scholarly journals The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

2018 ◽  
Author(s):  
Amy E. Rohlfing ◽  
Brian E. Eckenroth ◽  
Emily R. Forster ◽  
Yuzo Kevorkian ◽  
M. Lauren Donnelly ◽  
...  
Keyword(s):  
Bile Salt ◽  
Spore Form ◽  
Environmental Signals ◽  
Clostridioides Difficile ◽  
Gastrointestinal Pathogen ◽  
Increased Sensitivity ◽  
Dormant Spore ◽  
Hyper Sensitivity ◽  
Spore Forming Bacteria ◽  
Unique Mechanism

AbstractThe gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile uses the soluble pseudoprotease, CspC, to detect bile salt germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile salt germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile salt-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since the mechanism by which C. difficile spores sense co-germinants is unknown, our study provides the first evidence that CspC senses distinct classes of co-germinants in addition to bile salts. Since we observed that specific residues control CspC’s responsiveness to these different signals, CspC is critical for regulating C. difficile germination in response to multiple environmental signals.

scholarly journals Omadacycline compared to vancomycin when combined with germinants to disrupt the life cycle of Clostridioides difficile

2021 ◽  
Author(s):  
Noah Budi ◽  
Jared J. Godfrey ◽  
Nasia Safdar ◽  
Sanjay K. Shukla ◽  
Warren E. Rose
Keyword(s):  
Limit Of Detection ◽  
Disease Relapse ◽  
Vegetative Cells ◽  
Spore Form ◽  
Safety And Efficacy ◽  
Healthcare Environment ◽  
Clostridioides Difficile ◽  
Spore Shedding ◽  
Dormant Spore

Clostridioides difficile (C. difficile) infections (CDI) are commonly treated with antibiotics that do not impact the dormant spore form of the pathogen. CDI-directed antibiotics, such as vancomycin and metronidazole, can destroy the vegetative form of C. difficile and protective microbiota. After treatment, spores can germinate into vegetative cells causing clinical disease relapse and further spore shedding. This in vitro study compares the combination of germinants with vancomycin or omadacycline to antibiotics alone in eradicating C. difficile spores and vegetative cells. Among the four strains in this study, omadacycline minimum inhibitory concentrations (0.031-0.125 mg/L) were lower than vancomycin (1-4 mg/L). Omadacycline nor vancomycin in media alone reduced spore counts. In three of the four strains, including the epidemic ribotype 027, spore eradication with germinants was 94.8-97.4% with vancomycin and 99.4-99.8% with omadacycline (p<0.005). In ribotype 012, either antibiotic combined with germinants resulted in 100% spore eradication at 24 hours. The addition of germinants with either antibiotic did not result in significant toxin A or B production, which were below the limit of detection (<1.25 ng/mL) by 48 hours. Limiting the number of spores present in patient GI tracts at the end of therapy may be effective at preventing recurrent CDI and limiting spore shedding in the healthcare environment. These results with germinants warrant safety and efficacy evaluations in animal models.

scholarly journals Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4276-4291 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Shonna M. McBride
Keyword(s):  
Clostridium Difficile ◽  
Hamster Model ◽  
Content Type ◽  
Gastrointestinal Pathogen ◽  
Dormant Spore ◽  
Sporulation Initiation ◽  
Spore Forming Bacteria

ABSTRACTThe anaerobic gastrointestinal pathogenClostridium difficilemust form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by whichC. difficileinitiates and controls the early stages of sporulation inC. difficileare not highly conserved in otherClostridiumorBacillusspecies. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation inC. difficile. These permeases are known to positively affect sporulation inBacillusspecies through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases inC. difficileresulted in the earlier expression of early sporulation genes and increased sporulationin vitro. Furthermore, disruption ofoppandappresulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model ofC. difficileinfection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence inC. difficilethan inBacillus subtilisand suggest that nutrient availability plays a significant role in pathogenesis and sporulationin vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation inC. difficile.

scholarly journals Investigation of the Effectiveness of Disinfectants Used in Meat-Processing Facilities to Control Clostridium sporogenes and Clostridioides difficile Spores

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1436
Author(s):  
Siobhán McSharry ◽  
Leonard Koolman ◽  
Paul Whyte ◽  
Declan Bolton
Keyword(s):  
Test Tube ◽  
Meat Industry ◽  
Meat Processing ◽  
Clostridium Sporogenes ◽  
Safety Issues ◽  
Clostridioides Difficile ◽  
Plant Environment ◽  
D Values ◽  
Sulphamic Acid ◽  
Spore Forming Bacteria

Spore-forming bacteria are a major concern for the food industry as they cause both spoilage and food safety issues. Moreover, as they are more resistant than vegetative cells, their removal from the food processing environment may be difficult to achieve. This study investigated the efficacy of the ten most commonly used disinfectant agents (assigned 1–10), used at the recommended concentrations in the meat industry, for their ability to eliminate Clostridium sporogenes and Clostridioides difficile spores. Test-tube based suspension assays suggested that disinfectants 2 (10% v/v preparation of a mixture of hydrogen peroxide (10–30%), acetic acid (1–10%) and peracetic acid (1–10%)), 7 (4% w/v preparation of a mixture of peroxymonosulphate (30–50%), sulphamic acid (1–10%) and troclosene sodium (1–10%)) and 10 (2% v/v preparation of a mixture of glutaraldehyde (10–30%), benzalkonium chloride (1–10%)) were the most effective formulations. D-values for these ranged from 2.1 to 8.4 min at 20 °C for the target spores. Based on these findings, it is recommended that these disinfectants are used to control Clostridium spores in the meat plant environment.

An aniline-substituted bile salt analog protects both mice and hamsters from multiple Clostridioides difficile strains

2021 ◽  
Author(s):  
Jacqueline R. Phan ◽  
Dung M. Do ◽  
Minh Chau Truong ◽  
Connie Ngo ◽  
Julian H. Phan ◽  
...  
Keyword(s):  
Bile Salt ◽  
Spore Germination ◽  
Dependent Manner ◽  
Germination Inhibitors ◽  
New Methods ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Careful Screening ◽  
Strain Dependent

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active at preventing spore germination of other C. difficile ribotypes, including the hypervirulent strain R20291. Strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, (CaPA), was found to be a better anti-germinant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs in all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA’s efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify anti-germinants with broad CDI prophylaxis activity.

scholarly journals Treatment issues in recurrent Clostridioides difficile infections and the possible role of germinants

FEMS Microbes ◽  
2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Noah Budi ◽  
Nasia Safdar ◽  
Warren E Rose
Keyword(s):  
Antibiotic Therapy ◽  
Treatment Options ◽  
The United States ◽  
Current Treatment ◽  
Gi Tract ◽  
Spore Form ◽  
Environmental Cleaning ◽  
Clostridioides Difficile ◽  
Cleaning Procedures ◽  
Hospital Acquired

ABSTRACT Clostridioides difficile is the number one cause of hospital-acquired infections in the United States and one of the CDC's urgent-level pathogen threats. The inflammation caused by pathogenic C. difficile results in diarrhea and pseudomembranous colitis. Patients who undergo clinically successful treatment for this disease commonly experience recurrent infections. Current treatment options can eradicate the vegetative cell form of the bacteria but do not impact the spore form, which is impervious to antibiotics and resists conventional environmental cleaning procedures. Antibiotics used in treating C. difficile infections (CDI) often do not eradicate the pathogen and can prevent regeneration of the microbiome, leaving them vulnerable to recurrent CDI and future infections upon subsequent non-CDI-directed antibiotic therapy. Addressing the management of C. difficile spores in the gastrointestinal (GI) tract is important to make further progress in CDI treatment. Currently, no treatment options focus on reducing GI spores throughout CDI antibiotic therapy. This review focuses on colonization of the GI tract, current treatment options and potential treatment directions emphasizing germinant with antibiotic combinations to prevent recurrent disease.

scholarly journals Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Hector Benito de la Puebla ◽  
David Giacalone ◽  
Alexei Cooper ◽  
Aimee Shen
Keyword(s):  
Bacillus Subtilis ◽  
Atp Hydrolysis ◽  
Spore Formation ◽  
Spore Form ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Allele Specific ◽  
Coat Layer ◽  
Quality Control Mechanism

ABSTRACT The nosocomial pathogen Clostridioides difficile is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein SpoIVA and the clostridial-organism-specific spore protein SipL, which directly interact. Mutations that disrupt their interaction cause the coat to mislocalize and impair spore formation. In Bacillus subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in the C. difficile SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104-fold less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased the SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced the SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele specific, our data imply that SipL recognizes the ATP-bound form of SpoIVA and highlight the importance of this interaction for functional C. difficile spore formation. IMPORTANCE The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

scholarly journals Shifting mirrors: adaptive changes in retinal reflections to winter darkness in Arctic reindeer

2013 ◽  
Vol 280 (1773) ◽  
pp. 20132451 ◽  
Author(s):  
Karl-Arne Stokkan ◽  
Lars Folkow ◽  
Juliet Dukes ◽  
Magella Neveu ◽  
Chris Hogg ◽  
...  
Keyword(s):  
Retinal Sensitivity ◽  
Structural Adaptation ◽  
Deep Blue ◽  
Environmental Light ◽  
Tapetum Lucidum ◽  
Increased Sensitivity ◽  
Ocular Pressure ◽  
The Cost ◽  
Unique Mechanism ◽  
Scatter Light

Arctic reindeer experience extreme changes in environmental light from continuous summer daylight to continuous winter darkness. Here, we show that they may have a unique mechanism to cope with winter darkness by changing the wavelength reflection from their tapetum lucidum (TL). In summer, it is golden with most light reflected back directly through the retina, whereas in winter it is deep blue with less light reflected out of the eye. The blue reflection in winter is associated with significantly increased retinal sensitivity compared with summer animals. The wavelength of reflection depends on TL collagen spacing, with reduced spacing resulting in shorter wavelengths, which we confirmed in summer and winter animals. Winter animals have significantly increased intra-ocular pressure, probably produced by permanent pupil dilation blocking ocular drainage. This may explain the collagen compression. The resulting shift to a blue reflection may scatter light through photoreceptors rather than directly reflecting it, resulting in elevated retinal sensitivity via increased photon capture. This is, to our knowledge, the first description of a retinal structural adaptation to seasonal changes in environmental light. Increased sensitivity occurs at the cost of reduced acuity, but may be an important adaptation in reindeer to detect moving predators in the dark Arctic winter.

scholarly journals SpoIVA-SipL complex formation is essential for Clostridioides difficile spore assembly

2019 ◽  
Author(s):  
Megan H. Touchette ◽  
Hector Benito de la Puebla ◽  
Priyanka Ravichandran ◽  
Aimee Shen
Keyword(s):  
Molecular Mechanisms ◽  
Basic Mechanism ◽  
Spore Formation ◽  
Spore Coat ◽  
Coat Proteins ◽  
Nosocomial Pathogen ◽  
Spore Form ◽  
Infectious Particle ◽  
Lysm Domain ◽  
Clostridioides Difficile

AbstractSpores are the major infectious particle of the Gram-positive nosocomial pathogen, Clostridioides (formerly Clostridium) difficile, but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism, Bacillus subtilis, with only 25% of the ~70 spore coat proteins being conserved between the two organisms, and only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile. We previously identified SipL as a clostridia-specific coat protein essential for functional spore formation. Heterologous expression analyses in E. coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat morphogenetic protein conserved in all spore-forming organisms, through SipL’s C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL’s LysM domain to SpoIVA is required for SipL to localize to the forespore, while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.ImportanceThe metabolically dormant spore-form of the major nosocomial pathogen, Clostridioides difficile, is its major infectious particle. However, the mechanisms controlling the formation of these resistant cell types are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the Clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL’s C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and thus functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

scholarly journals Differential Effects of “Resurrecting” Csp Pseudoproteases during Clostridioides difficile Spore Germination

2019 ◽  
Author(s):  
M. Lauren Donnelly ◽  
Emily R. Forster ◽  
Amy E. Rohlfing ◽  
Aimee Shen
Keyword(s):  
Spore Germination ◽  
Protease Activity ◽  
Bacterial Pathogen ◽  
Catalytic Triad ◽  
Spore Form ◽  
Cellular Processes ◽  
Clostridioides Difficile ◽  
Serine Protease Family ◽  
The Stability ◽  
And Function

AbstractClostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the vertebrate gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently determined crystal structure of CspC revealed that its degenerate site residues align closely with the catalytic triad of CspB, so in this study we tested whether the ancestral protease activity of the CspC and CspA pseudoproteases could be “resurrected.” Restoring the catalytic triad to these pseudoproteases failed to resurrect their protease activity, although the mutations differentially affected the stability and function of these pseudoproteases. Degenerate site mutations destabilized CspC and impaired spore germination without impacting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since close homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results imply that bioinformatics predictions of enzyme activity may overlook pseudoenzymes in some cases.

scholarly journals c-di-GMP Inhibits Early Sporulation in Clostridioides difficile

mSphere ◽  
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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