Type 3 Immunity during Clostridioides difficile Infection: Too Much of a Good Thing?

ABSTRACT Clostridioides (formerly known as Clostridium) difficile is the leading cause of hospital-acquired gastrointestinal infections in the United States and one of three urgent health care threats identified by the Centers for Disease Control and Prevention. C. difficile disease is mediated by the production of toxins that disrupt the epithelial barrier and cause a robust host inflammatory response. Studies in humans as well as animal models of disease have shown that the type of immune response generated against the infection dictates the outcome of disease, often irrespective of bacterial burden. Much of the focus on immunity during C. difficile infection (CDI) has been on type 3 immunity because of the established role for this arm of the immune system in other gastrointestinal inflammatory conditions such as inflammatory bowel disease (IBD). For example, interleukin-22 (IL-22) production by group 3 innate lymphoid cells (ILC3s) protects against pathobionts translocating across the epithelium during CDI. On the other hand, interleukin-17 (IL-17) production by Th17 cells increases CDI-associated mortality. Additionally, neutropenia has been associated with increased susceptibility to CDI in humans, but increased neutrophilia in mouse models correlates with host pathology. Taking the data together, these findings suggest dual roles for type 3 immune responses during infection. Here, we review the complex role of type 3 immunity during CDI and delineate what is known about innate and adaptive cellular immunity as well as the downstream effector cytokines known to be important during this infection.

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Systemic Inflammatory Mediators Are Effective Biomarkers for Predicting Adverse Outcomes in Clostridioides difficile Infection

mBio ◽

10.1128/mbio.00180-20 ◽

2020 ◽

Vol 11 (3) ◽

Author(s):

Michael G. Dieterle ◽

Rosemary Putler ◽

D. Alexander Perry ◽

Anitha Menon ◽

Lisa Abernathy-Close ◽

...

Keyword(s):

Severe Disease ◽

The United States ◽

Human Infection ◽

Serum Biomarkers ◽

Adverse Outcomes ◽

Gastrointestinal Infections ◽

Content Type ◽

Clostridioides Difficile ◽

Increased Risk ◽

Patients At Risk

ABSTRACT Clostridioides difficile infection (CDI) can result in severe disease and death, with no accurate models that allow for early prediction of adverse outcomes. To address this need, we sought to develop serum-based biomarker models to predict CDI outcomes. We prospectively collected sera ≤48 h after diagnosis of CDI in two cohorts. Biomarkers were measured with a custom multiplex bead array assay. Patients were classified using IDSA severity criteria and the development of disease-related complications (DRCs), which were defined as ICU admission, colectomy, and/or death attributed to CDI. Unadjusted and adjusted models were built using logistic and elastic net modeling. The best model for severity included procalcitonin (PCT) and hepatocyte growth factor (HGF) with an area (AUC) under the receiver operating characteristic (ROC) curve of 0.74 (95% confidence interval, 0.67 to 0.81). The best model for 30-day mortality included interleukin-8 (IL-8), PCT, CXCL-5, IP-10, and IL-2Rα with an AUC of 0.89 (0.84 to 0.95). The best model for DRCs included IL-8, procalcitonin, HGF, and IL-2Rα with an AUC of 0.84 (0.73 to 0.94). To validate our models, we employed experimental infection of mice with C. difficile. Antibiotic-treated mice were challenged with C. difficile and a similar panel of serum biomarkers was measured. Applying each model to the mouse cohort of severe and nonsevere CDI revealed AUCs of 0.59 (0.44 to 0.74), 0.96 (0.90 to 1.0), and 0.89 (0.81 to 0.97). In both human and murine CDI, models based on serum biomarkers predicted adverse CDI outcomes. Our results support the use of serum-based biomarker panels to inform Clostridioides difficile infection treatment. IMPORTANCE Each year in the United States, Clostridioides difficile causes nearly 500,000 gastrointestinal infections that range from mild diarrhea to severe colitis and death. The ability to identify patients at increased risk for severe disease or mortality at the time of diagnosis of C. difficile infection (CDI) would allow clinicians to effectively allocate disease modifying therapies. In this study, we developed models consisting of only a small number of serum biomarkers that are capable of predicting both 30-day all-cause mortality and adverse outcomes of patients at time of CDI diagnosis. We were able to validate these models through experimental mouse infection. This provides evidence that the biomarkers reflect the underlying pathophysiology and that our mouse model of CDI reflects the pathogenesis of human infection. Predictive models can not only assist clinicians in identifying patients at risk for severe CDI but also be utilized for targeted enrollment in clinical trials aimed at reduction of adverse outcomes from severe CDI.

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Spo0A Suppresses sin Locus Expression in Clostridioides difficile

mSphere ◽

10.1128/msphere.00963-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Babita Adhikari Dhungel ◽

Revathi Govind

Keyword(s):

Diarrheal Disease ◽

Toxin Production ◽

The United States ◽

Upstream Region ◽

Pcr Analysis ◽

Bacterial Cells ◽

Master Regulator ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is the leading cause of nosocomial infection and is the causative agent of antibiotic-associated diarrhea. The severity of the disease is directly associated with toxin production, and spores are responsible for the transmission and persistence of the organism. Previously, we characterized sin locus regulators SinR and SinR′ (we renamed it SinI), where SinR is the regulator of toxin production and sporulation. The SinI regulator acts as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, controls SinR by regulating the expression of its antagonist, sinI. However, the role of Spo0A in the expression of sinR and sinI in C. difficile had not yet been reported. In this study, we tested spo0A mutants in three different C. difficile strains, R20291, UK1, and JIR8094, to understand the role of Spo0A in sin locus expression. Western blot analysis revealed that spo0A mutants had increased SinR levels. Quantitative reverse transcription-PCR (qRT-PCR) analysis of its expression further supported these data. By carrying out genetic and biochemical assays, we show that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates the sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile. IMPORTANCE Clostridioides difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. During infection, C. difficile spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. In C. difficile, the sin locus is known to regulate both sporulation and toxin production. In this study, we show that Spo0A, the master regulator of sporulation, controls sin locus expression. Results from our study suggest that Spo0A directly regulates the expression of this locus by binding to its upstream DNA region. This observation adds new detail to the gene regulatory network that connects sporulation and toxin production in this pathogen.

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Homologous Recombination in Clostridioides difficile Mediates Diversification of Cell Surface Features and Transport Systems

mSphere ◽

10.1128/msphere.00799-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Hannah D. Steinberg ◽

Evan S. Snitkin

Keyword(s):

Cell Wall ◽

Homologous Recombination ◽

Sugar Transport ◽

The United States ◽

Strain Typing ◽

Cell Wall Proteins ◽

Genetic Loci ◽

Content Type ◽

Clostridioides Difficile ◽

Host Pathogen

ABSTRACT Illness caused by the pathogen Clostridioides difficile is widespread and can range in severity from mild diarrhea to sepsis and death. Strains of C. difficile isolated from human infections exhibit great genetic diversity, leading to the hypothesis that the genetic background of the infecting strain at least partially determines a patient’s clinical course. However, although certain strains of C. difficile have been suggested to be associated with increased severity, strain typing alone has proved insufficient to explain infection severity. The limited explanatory power of strain typing has been hypothesized to be due to genetic variation within strain types, as well as genetic elements shared between strain types. Homologous recombination is an evolutionary mechanism that can result in large genetic differences between two otherwise clonal isolates, and also lead to convergent genotypes in distantly related strains. More than 400 C. difficile genomes were analyzed here to assess the effect of homologous recombination within and between C. difficile clades. Almost three-quarters of single nucleotide variants in the C. difficile phylogeny are predicted to be due to homologous recombination events. Furthermore, recombination events were enriched in genes previously reported to be important to virulence and host-pathogen interactions, such as flagella, cell wall proteins, and sugar transport and metabolism. Thus, by exploring the landscape of homologous recombination in C. difficile, we identified genetic loci whose elevated rates of recombination mediated diversification, making them strong candidates for being mediators of host-pathogen interaction in diverse strains of C. difficile. IMPORTANCE Infections with C. difficile result in up to half a million illnesses and tens of thousands of deaths annually in the United States. The severity of C. difficile illness is dependent on both host and bacterial factors. Studying the evolutionary history of C. difficile pathogens is important for understanding the variation in pathogenicity of these bacteria. This study examines the extent and targets of homologous recombination, a mechanism by which distant strains of bacteria can share genetic material, in hundreds of C. difficile strains and identifies hot spots of realized recombination events. The results of this analysis reveal the importance of homologous recombination in the diversification of genetic loci in C. difficile that are significant in its pathogenicity and host interactions, such as flagellar construction, cell wall proteins, and sugar transport and metabolism.

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γδ T cells regulate the intestinal response to nutrient sensing

Science ◽

10.1126/science.aba8310 ◽

2021 ◽

Vol 371 (6535) ◽

pp. eaba8310

Author(s):

Zuri A. Sullivan ◽

William Khoury-Hanold ◽

Jaechul Lim ◽

Chris Smillie ◽

Moshe Biton ◽

...

Keyword(s):

T Cells ◽

Nutrient Uptake ◽

Γδ T Cells ◽

Nutrient Sensing ◽

Lymphoid Cells ◽

Transcriptional Responses ◽

Interleukin 22 ◽

Epithelial Compartment ◽

A Site ◽

Type 3

The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The “on-demand” induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.

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A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates

Clinical and Vaccine Immunology ◽

10.1128/cvi.00150-16 ◽

2016 ◽

Vol 23 (7) ◽

pp. 586-600 ◽

Cited By ~ 10

Author(s):

Jian Sha ◽

Michelle L. Kirtley ◽

Curtis Klages ◽

Tatiana E. Erova ◽

Maxim Telepnev ◽

...

Keyword(s):

Fusion Gene ◽

The United States ◽

Pneumonic Plague ◽

Content Type ◽

Human Type ◽

Protective Antigens ◽

Type 3 Secretion System ◽

Needle Protein ◽

Trivalent Vaccine ◽

Type 3

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens ofYersinia pestis. We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimizedlcrVor the fusion gene designatedYFV(consisting ofycsF,caf1, andlcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged withY. pestisCO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

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Immune Profiling To Predict Outcome of Clostridioides difficile Infection

mBio ◽

10.1128/mbio.00905-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Mayuresh M. Abhyankar ◽

Jennie Z. Ma ◽

Kenneth W. Scully ◽

Andrew J. Nafziger ◽

Alyse L. Frisbee ◽

...

Keyword(s):

Prediction Model ◽

Risk Prediction ◽

Mortality Risk ◽

The United States ◽

Host Bacterium ◽

Clinical Parameters ◽

Content Type ◽

Clostridioides Difficile ◽

Tnf Α ◽

Immune Profiling

ABSTRACT There is a pressing need for biomarker-based models to predict mortality from and recurrence of Clostridioides difficile infection (CDI). Risk stratification would enable targeted interventions such as fecal microbiota transplant, antitoxin antibodies, and colectomy for those at highest risk. Because severity of CDI is associated with the immune response, we immune profiled patients at the time of diagnosis. The levels of 17 cytokines in plasma were measured in 341 CDI inpatients. The primary outcome of interest was 90-day mortality. Increased tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), C-C motif chemokine ligand 5 (CCL-5), suppression of tumorigenicity 2 receptor (sST-2), IL-8, and IL-15 predicted mortality by univariate analysis. After adjusting for demographics and clinical characteristics, the mortality risk (as indicated by the hazard ratio [HR]) was higher for patients in the top 25th percentile for TNF-α (HR = 8.35, P = 0.005) and IL-8 (HR = 4.45, P = 0.01) and lower for CCL-5 (HR = 0.18, P ≤ 0.008). A logistic regression risk prediction model was developed and had an area under the receiver operating characteristic curve (AUC) of 0.91 for 90-day mortality and 0.77 for 90-day recurrence. While limited by being single site and retrospective, our work resulted in a model with a substantially greater predictive ability than white blood cell count. In conclusion, immune profiling demonstrated differences between patients in their response to CDI, offering the promise for precision medicine individualized treatment. IMPORTANCE Clostridioides difficile infection is the most common health care-associated infection in the United States with more than 20% patients experiencing symptomatic recurrence. The complex nature of host-bacterium interactions makes it difficult to predict the course of the disease based solely on clinical parameters. In the present study, we built a robust prediction model using representative plasma biomarkers and clinical parameters for 90-day all-cause mortality. Risk prediction based on immune biomarkers and clinical variables may contribute to treatment selection for patients as well as provide insight into the role of immune system in C. difficile pathogenesis.

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Performance Evaluation of the Luminex Aries C. difficile Assay in Comparison to Two Other Molecular Assays within a Multihospital Health Care Center

Journal of Clinical Microbiology ◽

10.1128/jcm.01092-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 3

Author(s):

Stefan Juretschko ◽

Ryhana Manji ◽

Reeti Khare ◽

Shubhagata Das ◽

Sherry Dunbar

Keyword(s):

Care Center ◽

Hospital Setting ◽

The United States ◽

Turnaround Time ◽

Sample Number ◽

Content Type ◽

Health Care Center ◽

Clostridioides Difficile ◽

Comparison Results ◽

Hands On

ABSTRACT Clostridioides difficile infection (CDI) remain a serious issue in the United States. Fast and accurate diagnosis of CDI is paramount to achieve immediate infection control initiation, triaging, and isolation, as well as appropriate antibiotic treatment. However, both, over- and underdiagnosis can lead to adverse patient outcomes, such as unnecessary administration of antibiotics or unwanted spread of spores in any hospital setting, respectively. In this prospective study, we evaluated the FDA-cleared Aries C. difficile assay and compared its performance and workflow characteristics to those of the BD Max Cdiff and Xpert C. difficile/Epi assays. Out of 302 samples tested, 55 (18.2%) samples were positive, and 234 (77.5%) samples were negative for C. difficile by all three testing methods. Comparison results showed a positive and negative percent agreement (PPA and NPA, respectively) between the Aries and Xpert assays of 95.2% (59/62) and 99.2% (238/240), respectively. The PPA and NPA between the Aries and BD Max assays were 91.8% (56/61) and 96.6% (230/238), respectively. Invalid result rates were determined to be 2.6% for the BD Max assay, 1.0% for the Aries assay, and 0% for the Xpert assay. Hands-on time (HoT) and total turnaround time (TAT) varied considerably depending on the sample number and instrument throughput. The HoT ranged from 1.2 to 3.5 min per sample, and the TAT was 1 to 2.3 h. Overall, the results demonstrated that the Aries assay is a rapid and sensitive method for the diagnosis of CDI in clinical laboratories.

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Candida albicans Airway Exposure Primes the Lung Innate Immune Response against Pseudomonas aeruginosa Infection through Innate Lymphoid Cell Recruitment and Interleukin-22-Associated Mucosal Response

Infection and Immunity ◽

10.1128/iai.01085-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 306-315 ◽

Cited By ~ 34

Author(s):

Jean Baptiste Mear ◽

Philippe Gosset ◽

Eric Kipnis ◽

Emmanuel Faure ◽

Rodrigue Dessein ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Candida Albicans ◽

Lung Injury ◽

Molecular Mechanisms ◽

Lymphoid Cells ◽

Content Type ◽

Cell Recruitment ◽

Interleukin 22 ◽

Cellular Source ◽

Pseudomonas Aeruginosa Infection

ABSTRACTPseudomonas aeruginosaandCandida albicansare two pathogens frequently encountered in the intensive care unit microbial community. We have demonstrated thatC. albicansairway exposure protected againstP. aeruginosa-induced lung injury. The goal of the present study was to characterize the cellular and molecular mechanisms associated withC. albicans-induced protection. Airway exposure byC. albicansled to the recruitment and activation of natural killer cells, innate lymphoid cells (ILCs), macrophages, and dendritic cells. This recruitment was associated with the secretion of interleukin-22 (IL-22), whose neutralization abolishedC. albicans-induced protection. We identified, by flow cytometry, ILCs as the only cellular source of IL-22. Depletion of ILCs by anti-CD90.2 antibodies was associated with a decreased IL-22 secretion and impaired survival afterP. aeruginosachallenge. Our results demonstrate that the production of IL-22, mainly by ILCs, is a major and inducible step in protection againstP. aeruginosa-induced lung injury. This cytokine may represent a clinical target inPseudomonas aeruginosa-induced lung injury.

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In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials

mBio ◽

10.1128/mbio.00019-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 5

Author(s):

Kurt Selle ◽

Joshua R. Fletcher ◽

Hannah Tuson ◽

Daniel S. Schmitt ◽

Lana McMillan ◽

...

Keyword(s):

Gut Microbiota ◽

Broad Spectrum ◽

Bacterial Species ◽

Bacterial Genome ◽

The United States ◽

Type I ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms. IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

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