Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile

AbstractClostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

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CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile

Scientific Reports ◽

10.1038/s41598-021-86878-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Diogo Martins ◽

Michael A. DiCandia ◽

Aristides L. Mendes ◽

Daniela Wetzel ◽

Shonna M. McBride ◽

...

Keyword(s):

Life Cycle ◽

Biofilm Formation ◽

Regulatory Protein ◽

Well Being ◽

Human Intestine ◽

Healthy Humans ◽

Clostridioides Difficile ◽

Nutritional Conditions ◽

Sporulation Initiation

AbstractBacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

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Insights into the regulatory mechanisms of Clostridioides difficile biofilm formation

10.1101/2021.02.19.431970 ◽

2021 ◽

Author(s):

Anthony M. Buckley ◽

Duncan Ewin ◽

Ines B. Moura ◽

Mark H. Wilcox ◽

Gillian R. Douce

Keyword(s):

Metabolic Pathways ◽

Biofilm Formation ◽

Short Chain Fatty Acids ◽

Metabolic Reprogramming ◽

High Rate ◽

Regulatory Pathways ◽

Persistent Infections ◽

Clostridioides Difficile ◽

Transcriptional Changes ◽

Non Coding Rnas

AbstractMucosal biofilms play an important role in intestinal health; however, the mucosal bacterial community has been implicated in persistent infections. Clostridioides difficile is an important nosocomial pathogen, with an unacceptable high rate of recurrence following antibiotic treatment. As C. difficile is a known biofilm producer, a property which may contribute to this suboptimal therapeutic response, we have investigated the transcriptional changes and regulatory pathways during the transition from planktonic to biofilm mode of growth. Widespread metabolic reprogramming during biofilm formation was detected, characterised by an increased usage of glycine metabolic pathways to yield key metabolites, which are used for energy production and synthesis of short chain fatty acids. We detected the expression of 107 small non-coding RNAs that appear to, in some part, regulate these pathways; however, 25 of these small RNAs were specifically expressed during biofilm formation, indicating they may play a role in regulating biofilm-specific genes. Similar to Bacillus subtilis, biofilm formation is a multi-regulatory process and SinR negatively regulates biofilm formation independently of other known mechanisms. This comprehensive analysis furthers our understanding of biofilm formation in C. difficile, identifies potential targets for anti-virulence factors, and provides evidence of the link between metabolism and virulence traits.

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Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

PLoS Pathogens ◽

10.1371/journal.ppat.1009817 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009817

Author(s):

Megan G. Taggart ◽

William J. Snelling ◽

Patrick J. Naughton ◽

Roberto M. La Ragione ◽

James S. G. Dooley ◽

...

Keyword(s):

Biofilm Formation ◽

Gastrointestinal Disease ◽

Regulatory System ◽

Disease Recurrence ◽

The United States ◽

Extracellular Dna ◽

Published Data ◽

Bacterial Cells ◽

Healthcare Associated Infection ◽

Clostridioides Difficile

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

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c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

mSphere ◽

10.1128/msphere.00919-21 ◽

2021 ◽

Author(s):

Adrianne N. Edwards ◽

Caitlin L. Willams ◽

Nivedita Pareek ◽

Shonna M. McBride ◽

Rita Tamayo

Keyword(s):

Life Cycle ◽

Biofilm Formation ◽

Toxin Production ◽

Cyclic Diguanylate ◽

Content Type ◽

Critical Step ◽

Clostridioides Difficile ◽

Physiological Processes ◽

Gastrointestinal Pathogen ◽

The Impact

Many bacterial organisms utilize the small signaling molecule cyclic diguanylate (c-di-GMP) to regulate important physiological processes, including motility, toxin production, biofilm formation, and colonization. c-di-GMP inhibits motility and toxin production and promotes biofilm formation and colonization in the anaerobic, gastrointestinal pathogen Clostridioides difficile . However, the impact of c-di-GMP on C. difficile spore formation, a critical step in this pathogen’s life cycle, is unknown.

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Immobilizing hydrolytic active Papain on biodegradable PLLA for biofilm inhibition in cardiovascular applications

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2020-3044 ◽

2020 ◽

Vol 6 (3) ◽

pp. 172-175

Author(s):

Michael Teske ◽

Tina Kießlich ◽

Niels Grabow ◽

Sabine Illner ◽

Julia Fischer ◽

...

Keyword(s):

Biofilm Formation ◽

Antimicrobial Agents ◽

Hydrolytic Enzyme ◽

Bacterial Biofilm ◽

Biofilm Inhibition ◽

Biofilm Growth ◽

Clostridioides Difficile ◽

Adhesive Surface

AbstractThe use of biomaterials in medicine is becoming increasingly important. One of the main concerns is the foreign body associated infection caused by direct microbial contamination or clinical infections. The bacterial biofilm formation on biomaterials depends on their surface properties. Therefore, several anti-adhesive surface modifications were developed. Nevertheless, the demand for antimicrobial agents that prevent bacterial colonisation is still largely unmet. The immobilization of active antimicrobial agents, such as antibacterial peptides or enzymes, offers a potential approach to achieve long-lasting effectiveness. In this investigation, the hydrolytic enzyme papain with its published antibacterial activity was covalently immobilized on the well-established biodegradable biomaterial poly-L-lactic acid (PLLA). For the characterization of the enzymes on the PLLA surfaces, the protein content and enzyme activity were determined. A biofilm assay was performed to test the effect of the papain-modified PLLA samples on the biofilm-forming bacterial strain Clostridioides difficile, one of the most frequently occurring human nosocomial pathogens. The investigated hydrolytic enzyme papain could be immobilized by coupling via the crosslinker EDC to the PLLA surface. Detection was performed by determination of the amount of protein and the reduced biofilm growth after 24 h and 72 h compared to the reference.

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Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile

Scientific Reports ◽

10.1038/s41598-020-78437-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lisa F. Dawson ◽

Johann Peltier ◽

Catherine L. Hall ◽

Mark A. Harrison ◽

Maria Derakhshan ◽

...

Keyword(s):

Cell Surface ◽

Biofilm Formation ◽

Bacterial Species ◽

Surface Proteins ◽

Extracellular Dna ◽

Cell Surface Proteins ◽

Secondary Messenger ◽

Clostridioides Difficile ◽

Antibiotic Efficacy ◽

Biofilm Matrix

AbstractClostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

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What’s a Biofilm?—How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile

Frontiers in Microbiology ◽

10.3389/fmicb.2021.682111 ◽

2021 ◽

Vol 12 ◽

Author(s):

Madita Brauer ◽

Christian Lassek ◽

Christian Hinze ◽

Juliane Hoyer ◽

Dörte Becher ◽

...

Keyword(s):

Cell Surface ◽

Biofilm Formation ◽

Surface Proteins ◽

Type Iv ◽

Clostridioides Difficile ◽

Biofilm Model ◽

Proteomics Approach ◽

Surface Polysaccharides ◽

Mammalian Intestine

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.

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