Recombinant Fusion Protein Vaccine Containing Clostridioides difficile FliC and FliD Protects Mice against C. difficile Infection.

Bacterial flagella are involved in infection through their roles in host cell adhesion, cell invasion, auto-agglutination, colonization, the formation of biofilms, and the regulation and secretion of nonflagellar bacterial proteins that are involved in the virulence process. In this study, we constructed a fusion protein vaccine (FliCD) containing the Clostridioides difficile flagellar proteins FliC and FliD. The immunization of mice with FliCD induced potent IgG and IgA antibody responses against FliCD, protected mice against C. difficile infection (CDI), and decreased the C. difficile spore and toxin levels in the feces after infection. Additionally, the anti-FliCD serum inhibited the binding of C. difficile vegetative cells to HCT8 cells. These results suggest that FliCD may represent an effective vaccine candidate against CDI.

Inhibition of Hypoxia-Inducible Transcription Factor (HIF-1α) Signaling with Sulfonyl-γ-AApeptide Helices.

The development of inhibitors that selectively block protein-protein interactions (PPIs) is crucial for chemical biology, medicinal chemistry, and biomedical sciences. Herein, we reported the design, synthesis, and investigation of sulfonyl-γ-AApeptide as an alternative strategy of canonical peptide-based inhibitors to disrupt hypoxia-inducible factor 1α (HIF-1α) and p300 PPI by mimicking the helical domain of HIF-1α involved in the binding to p300. The designed molecules recognized the p300 protein with high affinity and potently inhibited the hypoxia-inducible signaling pathway. Gene expression profiling supported the idea that the lead molecules selectively inhibited hypoxia-inducible genes involved in the signaling cascade. Our studies also demonstrated that both helical faces consisting of either chiral side chains or achiral sulfonyl side chains of sulfonyl-γ-AApeptides could be adopted for mimicry of the α-helix engaging in PPIs. Furthermore, these sulfonyl-γ-AApeptides were cell-permeable and exhibited favorable stability and pharmacokinetic profiles. Our results could inspire the design of helical sulfonyl-γ-AApeptides as a general strategy to mimic the protein helical domain and modulate many other PPIs.

Regulatory transcription factors of Clostridioides difficile pathogenesis with a focus on toxin regulation.

Clostridioides difficile (CD), a nosocomial gut pathogen, produces two major exotoxins, TcdA and TcdB, which disrupt the gut epithelial barrier and induce inflammatory/immune responses, leading to symptoms ranging from mild diarrhoea to pseudomembranous colitis and potentially to death. The expression of toxins is regulated by various transcription factors (TFs) which are induced in response to CD physiological life stages, nutritional availability, and host environment. This review summarises our current understanding on the regulation of toxin expression by TFs that interconnect with pathways of flagellar synthesis, quorum sensing, motility, biofilm formation, sporulation, and phase variation. The pleiotropic roles of some key TFs suggest that toxin production is tightly linked to other cellular processes of the CD physiology.

This review summarises the current knowledge of the transcription factors involved in regulation of toxin production, which is affected by C. difficile physiological life stages, nutritional availability, and host environment in the gut.

Short, Lipidated Dendrimeric γ-AApeptides as New Antimicrobial Peptidomimetics.

Antibiotic resistance is one of the most significant issues encountered in global health. There is an urgent demand for the development of a new generation of antibiotic agents combating the emergence of drug resistance. In this article, we reported the design of lipidated dendrimeric γ-AApeptides as a new class of antimicrobial agents. These AApeptides showed excellent potency and broad-spectrum activity against both Gram-positive bacteria and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The mechanistic studies revealed that the dendrimeric AApeptides could kill bacteria rapidly through the permeabilization of bacterial membranes, analogous to host-defense peptides (HDPs). These dendrimers also did not induce antibiotic resistance readily. The easy access to the synthesis, together with their potent and broad-spectrum activity, make these lipidated dendrimeric γ-AApeptides a new generation of antibacterial agents.

Recent developments in systems biology and genetic engineering toward design of vaccines for TB.

Tuberculosis (TB) is one of the most prevalent diseases worldwide. The currently available Bacillus Calmette-Guérin vaccine is not sufficient in protecting against pulmonary TB. Although many vaccines have been evaluated in clinical trials, but none of them yet has proven to be more successful. Thus, new strategies are urgently needed to design more effective TB vaccines. The emergence of new technologies will undoubtedly accelerate the process of vaccine development. This review summarizes the potential and validated applications of emerging technologies, including: systems biology (genomics, proteomics, and transcriptomics), genetic engineering, and other computational tools to discover and develop novel vaccines against TB. It also discussed that the significant implementation of these approaches will play crucial roles in the development of novel vaccines to cure and control TB.

Chemistry and Bioactivity of the Deep-Water Antarctic Octocoral Alcyonium sp.

Chemical investigation of an Antarctic deep-water octocoral has led to the isolation of four new compounds, including three illudalane sesquiterpenoids (1-3) related to the alcyopterosins, a highly oxidized steroid, alcyosterone (5), and five known alcyopterosins (4, 6-9). The structures were established by extensive 1D and 2D NMR analyses, while 9 was verified by XRD. Alcyopterosins are unusual for their nitrate ester functionalization and have been characterized with cytotoxicity related to their DNA binding properties. Alcyopterosins V (3) and E (4) demonstrated single-digit micromolar activity against Clostridium difficile, an intestinal bacterium capable of causing severe diarrhea that is increasingly associated with drug resistance. Alcyosterone (5) and several alcyopterosins were similarly potent against the protist Leishmania donovani, the causative agent of leishmaniasis, a disfiguring disease that can be fatal if not treated. While the alcyopterosin family of sesquiterpenes is known for mild cytotoxicity, the observed activity against C. difficile and L. donovani is selective for the infectious agents.

Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.

Mechanisms of antibiotic resistance of Clostridioides difficile.

Clostridioides difficile (CD) is one of the top five urgent antibiotic resistance threats in USA. There is a worldwide increase in MDR of CD, with emergence of novel strains which are often more virulent and MDR. Antibiotic resistance in CD is constantly evolving with acquisition of novel resistance mechanisms, which can be transferred between different species of bacteria and among different CD strains present in the clinical setting, community, and environment. Therefore, understanding the antibiotic resistance mechanisms of CD is important to guide optimal antibiotic stewardship policies and to identify novel therapeutic targets to combat CD as well as other bacteria. Epidemiology of CD is driven by the evolution of antibiotic resistance. Prevalence of different CD strains and their characteristic resistomes show distinct global geographical patterns. Understanding epidemiologically driven and strain-specific characteristics of antibiotic resistance is important for effective epidemiological surveillance of antibiotic resistance and to curb the inter-strain and -species spread of the CD resistome. CD has developed resistance to antibiotics with diverse mechanisms such as drug alteration, modification of the antibiotic target site and extrusion of drugs via efflux pumps. In this review, we summarized the most recent advancements in the understanding of mechanisms of antibiotic resistance in CD and analysed the antibiotic resistance factors present in genomes of a few representative well known, epidemic and MDR CD strains found predominantly in different regions of the world.

Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.

TPL2 Is a Key Regulator of Intestinal Inflammation in Clostridium difficile Infection.

Tumor progression locus 2 (TPL2), a serine/threonine protein kinase, is a major inflammatory mediator in immune cells. The predominant inflammatory actions of TPL2 depend on the activation of mitogen-activated protein kinases (MAPK) and the upregulated production of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1ß (IL-1ß) in macrophages and dendritic cells in response to lipopolysaccharide (LPS). Significant increases in TNF-α, IL-6, IL-ß, and IL-8 levels in patients with Clostridium difficile infection (CDI) have been reported. Both TNF-α and IL-6 have been postulated to play key roles in the systemic inflammatory response in CDI, and IL-8 is essential for the development of local intestinal inflammatory responses in CDI. The objective of this study was to elucidate the role of TPL2 in the pathogenesis of CDI. We found that TPL2 was significantly activated in human and mouse intestinal tissues upon C. difficile toxin exposure or CDI. We further demonstrated that TPL2 knockout (TPL2-KO) mice were significantly more resistant to CDI than wild-type mice, with significantly reduced production of TNF-α, IL-6, IL-1ß, KC (a mouse homologue of IL-8), and myeloperoxidase (MPO) in the ceca and colons of TPL2-KO mice. Finally, we found that TPL2 inhibition by a specific inhibitor or TPL2 gene ablation significantly reduced TcdB-induced production of TNF-α, IL-6, IL-ß, and KC by inhibiting the activation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK). Taken together, our data suggest that TPL2 represents a potential therapeutic target for CDI treatment.

Oral Immunization with Nontoxigenic Clostridium difficile Strains Expressing Chimeric Fragments of TcdA and TcdB Elicits Protective Immunity against C. difficile Infection in Both Mice and Hamsters.

The symptoms of Clostridium difficile infection (CDI) are attributed largely to two C. difficile toxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However, C. difficile is an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target both C. difficile toxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenic C. difficile (NTCD) strain, resulting in strain NTCD_mTcd138. Oral immunization with spores of NTCD_mTcd138 provided mice full protection against infection with a hypervirulent C. difficile strain, UK6 (ribotype 027). The protective strength and efficacy of NTCD_mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD_mTcd138 also provided hamsters significant protection against infection with 2 × 104 UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenic C. difficile strain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI.

Facilely accessible quinoline derivatives as potent antibacterial agents.

Quinoline compounds have been extensively explored as anti-malaria and anti-cancer agents for decades and show profound functional bioactivities, however, the studies of these compounds in other medicinal fields have lagged dramatically. In this study, we report the development of a series of facilely accessible quinoline derivatives that display potent antibacterial activity against a panel of multidrug-resistant Gram-positive bacterial strains, especially C. difficile. We also demonstrated that these molecules are effective in vivo against C. difficile. These results revealed that these types of quinoline compounds could serve as prototypes for the development of an appealing class of antibiotic agents used to combat Gram-positive drug-resistant bacterial strains, including C. difficile.

Update on Antimicrobial Resistance in Clostridium difficile: Resistance Mechanisms and Antimicrobial Susceptibility Testing.

Oral antibiotics such as metronidazole, vancomycin and fidaxomicin are therapies of choice for Clostridium difficile infection. Several important mechanisms for C. difficile antibiotic resistance have been described, including the acquisition of antibiotic resistance genes via the transfer of mobile genetic elements, selective pressure in vivo resulting in gene mutations, altered expression of redox-active proteins, iron metabolism, and DNA repair, as well as via biofilm formation. This update summarizes new information published since 2010 on phenotypic and genotypic resistance mechanisms in C. difficile and addresses susceptibility test methods and other strategies to counter antibiotic resistance of C. difficile.

Immunogenicity and protective efficacy of Clostridium difficile spore proteins.

Clostridium difficile is a spore-forming, anaerobic, Gram-positive organism that is the leading cause of antibiotic-associated infectious diarrhea, commonly known as C. difficile infection (CDI). C. difficile spores play an important role in the pathogenesis of CDI. Spore proteins, especially those that are surface-bound may play an essential role in the germination, colonization and persistence of C. difficile in the human gut. In our current study, we report the identification of two surface-bound spore proteins, CdeC and CdeM that may be utilized as immunization candidates against C. difficile. These spore proteins are immunogenic in mice and are able to protect mice against challenge with C. difficile UK1, a clinically-relevant 027/B1/NAP1 strain. These spore proteins are also able to afford high levels of protection against challenge with C. difficile 630Δerm in golden Syrian hamsters. This unprecedented study shows the vaccination potential of C. difficile spore exosporium proteins.

Hyperimmune bovine colostrum as a novel therapy to combat Clostridium difficile infection.

BACKGROUND: Clostridium difficile is a primary cause of antibiotic-associated diarrhea that typically develops when gut microbiota is altered. Conventional treatment for C. difficile infection (CDI) is additional antimicrobial administration, which further disrupts normal intestinal microbiota, often resulting in poor treatment outcomes. METHODS: A pregnant dairy cow was repeatedly immunized with recombinant mutants of toxins A and B produced by C. difficile, and the resultant hyperimmune bovine colostrum (HBC) was evaluated for therapeutic efficacy in gnotobiotic piglets with diarrhea due to CDI. Control piglets received nonimmune colostrum. To determine the impact of HBC on gut microbiota, 1 of 2 groups of piglets transplanted with normal human gut microbiota was treated with HBC. RESULTS: Nonimmune colostrum-treated piglets developed moderate to severe diarrhea and colitis. In contrast, HBC-treated piglets had mild or no diarrhea and mild or no colitis. Lyophilization had no detectable impact on HBC efficacy. HBC had no discernible effect on the composition of normal human gut microbiota in the porcine intestinal tract. CONCLUSIONS: HBC provides an oral, cost-effective, and safe alternative to antibiotic therapy for CDI. By preserving intestinal microbiota, HBC may be more efficacious than antibiotics. Additional studies are warranted to establish HBC as a viable immunotherapeutic agent for human use against CDI.

Identification and functional characterization of Toll-like receptor 2-1 in geese.

BACKGROUND: Toll-like receptor 2 (TLR2), an important pattern recognition receptor, activates proinflammatory pathways in response to various pathogens. It has been reported in humans and chicken, but not in geese, an important waterfowl species in China. Since some vaccines stimulate robust immune responsesl in chicken but not in geeeses we speculated that their immune systems are different. RESULTS: In this study, we cloned the goose TLR2-1 gene using rapid amplification of cDNA ends (RACE)and showed that geese TLR2-1 encoded a 793-amino-acid protein, containing a signal secretion peptide, an extracellular leucine-rich repeat domain, a transmembrane domain and a Toll/interleukin-1 receptor signaling domain deduced from amino acid sequence. TLR2-1 shared 38.4%-93.5% homology with its homologues in other species. Tissue expression of geese TLR2-1 varied markedly, and was higher in kidney, cloacal bursa, skin and brain compared to other organs/tissues. HEK293 cells transfected with plasmids carrying goose TLR2-1 and NF-κB-luciferase responded significantly to stimulation with Mycoplasma fermentans lipopeptide. Furthermore, geese infected with Mycoplasma gallisepticum (MG) and Salmonella enteritidis (SE) showed significant upregulation of TLR2-1 in both in vivo and in vitro. CONCLUSION: Geese TLR2-1 is a functional homologue of TLR2 present in other species and plays an important role in bacterial recognition in geese.

The non-toxigenic Clostridium difficile CD37 protects mice against infection with a BI/NAP1/027 type of C. difficile strain.

Clostridium difficile CD37, a clinical isolate from the USA, does not produce toxin A, B or binary toxin. The aim of this study was to determine whether strain CD37 can protect mice against infection from a challenge with a toxigenic C. difficile strain. Three groups of mice (n = 10) were pretreated with a antibiotics cocktail for 5 days, switched to sterile water for 2 days, and given one dose of clindamycin (10 mg/kg) one day (day-1) before challenge (day 0) with a toxigenic C. difficile strain. Group 1 (CD37 + UK6) was given 10(7)C. difficile CD37 vegetative cells by gavage twice a day on days -1 and -2, followed by challenge with 10(6) spores of the toxigenic C. difficile UK6 (BI/NAPI/027) on day 0; Group 2 (UK6) was infected with 10(6)C. difficile UK6 spores on day 0; Group 3 (CD37) was challenged with 10(6) CD37 vegetative cells on day 0. Our data show that pre-inoculation of strain CD37 provided mice significant protection (survival, p < 0.001 between groups CD37 + UK6 and UK6) against subsequent infection with the strain UK6, while mice infected with CD37 only did not develop any symptoms of C. difficile infection (CDI). Our results highlight the potential use of CD37 as a therapeutic strain for the prevention of primary and recurrent CDI in humans.

Beneficial effect of oral tigecycline treatment on Clostridium difficile infection in gnotobiotic piglets.

The efficacy of oral tigecycline treatment (2 mg/kg of body weight for 7 days) of Clostridium difficile infection (CDI) was evaluated in the gnotobiotic pig model, and its effect on human gut microflora transplanted into the gnotobiotic pig was determined. Tigecycline oral treatment improved survival, clinical signs, and lesion severity and markedly decreased concentrations of Firmicutes but did not promote CDI. Our data showed that oral tigecycline treatment has a potential beneficial effect on the treatment of CDI.

Antimicrobial Guanidinylate Polycarbonates Show Oral In Vivo Efficacy Against Clostridioides Difficile.

The emerging antibiotic resistance has been named by the World Health Organization (WHO) as one of the top 10 threats to public health. Notably, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF) are designated as serious threats, whereas Clostridioides difficile (C. difficile) is recognized as one of the most urgent threats to human health and unmet medical need. Herein, they report the design and application of novel biodegradable polymers - the lipidated antimicrobial guanidinylate polycarbonates. These polymers showed potent antimicrobial activity against a panel of bacteria with fast-killing kinetics and low resistance development tendency, mainly due to their bacterial membrane disruption mechanism. More importantly, the optimal polymer showed excellent antibacterial activity against C. difficile infection (CDI) in vivo via oral administration. In addition, compared with vancomycin, the polymer demonstrated a much-prolonged therapeutic effect and virtually diminished recurrence rate of CDI. The convenient synthesis, easy scale-up, low cost, as well as biodegradability of this class of polycarbonates, together with their in vitro broad-spectrum antimicrobial activity and orally in vivo efficacy against CDI, suggest the great potential of lipidated guandinylate polycarbonates as a new class of antibacterial biomaterials to treat CDI and combat emerging antibiotic resistance.

Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models.

BACKGROUND: Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years. METHODS: Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI. RESULTS: We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI. CONCLUSION: Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.