Evaluation of Candidate Reference Genes Stability for Gene Expression Analysis by Reverse Transcription QPCR in Clostridium Perfringens

Identification of stable reference genes for normalization purposes is necessary for obtaining reliable and accurate results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. To our knowledge, no reference gene(s) have been validated for this purpose in Clostridium perfringens. In this study, the expression profile of ten candidate reference genes from three strains of C. perfringens were assessed for stability under various experimental conditions using geNorm in qbase+. These stability rankings were then compared to stability assessments evaluated by BestKeeper, NormFinder, delta Ct, and RefFinder algorithms. When comparing all the analyses; gyrA, ftsZ, and recA were identified within the most stable genes under the different experimental conditions and were further tested as a set of reference genes for normalization of alpha toxin gene expression over a 22-hour period. Depending on the condition, rpoA and rho might also be suitable to include as part of the reference set. Although commonly used for the purpose of normalizing RT-qPCR data, the 16S rRNA gene (rrs) was found to be an unsuitable gene to be used as a reference. This work provides a framework for the selection of a suitable stable reference gene set for data normalization of C. perfringens gene expression.

Reduced Toxicity of Centruroides vittatus (Say, 1821) May Result from Lowered Sodium β Toxin Gene Expression and Toxin Protein Production

Body tissue and venom glands from an eastern population of the scorpion Centruroides vittatus (Say, 1821) were homogenized and molecular constituents removed to characterize putative sodium β toxin gene diversity, RT-qPCR, transcriptomic, and proteomic variation. We cloned sodium β toxins from genomic DNA, conducted RT-qPCR experiments with seven sodium β toxin variants, performed venom gland tissue RNA-seq, and isolated venom proteins for mass spectrophotometry. We identified >70 putative novel sodium β toxin genes, 111 toxin gene transcripts, 24 different toxin proteins, and quantified sodium β toxin gene expression variation among individuals and between sexes. Our analyses contribute to the growing evidence that venom toxicity among scorpion taxa and their populations may be associated with toxin gene diversity, specific toxin transcripts variation, and subsequent protein production. Here, slight transcript variation among toxin gene variants may contribute to the major toxin protein variation in individual scorpion venom composition.

Elucidating the Venom Diversity in Sri Lankan Spectacled Cobra (Naja naja) through De Novo Venom Gland Transcriptomics, Venom Proteomics and Toxicity Neutralization

Inadequate effectiveness of Indian antivenoms in treating envenomation caused by the Spectacled Cobra/Indian Cobra (Naja naja) in Sri Lanka has been attributed to geographical variations in the venom composition. This study investigated the de novo venom-gland transcriptomics and venom proteomics of the Sri Lankan N. naja (NN-SL) to elucidate its toxin gene diversity and venom variability. The neutralization efficacy of a commonly used Indian antivenom product in Sri Lanka was examined against the lethality induced by NN-SL venom in mice. The transcriptomic study revealed high expression of 22 toxin genes families in NN-SL, constituting 46.55% of total transcript abundance. Three-finger toxins (3FTX) were the most diversely and abundantly expressed (87.54% of toxin gene expression), consistent with the dominance of 3FTX in the venom proteome (72.19% of total venom proteins). The 3FTX were predominantly S-type cytotoxins/cardiotoxins (CTX) and α-neurotoxins of long-chain or short-chain subtypes (α-NTX). CTX and α-NTX are implicated in local tissue necrosis and fatal neuromuscular paralysis, respectively, in envenomation caused by NN-SL. Intra-species variations in the toxin gene sequences and expression levels were apparent between NN-SL and other geographical specimens of N. naja, suggesting potential antigenic diversity that impacts antivenom effectiveness. This was demonstrated by limited potency (0.74 mg venom/ml antivenom) of the Indian polyvalent antivenom (VPAV) in neutralizing the NN-SL venom. A pan-regional antivenom with improved efficacy to treat N. naja envenomation is needed.

The C-Terminal Domain of Clostridioides difficile TcdC Is Exposed on the Bacterial Cell Surface

ABSTRACT Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (OB-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors. IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiTopt) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development.

Toxin A and B genes expression of Clostridium difficile in the sub-minimum inhibitory concentration of clindamycin, vancomycin and in combination with ceftazidime

Background and Objectives: Antibiotics prescribed for infections have diverse effects on microbiota and the pathogen Clostridium difficile (C. difficile) as the most important antibiotic-associated diarrhea. This study aims to determine the gene expression of toxins A and B at the transcription level in the sub-MIC of vancomycin (VAN), clindamycin (CLI), and cef- tazidime (CAZ) alone and in combination. Materials and Methods: The MIC and fractional inhibitory concentration (FIC) of two C. difficile samples (a clinical isolate and ATCC 9689) were determined by microdilution and checkerboard microdilution methods, respectively. The total RNA was extracted from the medium inoculated with ~106 CFU/mL of fresh bacteria in the pre-reduced medium containing ½ MIC of antibiotics alone and ½ FIC of antibiotics in combination. Real-time PCR was performed by sybrGreen methods in triplicate, and the data were analyzed by the comparative ∆∆CT method. Results: All antibiotics except CAZ (alone and in combination) decreased the gene expression of toxins A and B within 24 hours. VAN and CLI reduced toxin gene expression within 24 and 48 hours. However, CAZ alone and in combination with VAN as well as CLI increased the gene expression of toxins A and B. Conclusion: The results confirmed toxin gene transcription and toxin production are associated with the type of isolates and antibiotics, as well as the combined form of antibiotics. This could be the reason which can explain the occurrence of C. difficile infection among patients who were treated with the third generation of cephalosporins alone and in combination with another antibiotic in the form of combinational therapy.

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the OB-fold domain present at the C-terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the C-terminal OB-fold.In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action that is different from characterized anti-sigma factors.ImportanceTranscription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.Activity of TcdC has been mapped to its C-terminal OB fold domain. TcdC is anchored in the bacterial membrane, through its hydrophobic N-terminus and acting as an anti-sigma factor would require cytoplasmic localization of the C-terminal domain.Remarkably, topology predictions for TcdC suggest the N-terminus to be membrane localized and the C-terminal domain to be located extracellularly. Using independent assays, we show that the C-terminus of TcdC indeed is located in the extracellular environment, which is incompatible with its proposed role as anti-sigma factor in toxin regulation.

Detection of Abrin-Like and Prepropulchellin-Like Toxin Genes and Transcripts Using Whole Genome Sequencing and Full-Length Transcript Sequencing of Abrus precatorius

The sequenced genome and the leaf transcriptome of a near relative of Abrus pulchellus and Abrus precatorius was analyzed to characterize the genetic basis of toxin gene expression. From the high-quality genome assembly, a total of 26 potential coding regions were identified that contain genes with abrin-like, pulchellin-like, and agglutinin-like homology, with full-length transcripts detected in leaf tissue for 9 of the 26 coding regions. All of the toxin-like genes were identified within only five isolated regions of the genome, with each region containing 1 to 16 gene variants within each genomic region (<1 Mbp). The Abrus precatorius cultivar sequenced here contains genes which encode for proteins that are homologous to certain abrin and prepropulchellin genes previously identified, and we observed substantial diversity of genes and predicted gene products in Abrus precatorius and previously characterized toxins. This suggests diverse toxin repertoires within Abrus, potentially the results of rapid toxin evolution.

Strain-Dependent RstA Regulation of Clostridioides difficile Toxin Production and Sporulation

ABSTRACT The anaerobic spore former Clostridioides difficile causes significant diarrheal disease in humans and other mammals. Infection begins with the ingestion of dormant spores, which subsequently germinate within the host gastrointestinal tract. There, the vegetative cells proliferate and secrete two exotoxins, TcdA and TcdB, which cause disease symptoms. Although spore formation and toxin production are critical for C. difficile pathogenesis, the regulatory links between these two physiological processes are not well understood and are strain dependent. Previously, we identified a conserved C. difficile regulator, RstA, that promotes sporulation initiation through an unknown mechanism and directly and indirectly represses toxin and motility gene transcription in the historical isolate 630Δerm. To test whether perceived strain-dependent differences in toxin production and sporulation are mediated by RstA, we created an rstA mutant in the epidemic ribotype 027 strain R20291. RstA affected sporulation and toxin gene expression similarly but more robustly in R20291 than in 630Δerm. In contrast, no effect on motility gene expression was observed in R20291. Reporter assays measuring transcriptional regulation of tcdR, the sigma factor gene essential for toxin gene expression, identified sequence-dependent effects influencing repression by RstA and CodY, a global nutritional sensor, in four diverse C. difficile strains. Finally, sequence- and strain-dependent differences were evident in RstA negative autoregulation of rstA transcription. Altogether, our data suggest that strain-dependent differences in RstA regulation contribute to the sporulation and toxin phenotypes observed in R20291. Our data establish RstA as an important regulator of C. difficile virulence traits. IMPORTANCE Two critical traits of Clostridioides difficile pathogenesis are toxin production, which causes disease symptoms, and spore formation, which permits survival outside the gastrointestinal tract. The multifunctional regulator RstA promotes sporulation and prevents toxin production in the historical strain 630Δerm. Here, we show that RstA exhibits stronger effects on these phenotypes in an epidemic isolate, R20291, and additional strain-specific effects on toxin and rstA expression are evident. Our data demonstrate that sequence-specific differences within the promoter for the toxin regulator TcdR contribute to the regulation of toxin production by RstA and CodY. These sequence differences account for some of the variability in toxin production among isolates and may allow strains to differentially control toxin production in response to a variety of signals.