Human Pathogen Clostridium Difficile Research Articles

Type I Toxin-Antitoxin Systems in Clostridia.

Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.

Cellular Uptake and Mode-of-Action of Clostridium difficile Toxins.

Research on the human gut pathogen Clostridium difficile and its toxins has gained much attention, particularly as a consequence of the increasing threat to human health presented by emerging hypervirulent strains. Toxin A (TcdA) and B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (Clostridium difficile transferase). As C. difficile toxins are the causative agents of C. difficile-associated diseases (CDAD), such as antibiotics-associated diarrhea and pseudomembranous colitis, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Notably, a high proportion of studies on C. difficile toxins were performed in European laboratories. In this chapter we will highlight important recent advances in C. difficile toxins research.

Insights into the interactions between corrinoid iron-sulfur protein and methyl transferase from human pathogen Clostridium difficile

The human pathogen Clostridium difficile infection(CDI) is one of the most important healthcare-associated infections. Methyltransferase(MeTrCd) and corrinoid iron-sulfur protein(CoFeSPCd) are two key proteins in the acetyl-coenzyme A synthesis pathway of Clostridium difficile, which is essential for the survival of the pathogen and is absent in humans. Hence, the interaction between MeTrCd and CoFeSPCd can become innovative targets for the treatment of human CDI. In this study, the interaction between MeTrCd and CoFeSPCd was verified by fluorescence resonance energy transfer measurements. The influence of the interaction on the tertiary structure of MeTrCd and CoFeSPCd was studied by ANS-labeled fluorescence measurements. Molecular docking was also performed to understand the mechanism of the protein interactions. These results provide a molecular basis for innovative drug design and development to treat human CDI.

A genetic switch controls the production of flagella and toxins in Clostridium difficile.

In the human intestinal pathogen Clostridium difficile, flagella promote adherence to intestinal epithelial cells. Flagellar gene expression also indirectly impacts production of the glucosylating toxins, which are essential to diarrheal disease development. Thus, factors that regulate the expression of the flgB operon will likely impact toxin production in addition to flagellar motility. Here, we report the identification a “flagellar switch” that controls the phase variable production of flagella and glucosylating toxins. The flagellar switch, located upstream of the flgB operon containing the early stage flagellar genes, is a 154 bp invertible sequence flanked by 21 bp inverted repeats. Bacteria with the sequence in one orientation expressed flagellum and toxin genes, produced flagella, and secreted the toxins (“flg phase ON”). Bacteria with the sequence in the inverse orientation were attenuated for flagellar and toxin gene expression, were aflagellate, and showed decreased toxin secretion (“flg phase OFF”). The orientation of the flagellar switch is reversible during growth in vitro. We provide evidence that gene regulation via the flagellar switch occurs post-transcription initiation and requires a C. difficile-specific regulatory factor to destabilize or degrade the early flagellar gene mRNA when the flagellar switch is in the OFF orientation. Lastly, through mutagenesis and characterization of flagellar phase locked isolates, we determined that the tyrosine recombinase RecV, which catalyzes inversion at the cwpV switch, is also responsible for inversion at the flagellar switch in both directions. Phase variable flagellar motility and toxin production suggests that these important virulence factors have both advantageous and detrimental effects during the course of infection.

Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile

The human pathogen Clostridium difficile infection (CDI) is one of the most important healthcare-associated infections. The Wood-Ljungdahl pathway, which is responsible for Acetyl-CoA biosynthesis, is essential for the survival of the pathogen and is absent in humans. The key proteins and enzymes involved in the pathway are attractive targets for the treatment of CDI. Corrinoid iron-sulfur protein (CoFeSP) is a key protein and acts as a methyl transformer in the Wood-Ljungdahl pathway. In this study, CoFeSP from Clostridium difficile (CoFeSPCd) was cloned, expressed in E. coli and characterized for the first time. The structure and function of CoFeSPCd were investigated using homology structure modeling, spectroscopy, electrochemistry, steady state/pre-steady state kinetics and molecular docking. The two metal centers of CoFeSPCd, corrinoid cofactor and [4Fe-4S] cluster, were characterized using metal analysis, structural modeling, UV–Vis, EPR and direct electrochemistry. The methyl transfer activity between CH3-H4folate (CH3-THF) and CoFeSPCd catalyzed by methyl transferase (MeTrCd) was determined by kinetic studies. These results provide a molecular basis for innovative drug design and development to treat human CDI.

Synthesis of rationally designed tetrasaccharides for crystallographic and binding studies with Clostridium difficile toxins and unexpected partial N-methylations during catalytic hydrogenation of azides in methanol

A β-GlcNAc-LeX tetrasaccharides and another α-Gal-LeX analog have been synthesized for studying interaction with toxins produced by the human pathogen Clostridium difficile. LeX-based trisaccharides bearing either a 2-azidoethyl or a 6-azidohexyl aglycone have been employed in the total synthesis to afford the desired tetrasaccharides. Interestingly, during the final catalytic hydrogenation step to remove benzyl and benzylidene protecting groups and simultaneously reduce azide functionality, partial N-methylations were observed. The N-methylations appear to be a general issue with catalytic hydrogenation of azides in methanol.

Cell Lysing and DNA Fragmentation of Listeria Monocytogenes in One Step

Listeria is a gram-positive, deadly food borne bacterial pathogen with a mortality of 20-30% of those who get infected. People with a compromised immune system and pregnant women are more likely to suffer the effects of Listeriosis.1 The Listeria bacterium can grow under extreme conditions such as low pH and high temperatures. Places that are most likely to transmit the bacterium are food processing plants during any food processing step. Once Listeria is contracted in the human body, it can cross in to the blood, through the blood brain barrier, and into the cerebral spinal fluid.2 The most common DNA extraction method, PROMEGA, and detection method, PCR, are slow, costly, and require multiple steps for cell lysis, DNA fragmentation and purification.We demonstrate Listeria DNA can be efficiently and rapidly extracted and fragmented using metal microwave focusing lysing triangles and a conventional microwave. Our microwave based lysing approach2 has the particular advantages in that it can fragment the Listeria genome to much smaller DNA fragments, which is ideal for rapid detection using the MAMEF (Microwave-Accelerated Metal-Enhanced Fluorescence) platform3, as well as combining the two step process of cell lysing and DNA fragmentation.1.Painter J & Slutsker L. Listeriosis in humans. In: E. T. Ryser & E. H. Marth., editor. Listeria, Listeriosis and Food Safety 3rd ed Boca Raton, Florida: Taylor and Francis Group; 2007. p. 85-110.2.Melendez, J.H., et. al. (2013). Blind Evaluation of the Microwave-Accelerated Metal-Enhanced Fluorescence Ultrarapid and Sensitive Chlamydia Trachomatis test by use of Clinical Samples, Journal of Clinical Microbiology, 51(9), 2913-2920.3.Joshi, T., et. al. (2014). Extraction and Sensitive Detection of Toxins A and B from the human pathogen Clostridium difficile in 40 seconds using Microwave Accelerated Metal Enhanced Fluorescence, Plos One, 9,8,e104334.

A Fluorescent Reporter for Single Cell Analysis of Gene Expression in Clostridium difficile.

Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.

Isolation, Fragmentation and the Detection of Listeria DNA from Ground Beef

Listeria is a gram-positive, rod shaped food borne bacterial pathogen with a mortality of 20-30% of those who get infected. People with a compromised immune system and pregnant women are more likely to suffer effects of Listeriosis.[1] Listeria bacterium can grow under extreme conditions such as low pH and high temperatures. Places that are most likely to transmit the bacterium are food processing plants during any food processing step. Once Listeria is contracted in the human body, it can cross in to the blood, through the blood brain barrier, and into the cerebral spinal fluid. Current detection such as PCR methods are slow and costly and the extraction methods to see if Listeria is present is blood work, spinal tap, or a biopsy of the placenta.In this paper we show that Listeria DNA can be efficiently and rapidly extracted from ground bovine meat and lysed. Our microwave based lysing approach[2] has particular advantages in that it can fragment the Listeria genome to smaller DNA fragments, which is ideal for rapid detection using the MAMEF (Microwave-Accelerated Metal-Enhanced Fluorescence) platform[3], as well as diminishing the effects of DNases due to the elevated lysing temperatures.1.Painter J et. al, Listeriosis in humans. In: E. T. Ryser & E. H. Marth., editor. Listeria, Listeriosis and Food Safety 3rd ed Boca Raton, Florida: Taylor and Francis Group; 2007. p. 85-110.2.Melendez, J.H., et. al, (2013). Blind Evaluation of the Microwave-Accelerated Metal-Enhanced Fluorescence Ultrarapid and Sensitive Chlamydia Trachomatis test by use of Clinical Samples, Journal of Clinical Microbiology, 51(9), 2913-2920.3.Joshi, T., et. al, (2014). Extraction and Sensitive Detection of Toxins A and B from the human pathogen Clostridium difficile in 40 seconds using Microwave-Accelerated Metal-Enhanced Fluorescence, Plos One, 9,8,e104334.

Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation

Covalent attachment of surface proteins to the cell wall of Gram-positive bacteria requires a sortase-mediated transpeptidation reaction. In almost all Gram-positive bacteria, the housekeeping sortase, sortase A, recognizes the canonical recognition sequence LPXTG (X=any amino acid). The human pathogen Clostridium difficile carries a single putative sortase gene (cd2718) but neither transpeptidation activity nor specificity of CD2718 has been investigated. We produced recombinant CD2718 and examined its transpeptidation activity in vitro using synthetic peptides and MALDI-ToF(-ToF) MS analysis. We demonstrate that CD2718 has sortase activity with specificity for a (S/P)PXTG motif and can accommodate diaminopimelic acid as a substrate for transpeptidation.

A mother cell-to-forespore channel: current understanding and future challenges

Formation of endospores allows some bacteria to survive extreme nutrient limitation. The resulting dormant cell, the spore, persists in the environment and is highly resistant to physical and chemical stresses. During spore formation, cells divide asymmetrically and the mother cell engulfs the developing spore, encasing it within a double membrane and isolating it from the medium. Communication between mother cell and isolated forespore involves a specialised connection system that allows nurturing of the forespore and continued macromolecular synthesis, required to finalise spore maturation. Here, we review current understanding of this feeding channel formed by a forespore protein, SpoIIQ, and a mother cell protein, SpoIIIAH, in the model organism Bacillus subtilis and the important human pathogen Clostridium difficile. We also analyse the presence of this channel across endospore-forming bacteria and highlight the main questions still remaining.

Extraction and sensitive detection of toxins A and B from the human pathogen Clostridium difficile in 40 seconds using microwave-accelerated metal-enhanced fluorescence.

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile.

Direct electrochemistry of superoxide dismutases (Mn-, Fe-, and Ni-) from human pathogen Clostridium difficile: Toward application to superoxide biosensor

In this work, direct electrochemistry and electrocatalytic activity of four new kinds of artificially prepared superoxide dismutases (SODs) from human pathogen Clostridium difficile with different metal centers and microenvironments, that is, manganese superoxide dismutase (Mn-SOD), azide ion-coordinated manganese superoxide dismutase (Mn(N3−)-SOD), iron superoxide dismutase (Fe-SOD), and nickel superoxide dismutase (Ni-SOD), are systematically investigated. The direct electron transfer of the SODs is realized by 3-mercaptopropionic acid (MPA) confined on gold flowers electrodeposited onto glassy carbon electrode. The electrochemical parameters are dependent on the metal centers and microenvironments of the SODs with respect to the formal potential, reversibility of electrode reactions, and kinetic rate constant. Furthermore, except Ni-SOD, the other three kinds of SODs all exhibit a bifunctional electrocatalytic activity toward O2−. The combination of the enhanced electron transfer and the enzyme catalytic activities of SODs enables a sensitive and selective determination of O2−.

Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

Probing the metal specificity mechanism of superoxide dismutase from human pathogen Clostridium difficile

The molecular mechanism of the metal specificity to superoxide dismutase from human pathogen C. difficile (SODcd) was investigated by X-ray crystallography, spectroscopy, SOD activity assay, electrochemistry, and DFT calculations, and the results indicate that the cognate metal characters tuned by the metal micro-environment dominate the metal specificity of the SODcd.

Structural, spectroscopic and functional investigation into Fe-substituted MnSOD from human pathogen Clostridium difficile

Clostridium difficile, which inhabits the human digestive tract, is an etiological agent that causes pseudomembranous colitis and antibiotic-associated diarrhea. The oxidative stress tightly relates to its virulence, which highlights the function of its superoxide dismutase (SOD). The SOD from Clostridium difficile (SODcd) is a Mn/Fe cambialistic SOD with MnSODcd exhibiting an optimal activity while Fe-sub-MnSODcd showing 10-fold less activity. To explain why the Fe-loaded protein exhibits a much lower activity than the Mn-loaded form, Fe-sub-MnSODcd and MnSODcd were expressed in E. coli using M9 minimal medium, and characterized by X-ray crystallography, metal analysis, optical and EPR pH titration, azide binding affinity, etc. The pKa values of the active site residues and substrate affinities determined by spectroscopic titrations indicated that MnSODcd has a higher affinity for the substrate compared to Fe-sub-MnSODcd, while Fe-sub-MnSODcd has more affinity for OH(-). The different tendency of the anion ligation may be ascribed to the electronic configurations of Fe(3+) in d(5)vs. Mn(3+) in d(4), and it could be tuned by the hydrogen-bonding network around the active site of SODcd. Furthermore, the free energy for the O2˙(-) oxidation-reduction transition state from DFT calculation demonstrated that MnSODcd could disproportionate O2˙(-) more easily than Fe-sub-MnSODcd. These results revealed that SODcd could exquisitely differentiate between the Mn- and Fe-based activity. This metal specificity for SODcd may benefit the pathogenicity of C. difficile and pave a fundamental way for retarding C. difficile associated diseases.

Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile

Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA–seq and differential 5′-end RNA–seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA–based regulation of gene expression in this emergent enteropathogen.

Structural and functional investigation into acetyl-coenzyme A synthase and methyltransferase from human pathogen Clostridium difficile

Methyltransferase (MeTrCd) and acetyl-coenzyme A synthase (ACSCd) are two key enzymes in the acetyl-coenzyme A synthesis pathway of the human pathogen Clostridium difficile. The pathway is absent in humans and is essential for the survival of the pathogen. MeTrCd and ACSCd were cloned, expressed in E. coli, and characterized for the first time. Structural and functional investigations of the two enzymes were performed using homology structure modeling, fluorescence spectroscopy, and steady state/pre-steady state kinetics. The conformational change and methyl transfer activity of MeTrCd were shown to be pH dependent. The kinetic studies of MeTrCd at the optimal pH 5.1 yield the parameters kcat (2.63 s(-1)), Km (17.8 μM) and kcat/Km (0.15 μM(-1) s(-1)). The active site metal cluster (A-cluster) of ACSCd, [Fe4S4][NipNid], was characterized using metal analysis, structural modeling, and UV/Vis spectra of the characteristic features of [Fe4S4] cubane. Nip, as a labile metal, can be removed by treatment with chelators, resulting in the loss of ACS activity. Three bidentate chelators (1,10-phenanthroline, 8-hydroxyquinoline, and 2,2-dipyridyl) exhibited excellent inhibition effects on ACSCd methyl group transfer and acetyl-coenzyme A synthesis activity. These inhibitory effects were further examined using antibacterial activity assays against Clostridium difficile. These results provide a new strategy to find new potential antibiotics for the treatment of CDI.

In silicoanalysis of sequenced strains ofClostridium difficilereveals a related set of conjugative transposons carrying a variety of accessory genes

The human gut pathogen Clostridium difficile contains many conjugative transposons that have an array of accessory genes. In the current study, recently sequenced genomes were analyzed to identify new putative conjugative transposons. Eleven new elements in 5 C. difficile strains were identified and all had a similar structure to the previously described elements CTn1, CTn5 and CTn7 in C. difficile strain 630. Each element identified did however contain a new set of accessory genes compared with those previously reported; including those predicted to encode ABC transporters, a toxin/antitoxin system and multiple antibiotic resistance genes.

Structural Basis for a Kolbe-Type Decarboxylation Catalyzed by a Glycyl Radical Enzyme

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 Å resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (βγ)(4) tetramer of heterodimers composed of a catalytic β-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 Å distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the βγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.