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

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

Cell lysis in <i>C. difficile</i> A⁻B⁻ double mutant strain.

<p><b>A.</b> Growth curves of strains, 630A⁻B⁻ (<i>C. difficile tcd</i>AB double mutant), 630E (wild type) and a PaLoc negative strain. <i>C. difficile</i> strains were grown in TY medium in a 100 ml Erlenmeyer flask and the optical density at 600 nms was recorded at regular time interval. <b>B.</b> Bacterial cultures were harvested at a 30 hour time point for FACS analysis after propidium iodide (PI) and SYTO staining.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Complementation of TcdE mutant.

<p><b>A</b> Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. <b>B.</b> Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. <b>C.</b> Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. <b>D.</b> Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.</p

Bacterial strains, plasmids and phages used in this study.

<p>Bacterial strains, plasmids and phages used in this study.</p