Elucidation of the Glycan Structure of the b-type Flagellin of PAO1.

Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin (FliC), often undergoes post-translational modifications, with glycosylation being a common occurrence. In PAO1, the b-type flagellin is -glycosylated with a structure that includes a deoxyhexose, a phospho-group, and a previous unknown moiety.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

is the leading cause of antibiotic-associated infections worldwide. Within the host, can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds.

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

To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein.

Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity.

Revised Model for the Type A Glycan Biosynthetic Pathway in Strain 630Δ Based on Quantitative Proteomics of - Mutant Strains.

The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the strain 630Δ, the main filamentous component, FliC, is post-translationally modified with an -linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan.

In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry.

Proteases comprise the class of enzymes that catalyzes the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility toward protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors.

New insights into the type A glycan modification of Clostridioides difficile flagellar protein flagellin C by phosphoproteomics analysis.

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility.

Development and Application of a Mechanistic Cooling and Freezing Model of the Spin Freezing Step within the Framework of Continuous Freeze-Drying.

During the spin freezing step of a recently developed continuous spin freeze-drying technology, glass vials are rapidly spun along their longitudinal axis. The aqueous drug formulation subsequently spreads over the inner vial wall, while a cold gas flow is used for cooling and freezing the product. In this work, a mechanistic model was developed describing the energy transfer during each phase of spin freezing in order to predict the vial and product temperature change over time.

Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile.

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail.

Phylogenetic analysis of the bacterial Pro-Pro-endopeptidase domain reveals a diverse family including secreted and membrane anchored proteins.

Pro-Pro-endopeptidases (PPEP, EC 3.4.24.

A Bioluminescent Sensor for Rapid Detection of PPEP-1, a Biomarker.

Current assays for in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a "turn-on" bioluminescent readout of a -specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was "caged" with a PPEP-1-responsive peptide tail that inhibited luminescence.

Plasmids of Clostridioides difficile.

Plasmids are ubiquitous in the bacterial world. In many microorganisms, plasmids have been implicated in important aspects of bacterial physiology and contribute to horizontal gene transfer. In contrast, knowledge on plasmids of the enteropathogen Clostridioides difficile is limited, and there appears to be no phenotypic consequence to carriage of many of the identified plasmids.

A primary drying model-based comparison of conventional batch freeze-drying to continuous spin-freeze-drying for unit doses.

An innovative continuous spin-freeze-drying technology for unit doses was recently developed. For this technology, a mechanistic primary drying model was developed allowing the calculation of the optimal dynamic drying trajectory for spin-frozen formulations. In this work, a model-based and experimentally verified comparison was made between conventional batch freeze-drying and spin-freeze-drying by analyzing the outputs (i.

Redefining the Clostridioides difficile σ Regulon: σ 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 (σ). In , σ has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σ antibody to demonstrate time-limited overproduction of σ in despite its toxicity at higher cellular concentrations.

The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface.

is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the genotype and toxin production.

Plasmid-mediated metronidazole resistance in Clostridioides difficile.

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L).

Clostridium difficile clade 3 (RT023) have a modified cell surface and contain a large transposable island with novel cargo.

The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

Objectives: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade.

Methods: In this cohort study the clinical presentation of C.

Dual chamber cartridges in a continuous pharmaceutical freeze-drying concept: Determination of the optimal dynamic infrared heater temperature during primary drying.

The applicability of DCCs in a continuous freeze-drying concept based on spin freezing and infrared heating was evaluated. Maximum applicable filling volume was evaluated. Secondly the mechanistic model for the determination of the optimal dynamic infrared heater temperature during primary drying of regular vials during continuous freeze-drying was adapted and validated for DCCs.

Identification and validation of two peptide markers for the recognition of Clostridioides difficile MLST-1 and MLST-11 by MALDI-MS.

Objectives: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests.

Thermal Imaging as a Noncontact Inline Process Analytical Tool for Product Temperature Monitoring during Continuous Freeze-Drying of Unit Doses.

Freeze-drying is a well-established technique to improve the stability of biopharmaceuticals which are unstable in aqueous solution. To obtain an elegant dried product appearance, the temperature at the moving sublimation interface T should be kept below the critical product temperature T during primary drying. The static temperature sensors applied in batch freeze-drying provide unreliable T data due to their invasive character.

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

Fecal microbiota transplantation has proven to be an effective treatment for infections with the gram-positive enteropathogen . Despite its effectiveness, the exact mechanisms that underlie its success are largely unclear. In this review, we highlight the pleiotropic effectors that are transferred during fecal microbiota transfer and relate this to the lifecycle.

Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacter xylosoxidans.

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans.

Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family.

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen (PPEP-1) cleaves two cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family.