Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

Citation: Girinathan, B. P., Braun, S., Sirigireddy, A. R., Lopez, J. E., & Govind, R. (2016). Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo. Plos One, 11(7), 18. doi:10.1371/journal.pone.0160107Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis

Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291

Citation: Girinathan, B. P., Monot, M., Boyle, D., McAllister, K. N., Sorg, J. A., Dupuy, B., & Govind, R. (2017). Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291. Msphere, 2(1), 14. doi:10.1128/mSphere.00383-16Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than to germinate the spores prepared from the WT strain. Transmission electron microscopic analysis of the spores also revealed a weakly assembled exosporium on the tcdR mutant spores. Accordingly, comparative transcriptome analysis showed many differentially expressed sporulation genes in the tcdR mutant compared to the WT strain. These data suggest that regulatory networks of toxin production and sporulation in C. difficile strain R20291 are linked with each other. IMPORTANCE C. difficile infects thousands of hospitalized patients every year, causing significant morbidity and mortality. C. difficile spores play a pivotal role in the transmission of the pathogen in the hospital environment. During infection, the spores germinate, and the vegetative bacterial cells produce toxins that damage host tissue. Thus, sporulation and toxin production are two important traits of C. difficile. In this study, we showed that a mutation in tcdR, the toxin gene regulator, affects both toxin production and sporulation in epidemic-type C. difficile strain R20291

Assessment of Anti-bacterial Activity of Herbal Silver Nanoparticles of Peristrophe bicalyculata (R.) Nees

Herbal medicinal plants have been used in traditional medicine for generations, and it\u27s fascinating to see how contemporary research has found the key components responsible for their healing properties. Silver nanoparticles were synthesised utilising a hydroalcoholic extract of Peristrophe bicalyculata (R.) Nees leaves (HAEPBL) and various concentrations of silver nitrate. The antibacterial activity of plant extracts and silver nanoparticles was then tested against several pathogenic bacteria using the disc diffusion method.The results show that herbal silver nanoparticles have promising antibacterial properties

Biosynthesis of Silver nanoparticles Using Rosaceae Petal extract and analysing its antimicrobial assay

Recent developments in nanoscience and nanotechnology have brought about a fundamental shift in the way we identify, treat, and prevent numerous diseases in all aspects of human life. Silver nanoparticles (AgNPs) are among the most significant and intriguing metallic nanoparticles employed in biomedical applications. AgNPs are very important for the domains of nanomedicine, nanoscience, and nanotechnology. Although numerous noble metals have been used for a wide range of applications, AgNPs have drawn special attention because of their potential for use in cancer treatment and diagnosis. The study showed an efficient method for the successful synthesis of AgNPs using petal extract from Rosaceae plants and characterizes them using a UV spectrometer and SEM. The produced AgNPs showed notable antibacterial activity against a variety of microbes, suggesting that they could find use as an antimicrobial agent in a number of different contexts. The work offers insightful information about how AgNPs might be used as a robust antibacterial agent against a variety of microbes

Secretion of Clostridium difficile Toxins A and B Requires the Holin-like Protein TcdE

The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system

Bacteriophage mediated toxin gene regulation in Clostridium difficle

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Spo0A suppresses <i>sin</i> locus expression in <i>Clostridioides difficile</i>

AbstractClostridioides 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 the production of toxins, and spores are responsible for the transmission and persistence of the organism. Previously we characterized sin locus regulators SinR and SinR’, where SinR is the regulator of toxin production and sporulation, while the SinR’ acting as its antagonist. In Bacillus subtilis, Spo0A, the master regulator of sporulation, regulates SinR, by regulating the expression of its antagonist sinI. However, the role of Spo0A in the expression of sinR and sinR’ in C. difficile is not yet 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. The qRT-PCR analysis for its expression further supported this data. By carrying out genetic and biochemical assays, we have shown that Spo0A can bind to the upstream region of this locus to regulates its expression. This study provides vital information that Spo0A regulates sin locus, which controls critical pathogenic traits such as sporulation, toxin production, and motility in C. difficile.IMPORTANCEClostridioides 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, sin locus is known to regulate both sporulation and toxin production. In this study, we have shown that Spo0A, the master regulator of sporulation to control the sin locus expression. We performed various genetic and biochemical experiments to show 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.</jats:sec

Phase variable expression of <i>pdcB</i>, a phosphodiesterase influences sporulation in <i>Clostridioides difficile</i>

AbstractClostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to hypervirulence strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovered that PdcB, a phosphodiesterase to influence sporulation in C. difficile UK1 strain positively. Through genetic and biochemical assays, we have shown that phase variable expression of pdcB results in hypo- and hyper-sporulation phenotype. In the “ON” orientation, the identified promotor is the right orientation to drive the expression of pdcB. Production of PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP concentration, resulting in hyper-sporulation phenotype. The OFF orientation of pdcB switch or mutating pdcB results in increased cyclic-di-GMP and hypo-sporulating phenotype. Additionally, we demonstrated that CodY binds to the upstream region of pdcB to represses its expression, and CodY mediated repression is relieved by the DNA inversion.</jats:p