Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production

ABSTRACTThe Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action.IMPORTANCEC. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agr1 genes exhibit distinct phenotypes in C. difficile. These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr’s mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.

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Finding of Agr Phase Variants in Staphylococcus aureus

mBio ◽

10.1128/mbio.00796-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 3

Author(s):

Vishal Gor ◽

Aya J. Takemura ◽

Masami Nishitani ◽

Masato Higashide ◽

Veronica Medrano Romero ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Virulence Factors ◽

Phase Variation ◽

Accessory Gene ◽

Content Type ◽

Accessory Gene Regulator ◽

Immune Mediated ◽

A Minor ◽

First Time ◽

Phase Variants

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

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Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile

Access Microbiology ◽

10.1099/acmi.0.000134 ◽

2020 ◽

Vol 2 (7) ◽

Author(s):

Yuta Okada ◽

Shu Okugawa ◽

Mahoko Ikeda ◽

Tatsuya Kobayashi ◽

Ryoichi Saito ◽

...

Keyword(s):

Genetic Diversity ◽

In Silico ◽

Type Species ◽

Clinical Samples ◽

Pcr Analysis ◽

Accessory Gene ◽

Content Type ◽

Link Type ◽

Accessory Gene Regulator ◽

Clostridioides Difficile

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

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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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Impact of agr Functionality on the Outcome of Patients with Methicillin-Susceptible Staphylococcus aureus Bacteremia

Microbiology Spectrum ◽

10.1128/spectrum.00116-21 ◽

2021 ◽

Author(s):

Jeong Eun Lee ◽

Shinwon Lee ◽

Sohee Park ◽

Soon O. Lee ◽

Sun H. Lee

Keyword(s):

Staphylococcus Aureus ◽

Accessory Gene ◽

Content Type ◽

Accessory Gene Regulator ◽

Staphylococcus Aureus Bacteremia ◽

Mssa Infection

Few studies have examined the association between methicillin-susceptible Staphylococcus aureus (MSSA) infection and accessory gene regulator ( agr ) functionality. We evaluated the association between agr dysfunction and mortality in patients with MSSA bacteremia.

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RstA Is a Major Regulator ofClostridioides difficileToxin Production and Motility

mBio ◽

10.1128/mbio.01991-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 10

Author(s):

Adrianne N. Edwards ◽

Brandon R. Anjuwon-Foster ◽

Shonna M. McBride

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Toxin Production ◽

Toxin Gene ◽

Dna Binding Domain ◽

Content Type ◽

Toxin Genes ◽

Clostridioides Difficile ◽

Species Specific ◽

Toxin Gene Expression

ABSTRACTClostridioides difficileinfection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two majorC. difficiletoxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified aC. difficileregulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, andrstAtranscription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds therstApromoter via the predicted DNA-binding domain. Through mutational analysis of therstApromoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genestcdAandtcdB, as well as the promoters for thesigDandtcdRgenes, which encode regulators of toxin gene expression. Complementation analyses with theClostridium perfringensRstA ortholog and a multispecies chimeric RstA protein revealed that theC. difficileC-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal pathogen of humans and other mammals.C. difficileproduces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link betweenC. difficilesporulation and toxin production. Further, our data suggest thatC. difficiletoxin production is regulated through a direct, species-specific sensing mechanism.

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ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

mSphere ◽

10.1128/msphere.00061-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 3

Author(s):

Joseph P. Zackular ◽

Reece J. Knippel ◽

Christopher A. Lopez ◽

William N. Beavers ◽

C. Noel Maxwell ◽

...

Keyword(s):

Immune Response ◽

Therapeutic Potential ◽

Neutrophil Infiltration ◽

Toxin Production ◽

Host Protein ◽

Content Type ◽

Nutritional Immunity ◽

Clostridioides Difficile ◽

Therapeutic Development ◽

Dependent Growth

ABSTRACT Clostridioides difficile is a spore-forming bacterium that causes severe colitis and is a major public health threat. During infection, C. difficile toxin production results in damage to the epithelium and a hyperinflammatory response. The immune response to CDI leads to robust neutrophil infiltration at the sight of infection and the deployment of numerous antimicrobials. One of the most abundant host immune factors associated with CDI is calprotectin, a metal-chelating protein with potent antimicrobial activity. Calprotectin is essential to the innate immune response to C. difficile and increasing levels of calprotectin correlate with disease severity in both adults and children with CDI. The fact that C. difficile persists in the presence of high levels of calprotectin suggests that this organism may deploy strategies to compete with this potent antimicrobial factor for essential nutrient metals during infection. In this report, we demonstrate that a putative zinc (Zn) transporter, ZupT, is employed by C. difficile to survive calprotectin-mediated metal limitation. ZupT is highly expressed in the presence of calprotectin and is required to protect C. difficile against calprotectin-dependent growth inhibition. When competing against wild-type C. difficile, zupT mutants show a defect in colonization and persistence in a murine model of infection. Together these data demonstrate that C. difficile utilizes a metal import system to combat nutritional immunity during CDI and suggest that strategies targeting nutrient acquisition in C. difficile may have therapeutic potential. IMPORTANCE During infection, pathogenic organisms must acquire essential transition metals from the host environment. Through the process of nutritional immunity, the host employs numerous strategies to restrict these key nutrients from invading pathogens. In this study, we describe a mechanism by which the important human pathogen Clostridioides difficile resists transition-metal limitation by the host. We report that C. difficile utilizes a zinc transporter, ZupT, to compete with the host protein calprotectin for nutrient zinc. Inactivation of this transporter in C. difficile renders this important pathogen sensitive to host-mediated metal restriction and confers a fitness disadvantage during infection. Our study demonstrates that targeting nutrient metal transport proteins in C. difficile is a potential avenue for therapeutic development.

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Mini droplets to super droplets: evolution of self-assembled Au droplets on GaAs(111)B and (110)

Journal of Applied Crystallography ◽

10.1107/s1600576713034316 ◽

2014 ◽

Vol 47 (2) ◽

pp. 505-510 ◽

Cited By ~ 12

Author(s):

Mao Sui ◽

Ming-Yu Li ◽

Eun-Soo Kim ◽

Jihoon Lee

Keyword(s):

Growth Parameters ◽

Average Diameter ◽

Growth Conditions ◽

Average Density ◽

Minor Effect ◽

Average Height ◽

Deposition Amount ◽

Self Assembled ◽

A Minor ◽

20 Nm

In this article, the effect of deposition amount on self-assembled Au droplets fabricated on GaAs(111)B and (110) is presented. The investigation is systematically performed by the variation of the Au deposition amount from 2 to 20 nm while fixing the other growth parameters such as annealing temperature and duration to clearly observe the effect. Under identical growth conditions, the self-assembled Au droplets show significantly different size and density depending on the amount of Au deposition:i.e.the average height varies by 436% from 21.8 to 95.5 nm and the average diameter swings by 827% from 52 to 430 nm, showing that the size increase is dominated by the lateral expansion. Meanwhile the average density varies by over two orders of magnitude from 1.24 × 108to 4.48 × 1010 cm−2on GaAs(111)B. With relatively low Au deposition amounts, below 3 nm, round dome-shaped mini Au droplets with high packing density can be fabricated, while super large Au droplets result with higher deposition amounts, above 10 nm, with a density two orders of magnitude lower. It is also found that the surface index has a minor effect on the fabrication of self-assembled Au droplets with the variation of deposition amount. The results are systematically analyzed and discussed in terms of atomic force microscopy and scanning electron microscopy images, line profiles, power spectrums, r.m.s. surface roughness, and size and density plots.

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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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Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01401-20 ◽

2020 ◽

Vol 65 (1) ◽

pp. e01401-20

Author(s):

Hannah C. Harris ◽

Emma L. Best ◽

Charmaine Normington ◽

Nathalie Saint-Lu ◽

Frédérique Sablier-Gallis ◽

...

Keyword(s):

Healthy Subjects ◽

Toxin Production ◽

Antibiotic Exposure ◽

Anaerobic Incubation ◽

Colonization Resistance ◽

Fecal Samples ◽

Content Type ◽

Clostridioides Difficile ◽

Gastrointestinal Pathogens

ABSTRACTA healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or −80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

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Virulence Factors of Clostridioides (Clostridium) difficile Linked to Recurrent Infections

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/7127850 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7

Author(s):

Laura Tijerina-Rodríguez ◽

Licet Villarreal-Treviño ◽

Rayo Morfín-Otero ◽

Adrián Camacho-Ortíz ◽

E. Garza-González

Keyword(s):

Virulence Factors ◽

Toxin Production ◽

Negative Regulator ◽

Binary Toxin ◽

Recurrent Infections ◽

Accessory Gene ◽

Accessory Gene Regulator ◽

Cell Internalization ◽

Adhesion Efficiency ◽

First Recurrence

From 20 to 30% of Clostridioides (Clostridium) difficile infection (CDI), patients might develop recurrence of the infection (RCDI) and, after the first recurrence, the risk of further episodes increases up to 60%. Several bacterial virulence factors have been associated with RCDI, including the elevated production of toxins A and B, the presence of a binary toxin CDT, and mutations in the negative regulator of toxin expression, tcdC. Additional factors have shown to regulate toxin production and virulence in C. difficile in RCDI, including the accessory-gene regulator agr, which acts as a positive switch for toxin transcription. Furthermore, adhesion and motility-associated factors, such as Cwp84, SlpA, and flagella, have shown to increase the adhesion efficiency to host epithelia, cell internalization, and the formation of biofilm. Finally, biofilm confers to C. difficile protection from antibiotics and acts as a reservoir for spores that allow the persistence of the infection in the host. In this review, we describe the key virulence factors of C. difficile that have been associated with recurrent infections.

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