demonstrate that low pH conditions reduce C. difficile growth, sporulation, and motility. However, toxin production and spore morphology were differentially impacted in the two strains at low pH. In addition, we observed that alkaline environments reduce C. difficile growth, but increase cell motility. When pH was adjusted rapidly during growth, we observed similar impacts on both strains. This study provides new insights into the phenotypic diversity of C. difficile grown under diverse pH conditions present in the intestinal tract, and demonstrates similarities and differences in the pH responses of different C. difficile isolates.

Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori.

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His(6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5-6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30-40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants.

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge.

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

Toxin production spontaneously becomes regulated by local cell density in evolving bacterial populations.

The production of anticompetitor toxins is widespread among bacteria. Because production of such toxins is costly, it is typically regulated. In particular, many toxins are produced only when the local cell density is high. It is unclear which selection pressures shaped the evolution of density-dependent regulation of toxin production. Here, we study the evolution of toxin production, resistance and the response to a cell-density cue in a model of an evolving bacterial population with spatial structure. We present results for two growth regimes: (i) an undisturbed, fixed habitat in which only small fluctuations of cell density occur, and (ii) a serial-transfer regime with large fluctuations in cell density. We find that density-dependent toxin production can evolve under both regimes. However, the selection pressures driving the evolution of regulation differ. In the fixed habitat, regulation evolves because it allows cells to produce toxin only when opportunities for reproduction are highly limited (because of a high local cell density), and the effective fitness costs of toxin production are hence low. Under serial transfers, regulation evolves because it allows cells to switch from a fast-growing non-toxic phenotype when colonising a new habitat, to a slower-growing competitive toxic phenotype when the cell density increases. Colonies of such regulating cells rapidly expand into unoccupied space because their edges consist of fast-growing, non-toxin-producing cells, but are also combative because cells at the interfaces with competing colonies do produce toxin. Because under the two growth regimes different types of regulation evolve, our results underscore the importance of growth conditions in the evolution of social behaviour in bacteria.