SNP-ing out the differences: Investigating differences between Clostridium difficile lab strains

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

DNA replication proteins as potential targets for antimicrobials in drug-resistant bacterial pathogens

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Host immune responses to clostridioides difficile: toxins and beyond

Clostridioides difficile is often resistant to the actions of antibiotics to treat other bacterial infections and the resulting C. difficile infection (CDI) is among the leading causes of nosocomial infectious diarrhea worldwide. The primary virulence mechanism contributing to CDI is the production of toxins. Treatment failures and recurrence of CDI have urged the medical community to search for novel treatment options. Strains that do not produce toxins, so called non-toxigenic C. difficile, have been known to colonize the colon and protect the host against CDI. In this review, a comprehensive description and comparison of the immune responses to toxigenic C. difficile and non-toxigenic adherence, and colonization factors, here called non-toxin proteins, is provided. This revealed a number of similarities between the host immune responses to toxigenic C. difficile and non-toxin proteins, such as the influx of granulocytes and the type of T-cell response. Differences may reflect genuine variation between the responses to toxigenic or non-toxigenic C. difficile or gaps in the current knowledge with respect to the immune response toward non-toxigenic C. difficile. Toxin-based and non-toxin-based immunization studies have been evaluated to further explore the role of B cells and reveal that plasma cells are important in protection against CDI. Since the success of toxin-based interventions in humans to date is limited, it is vital that future research will focus on the immune responses to non-toxin proteins and in particular non-toxigenic strains.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Redefining the clostridioides difficile sigma(B) regulon: sigma(B) activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (sigma(B)). In C. difficile, sigma(B) has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-us antibody to demonstrate time-limited overproduction of sigma(B) in C difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced sigma(B) gene expression. Inducible sigma(B) overproduction uncouples sigma(B) expression from its native regulatory network and allows for the refinement of the previously proposed sigma(B) regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile 0 8 was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to sigma(B)-dependent gene activation.IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of sigma(B) by evaluating the transcriptional effects of sigma(B) overproduction, provide biochemical evidence of direct transcriptional activation by sigma(B), and show that sigma(B)-dependent genes can be activated by antimicrobials. Together, our data suggest that sigma(B) is a key player in dealing with toxic radicals.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Mechanistic Insights in the Success of Fecal Microbiota Transplants for the Treatment of Clostridium difficile Infections

Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Cyclodextrin/adamantane-mediated targeting of inoculated bacteria in mice

Cyclodextrin (CD)-based host-guest interactions with adamantane (Ad) have demonstrated use for functionalizing living cells in vitro. The next step in this supramolecular functionalization approach is to explore the concept to deliver chemical cargo to living cells in vivo, e.g., inoculated bacteria, in order to study their dissemination. We validated this concept in two rodent Staphylococcus aureus models. Bacteria (1 X 10(8) viable S. aureus) were inoculated by (1) intramuscular injection or (2) intrasplenic injection followed by dissemination throughout the liver. The bacteria were prefunctionalized with Tc-99m-UBI29-41-Ad(2) (primary vector), which allowed us to both determine the bacterial load and create an in vivo target for the secondary host-vector (24 h post-inoculation). The secondary vector, i.e., chemical cargo delivery system, made use of a In-111-Cy5(0)(.5)CD(9)PIBMA(39 )polymer that was administered intravenously. Bacteria-specific cargo delivery as a result of vector complexation was evaluated by dual-isotope SPECT imaging and biodistribution studies (In-111), and by fluorescence (Cy5); these evaluations were performed 4 h post-injection of the secondary vector. Mice inoculated with nonfunctionalized S. aureus and mice without an infection served as controls. Dual-isotope SPECT imaging demonstrated that In-111-Cy5(0)(.5)CD(9)PIBMA(3)(9) colocalized with Tc-99m-UBI29-41-Ad(2)-labeled bacteria in both muscle and liver. In inoculated muscle, a 2-fold higher uptake level (3.2 +/- 1.0%ID/g) was noted compared to inoculation with nonfunctionalized bacteria (1.9 +/- 0.4%ID/g), and a 16-fold higher uptake level compared to noninfected muscle (0.2 +/- 0.1%ID/g). The hepatic accumulation of the host-vector was nearly 10-fold higher (27.1 +/- 11.1%ID/g) compared to the noninfected control (2.7 +/- 0.3%ID/g; p < 0.05). Fluorescence imaging of the secondary vector corroborated SPECT-imaging and biodistribution findings. We have demonstrated that supramolecular host-guest complexation can be harnessed to achieve an in vivo cargo delivery strategy, using two different bacterial models in soft tissue and liver. This proof-of-principle study paves a path toward developing innovative drug delivery concepts via cell functionalization techniques.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc