- Email: [email protected]
Small Molecules Take A Big Step Against Clostridium difficile
bacteriophages with swapped receptors can specifically eliminate unwanted bacterial species within a mixed bacterial population with a minimal effect on adjacent species. A platform for phage and bacterial engineering had been demonstrated previously [4,5]. Extension of phage host range by isolating mutant phages with altered ligand-recognition properties, crossing species barriers, had also been shown previously [6]. However, combining the powerful engineering technologies to carry out specificity swapping between phages by complete swapping of entire sets of proteins is novel. Demonstrating the modularity of tail and tail-fiber proteins from various members of the T7 phage group and their ability to be assembled and function together despite significant sequence divergence is a significant achievement.
encode their own RNA polymerase, and depend on their host's genes. Thus, the proof of concept presented here is limited to the repertoire of hosts infected by the T7 group and, for example, exclude the entire group of Gram-positive bacteria [6]. Further work is required to extend the study to a variety of bacteria that are infected by a single phage scaffold with alternative specificity domains. The timeliness of the study is reflected in the recent information gained from microbiome studies suggesting ways to shape the microbiome, and the fact that the CRISPR-Cas system has also been recently used for eliminating and selecting desired populations in bacterial mixtures [8–10]. Combining the specificity of the CRISPR-Cas system with that of phages may prove to be synergistic in shaping bacterial populations. 1
These studies should make it easier to obtain regulatory approval for phages used in therapeutics or bacterial population editing. Typically, a cocktail of phages is required for efficient treatment (e.g., [7]). Each phage of the cocktail must undergo strict regulatory approval that requires labor and associated costs. The new study should allow the use of a single phage with many tail variations, thus simplifying the approval process for various applications and allowing easy adaptation of an already approved phage for alternative applications. This study proved to be efficient on various phages of the T7 group. Following transformation of the YAC encoding the desired phage DNA, phage capsids were produced in the E. coli host, even for phages whose natural host is not E. coli. This ability to produce infective particles in a non-host strain is due mainly to the fact that members of the T7 group are almost independent of their host's genes. Most of the required components for infective particles, including their own RNA polymerase, are encoded in their genome. However, most bacteriophages do not
746
Department of Clinical Microbiology and Immunology,
Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
Spotlight
Small Molecules Take A Big Step Against Clostridium difficile Greg L. Beilhartz,1 John Tam,1 and Roman A. Melnyk1,2,* Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile.
z
Equal contribution.
*Correspondence: [email protected] (U. Qimron). http://dx.doi.org/10.1016/j.tim.2015.10.006 References 1. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr. Opin. Microbiol. 14, 524–531 2. Round, J.L. and Mazmanian, S.K. (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 3. Ando, H. et al. (2015) Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems 1, 187–196 4. Gibson, D.G. et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 5. Jaschke, P.R. et al. (2012) A fully decompressed synthetic bacteriophage oX174 genome assembled and archived in yeast. Virology 434, 278–284 6. Molineux, I.J. (2005) The T7 Group. In The Bacteriophages (Abedon, S.T. and Calendar, R.L., eds), pp. 275–299, Oxford University Press 7. Abuladze, T. et al. (2008) Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230–6238 8. Bikard, D. et al. (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 9. Yosef, I. et al. (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl. Acad. Sci. U.S.A. 112, 7267–7272 10. Citorik, R.J. et al. (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145
Trends in Microbiology, December 2015, Vol. 23, No. 12
True to its name, Clostridium difficile has proven to be a difficult pathogen to manage therapeutically. Recently placed at the top of the notorious list of urgent antibiotic resistant threats by the CDC (http://www. cdc.gov/drugresistance/threat-report2013/index.html), C. difficile continues to infect and re-infect susceptible individuals with apparent impunity, and with everincreasing frequency. Paradoxically, the problematic resistance is not to the antibiotics currently used to treat C. difficile infection (CDI), but rather to the oft-prescribed fluoroquinolone antibiotics used to treat other infections. The collateral damage to the microbiome resulting from a broad-spectrum antibiotic ‘carpetbombing’ has the unfortunate consequence of creating ideal conditions within the gut for opportunistic C. difficile to flourish. Once established, C. difficile then secretes homologous protein toxins (i.e., TcdA and TcdB), which cause extensive damage to the intestinal epithelia, producing a range of downstream symptoms from diarrhea and pseudomembranous
colitis to life-threatening toxic megacolon. These factors complicate matters for clinicians deciding the best course of action for patients that test positive for toxigenic C. difficile. What is clear is that antibiotics are, at best, an inadequate treatment, and, at worst, introduce the possibility of creating an even bigger problem if and when C. difficile develops resistance to these targeted antibiotics. The idea of targeting toxins – instead of bacterial survival – as a strategy to treat CDI has gained momentum in recent years, fueled by studies examining the role of toxins in disease pathogenesis. Using isogenic toxin gene knockout experiments for instance, it was shown that strains lacking toxin (i.e., A—B—) were completely avirulent and unable to cause any discernable disease phenotype in preclinical animal models [1]. Results from a recently completed Phase 3 clinical trial showed that neutralization of TcdB with the monoclonal antibody bezlotoxumab reduced CDI recurrence (https://idsa. confex.com/idsa/2015/webprogram/ Paper52984.html). A new question arises as to whether targeting toxins earlier, particularly in higher risk patients, could have an even greater impact as they might prevent or lessen the occurrence, severity, and recurrence. Toxoid vaccines, currently in Phase 3 clinical trials for this purpose offer one potential solution, yet questions remain about who would receive such a vaccine, if proven successful. An argument could be made that a small molecule inhibitor of toxin action would offer the most ideal treatment modality for treating CDI. In addition to the clear practical advantages of small molecules related to cost and convenience, there are potential therapeutic benefits. Given that the site-of-action for the toxins is in the gut, it may be advantageous to deliver the toxin-neutralizing agents orally to intercept the toxins on the apical side of the gut, rather than wait for them to breach
Glucosyltransferase Autoprocessing doman doman
Delivery/Translocaon doman
Receptor-binding doman
Large clostridial toxins Hydrophobic region
Small molecule Inhibitors Target cell
Cell death
H+
vATPase
H+
H+ +
H
H+ H+
UDP-G
UDP Glucose
Rho Inacve
Inositol-P6
Figure 1. Host and Toxin Targets of Small Molecules that Protect Cells from Intoxication. TcdA and TcdB have four distinct functional domains; a glucosyltransferase domain (red); an autoprocessing domain (blue), a delivery/translocation domain (yellow); and a receptor-binding domain (green). Toxin entry begins with binding of toxins to cell-surface receptors, which triggers receptor-mediated endocytosis into vesicles. vATPases then acidify endosomes, triggering conformational changes in the toxin, leading most significantly to the formation of a transmembrane pore through which the glucosyltransferase and autoprocessing domains translocate through to the cytosol. Once in the cytosol, intracellular inositol hexakisphosphate allosterically activates the autoprocessing domain, releasing the glucosyltransferase domain, which then glucosylates RhoGTPases leading ultimately to cell death. Small molecule inhibitors have previously been shown to block receptor binding, endosomal acidification, and glucosyltransferase activity (red broken lines). In the recent study by Bender et al., ebselen, a potent inhibitor of autoprocessing (red unbroken line,) protected cells from intoxication and reduced disease pathology in a mouse model of CDI.
the barrier and enter the bloodstream. The multi-domain architecture of TcdA and TcdB (Figure 1) provides a number of different options for how to best block these toxins; but which approach is best? In a screen to identify small molecules that protected cells from anthrax lethal toxin, Slater et al. identified a number of hosttargeted compounds largely affecting endosomal maturation that were also able to protect cells from TcdB intoxication [2], consistent with a shared uptake mechanism for both toxins. In an attempt to identify additional host and toxin targets, a high content phenotypic screen was recently performed to identify small
molecules that protected cells from the cytopathic effects of TcdB (i.e., cell rounding) [3]. In addition to the expected preponderance of endosomal acidification inhibitors, the first direct toxin inhibitors were identified, including inhibitors of receptorbinding and glucosyltransferase activity. Now, in an exciting new study published in Science Translational Medicine, Bender et al. show for the first time that a small molecule inhibitor of toxin function was capable of preventing C. difficile induced disease pathology in vivo [4]. Using an activity-based screen on the isolated TcdB cysteine protease domain, the
Trends in Microbiology, December 2015, Vol. 23, No. 12
747
authors uncovered a number of activators and inhibitors of the autoprocessing domain. Among those that also protected cells from full length TcdB was the low molecular weight compound ebselen, a molecule with unique properties that that readily covalently modifies accessible cysteine residues by virtue of its reactive selenium moiety. In relation to the potent inhibition of cysteine protease domain activation and protection from cell rounding, mass spectrometry indicated at least one site was covalently modified, with competitive labeling assays pointing towards the active site cysteine. Most importantly, ebselen performed well in both a mouse toxigenic model of intraperitoneally injected TcdB as well as a clinically relevant infection model at doses up to 100 mg/kg/day. Their report of favorable clinical, survival, and histopathology scores, coupled with reduced TcdB potency in feces, point to the therapeutic potential of this toxin-neutralizing compound. This new discovery adds to the impressive growing resume of ebselen, which is currently undergoing clinical trials for the prevention of disparate disorders such as cardiovascular diseases, arthritis, stroke, atherosclerosis, and cancer [5].
intracellular targets [5] – may exert protection through happenstance polypharmacology. As the authors note, plausible intersections between the cellular pharmacology of ebselen and the cell biology of toxins exist, which may contribute to the observed protection. Nevertheless, this work shows for the first time that a small molecule is capable of halting toxin pathogenesis in vivo. Period.
The implications of this study are many. First, this work appears to provide in vivo validation of autoprocessing as a target for blocking TcdB. Given that completely ablating autoprocessing through mutagenesis of the active site cysteine (or the proteolytic cut-site) merely delays intoxication [6,7], this comes as somewhat of a surprise, and suggests that the degree of blockade required for protection in vitro might overestimate that required for in vivo protection. If true, it may be that other targets, such as the glucosyltransferase domain, in which active-site mutations have a more crippling effect on the toxins, may offer similar if not greater protection in vivo. Another possibility that cannot be excluded at this point is that ebselen – a promiscuous molecule with many
http://dx.doi.org/10.1016/j.tim.2015.10.009
748
As CDI rages on in hospitals around the world and the search for new non-antibiotic approaches heats up, small molecule antitoxins appear poised to enter the ring alongside exciting new therapies in the pipeline to battle this devastating pathogen. Acknowledgments G.L.B. is the recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR). Owing to space limitations, the authors apologize for not being able to cite all of the relevant papers. 1 Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada 2 Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
*Correspondence: [email protected] (R.A. Melnyk).
References 1. Kuehne, S.A. et al. (2014) Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 2. Slater, L.H. et al. (2013) Identification of novel host-targeted compounds that protect from anthrax lethal toxin-induced cell death. ACS Chem. Biol. 8, 812–822 3. Tam, J. et al. (2015) Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage. Chem. Biol. 22, 175–185 4. Bender, K.O. et al. (2015) A small-molecule antivirulence agent for treating Clostridium difficile infection. Sci. Transl. Med. 7, 306ra148 5. Azad, G.K. and Tomar, R.S. (2014) Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Mol. Biol. Rep. 41, 4865–4879 6. Li, S. et al. (2013) Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol. Pathog. Dis. 67, 11–18 7. Chumbler, N.M. et al. (2012) Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessingindependent mechanism. PLoS Pathog. 8, e1003072
Trends in Microbiology, December 2015, Vol. 23, No. 12
Spotlight
Adipose Tissue: Sanctuary for HIV/ SIV Persistence and Replication Suresh Pallikkuth1 and Mahesh Mohan2,* This commentary highlights new findings from a recent study identifying adipose tissue as a potential HIV reservoir and a major site of inflammation during chronic human/simian immunodeficiency virus (HIV/SIV) infection. A concise discussion about upcoming challenges and new research avenues for reducing chronic adipose inflammation during HIV/SIV infection is presented.
Antiretroviral therapy (ART) during HIV infection effectively reduces viremia, allows immune reconstitution and has increased the lifespan of HIV-infected individuals. Nevertheless, complete eradication of HIV-1 remains an unresolved task due to the persistence of long-lived viral reservoirs in different anatomical compartments coupled with the largely unknown nature of the extent and magnitude of these viral sanctuaries [1,2]. Accordingly, studies providing insights into HIV cellular reservoirs in previously unexplored anatomical compartments, and their contribution to HIV pathogenesis have great relevance for defining novel therapeutic strategies to achieve the goals of HIV cure. In this context, a recent study by Damouche and colleagues published in the September 2015 issue of PLoS Pathogens has extensively addressed the contribution of adipose tissue to maintenance of viral reservoirs and as a site of persistent
encode their own RNA polymerase, and depend on their host's genes. Thus, the proof of concept presented here is limited to the repertoire of hosts infected by the T7 group and, for example, exclude the entire group of Gram-positive bacteria [6]. Further work is required to extend the study to a variety of bacteria that are infected by a single phage scaffold with alternative specificity domains. The timeliness of the study is reflected in the recent information gained from microbiome studies suggesting ways to shape the microbiome, and the fact that the CRISPR-Cas system has also been recently used for eliminating and selecting desired populations in bacterial mixtures [8–10]. Combining the specificity of the CRISPR-Cas system with that of phages may prove to be synergistic in shaping bacterial populations. 1
These studies should make it easier to obtain regulatory approval for phages used in therapeutics or bacterial population editing. Typically, a cocktail of phages is required for efficient treatment (e.g., [7]). Each phage of the cocktail must undergo strict regulatory approval that requires labor and associated costs. The new study should allow the use of a single phage with many tail variations, thus simplifying the approval process for various applications and allowing easy adaptation of an already approved phage for alternative applications. This study proved to be efficient on various phages of the T7 group. Following transformation of the YAC encoding the desired phage DNA, phage capsids were produced in the E. coli host, even for phages whose natural host is not E. coli. This ability to produce infective particles in a non-host strain is due mainly to the fact that members of the T7 group are almost independent of their host's genes. Most of the required components for infective particles, including their own RNA polymerase, are encoded in their genome. However, most bacteriophages do not
746
Department of Clinical Microbiology and Immunology,
Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
Spotlight
Small Molecules Take A Big Step Against Clostridium difficile Greg L. Beilhartz,1 John Tam,1 and Roman A. Melnyk1,2,* Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile.
z
Equal contribution.
*Correspondence: [email protected] (U. Qimron). http://dx.doi.org/10.1016/j.tim.2015.10.006 References 1. Lu, T.K. and Koeris, M.S. (2011) The next generation of bacteriophage therapy. Curr. Opin. Microbiol. 14, 524–531 2. Round, J.L. and Mazmanian, S.K. (2009) The gut microbiota shapes intestinal immune responses during health and disease. Nat. Rev. Immunol. 9, 313–323 3. Ando, H. et al. (2015) Engineering modular viral scaffolds for targeted bacterial population editing. Cell Systems 1, 187–196 4. Gibson, D.G. et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 5. Jaschke, P.R. et al. (2012) A fully decompressed synthetic bacteriophage oX174 genome assembled and archived in yeast. Virology 434, 278–284 6. Molineux, I.J. (2005) The T7 Group. In The Bacteriophages (Abedon, S.T. and Calendar, R.L., eds), pp. 275–299, Oxford University Press 7. Abuladze, T. et al. (2008) Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230–6238 8. Bikard, D. et al. (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 9. Yosef, I. et al. (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl. Acad. Sci. U.S.A. 112, 7267–7272 10. Citorik, R.J. et al. (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145
Trends in Microbiology, December 2015, Vol. 23, No. 12
True to its name, Clostridium difficile has proven to be a difficult pathogen to manage therapeutically. Recently placed at the top of the notorious list of urgent antibiotic resistant threats by the CDC (http://www. cdc.gov/drugresistance/threat-report2013/index.html), C. difficile continues to infect and re-infect susceptible individuals with apparent impunity, and with everincreasing frequency. Paradoxically, the problematic resistance is not to the antibiotics currently used to treat C. difficile infection (CDI), but rather to the oft-prescribed fluoroquinolone antibiotics used to treat other infections. The collateral damage to the microbiome resulting from a broad-spectrum antibiotic ‘carpetbombing’ has the unfortunate consequence of creating ideal conditions within the gut for opportunistic C. difficile to flourish. Once established, C. difficile then secretes homologous protein toxins (i.e., TcdA and TcdB), which cause extensive damage to the intestinal epithelia, producing a range of downstream symptoms from diarrhea and pseudomembranous
colitis to life-threatening toxic megacolon. These factors complicate matters for clinicians deciding the best course of action for patients that test positive for toxigenic C. difficile. What is clear is that antibiotics are, at best, an inadequate treatment, and, at worst, introduce the possibility of creating an even bigger problem if and when C. difficile develops resistance to these targeted antibiotics. The idea of targeting toxins – instead of bacterial survival – as a strategy to treat CDI has gained momentum in recent years, fueled by studies examining the role of toxins in disease pathogenesis. Using isogenic toxin gene knockout experiments for instance, it was shown that strains lacking toxin (i.e., A—B—) were completely avirulent and unable to cause any discernable disease phenotype in preclinical animal models [1]. Results from a recently completed Phase 3 clinical trial showed that neutralization of TcdB with the monoclonal antibody bezlotoxumab reduced CDI recurrence (https://idsa. confex.com/idsa/2015/webprogram/ Paper52984.html). A new question arises as to whether targeting toxins earlier, particularly in higher risk patients, could have an even greater impact as they might prevent or lessen the occurrence, severity, and recurrence. Toxoid vaccines, currently in Phase 3 clinical trials for this purpose offer one potential solution, yet questions remain about who would receive such a vaccine, if proven successful. An argument could be made that a small molecule inhibitor of toxin action would offer the most ideal treatment modality for treating CDI. In addition to the clear practical advantages of small molecules related to cost and convenience, there are potential therapeutic benefits. Given that the site-of-action for the toxins is in the gut, it may be advantageous to deliver the toxin-neutralizing agents orally to intercept the toxins on the apical side of the gut, rather than wait for them to breach
Glucosyltransferase Autoprocessing doman doman
Delivery/Translocaon doman
Receptor-binding doman
Large clostridial toxins Hydrophobic region
Small molecule Inhibitors Target cell
Cell death
H+
vATPase
H+
H+ +
H
H+ H+
UDP-G
UDP Glucose
Rho Inacve
Inositol-P6
Figure 1. Host and Toxin Targets of Small Molecules that Protect Cells from Intoxication. TcdA and TcdB have four distinct functional domains; a glucosyltransferase domain (red); an autoprocessing domain (blue), a delivery/translocation domain (yellow); and a receptor-binding domain (green). Toxin entry begins with binding of toxins to cell-surface receptors, which triggers receptor-mediated endocytosis into vesicles. vATPases then acidify endosomes, triggering conformational changes in the toxin, leading most significantly to the formation of a transmembrane pore through which the glucosyltransferase and autoprocessing domains translocate through to the cytosol. Once in the cytosol, intracellular inositol hexakisphosphate allosterically activates the autoprocessing domain, releasing the glucosyltransferase domain, which then glucosylates RhoGTPases leading ultimately to cell death. Small molecule inhibitors have previously been shown to block receptor binding, endosomal acidification, and glucosyltransferase activity (red broken lines). In the recent study by Bender et al., ebselen, a potent inhibitor of autoprocessing (red unbroken line,) protected cells from intoxication and reduced disease pathology in a mouse model of CDI.
the barrier and enter the bloodstream. The multi-domain architecture of TcdA and TcdB (Figure 1) provides a number of different options for how to best block these toxins; but which approach is best? In a screen to identify small molecules that protected cells from anthrax lethal toxin, Slater et al. identified a number of hosttargeted compounds largely affecting endosomal maturation that were also able to protect cells from TcdB intoxication [2], consistent with a shared uptake mechanism for both toxins. In an attempt to identify additional host and toxin targets, a high content phenotypic screen was recently performed to identify small
molecules that protected cells from the cytopathic effects of TcdB (i.e., cell rounding) [3]. In addition to the expected preponderance of endosomal acidification inhibitors, the first direct toxin inhibitors were identified, including inhibitors of receptorbinding and glucosyltransferase activity. Now, in an exciting new study published in Science Translational Medicine, Bender et al. show for the first time that a small molecule inhibitor of toxin function was capable of preventing C. difficile induced disease pathology in vivo [4]. Using an activity-based screen on the isolated TcdB cysteine protease domain, the
Trends in Microbiology, December 2015, Vol. 23, No. 12
747
authors uncovered a number of activators and inhibitors of the autoprocessing domain. Among those that also protected cells from full length TcdB was the low molecular weight compound ebselen, a molecule with unique properties that that readily covalently modifies accessible cysteine residues by virtue of its reactive selenium moiety. In relation to the potent inhibition of cysteine protease domain activation and protection from cell rounding, mass spectrometry indicated at least one site was covalently modified, with competitive labeling assays pointing towards the active site cysteine. Most importantly, ebselen performed well in both a mouse toxigenic model of intraperitoneally injected TcdB as well as a clinically relevant infection model at doses up to 100 mg/kg/day. Their report of favorable clinical, survival, and histopathology scores, coupled with reduced TcdB potency in feces, point to the therapeutic potential of this toxin-neutralizing compound. This new discovery adds to the impressive growing resume of ebselen, which is currently undergoing clinical trials for the prevention of disparate disorders such as cardiovascular diseases, arthritis, stroke, atherosclerosis, and cancer [5].
intracellular targets [5] – may exert protection through happenstance polypharmacology. As the authors note, plausible intersections between the cellular pharmacology of ebselen and the cell biology of toxins exist, which may contribute to the observed protection. Nevertheless, this work shows for the first time that a small molecule is capable of halting toxin pathogenesis in vivo. Period.
The implications of this study are many. First, this work appears to provide in vivo validation of autoprocessing as a target for blocking TcdB. Given that completely ablating autoprocessing through mutagenesis of the active site cysteine (or the proteolytic cut-site) merely delays intoxication [6,7], this comes as somewhat of a surprise, and suggests that the degree of blockade required for protection in vitro might overestimate that required for in vivo protection. If true, it may be that other targets, such as the glucosyltransferase domain, in which active-site mutations have a more crippling effect on the toxins, may offer similar if not greater protection in vivo. Another possibility that cannot be excluded at this point is that ebselen – a promiscuous molecule with many
http://dx.doi.org/10.1016/j.tim.2015.10.009
748
As CDI rages on in hospitals around the world and the search for new non-antibiotic approaches heats up, small molecule antitoxins appear poised to enter the ring alongside exciting new therapies in the pipeline to battle this devastating pathogen. Acknowledgments G.L.B. is the recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR). Owing to space limitations, the authors apologize for not being able to cite all of the relevant papers. 1 Molecular Structure & Function, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada 2 Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
*Correspondence: [email protected] (R.A. Melnyk).
References 1. Kuehne, S.A. et al. (2014) Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain. J. Infect. Dis. 209, 83–86 2. Slater, L.H. et al. (2013) Identification of novel host-targeted compounds that protect from anthrax lethal toxin-induced cell death. ACS Chem. Biol. 8, 812–822 3. Tam, J. et al. (2015) Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage. Chem. Biol. 22, 175–185 4. Bender, K.O. et al. (2015) A small-molecule antivirulence agent for treating Clostridium difficile infection. Sci. Transl. Med. 7, 306ra148 5. Azad, G.K. and Tomar, R.S. (2014) Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Mol. Biol. Rep. 41, 4865–4879 6. Li, S. et al. (2013) Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol. Pathog. Dis. 67, 11–18 7. Chumbler, N.M. et al. (2012) Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessingindependent mechanism. PLoS Pathog. 8, e1003072
Trends in Microbiology, December 2015, Vol. 23, No. 12
Spotlight
Adipose Tissue: Sanctuary for HIV/ SIV Persistence and Replication Suresh Pallikkuth1 and Mahesh Mohan2,* This commentary highlights new findings from a recent study identifying adipose tissue as a potential HIV reservoir and a major site of inflammation during chronic human/simian immunodeficiency virus (HIV/SIV) infection. A concise discussion about upcoming challenges and new research avenues for reducing chronic adipose inflammation during HIV/SIV infection is presented.
Antiretroviral therapy (ART) during HIV infection effectively reduces viremia, allows immune reconstitution and has increased the lifespan of HIV-infected individuals. Nevertheless, complete eradication of HIV-1 remains an unresolved task due to the persistence of long-lived viral reservoirs in different anatomical compartments coupled with the largely unknown nature of the extent and magnitude of these viral sanctuaries [1,2]. Accordingly, studies providing insights into HIV cellular reservoirs in previously unexplored anatomical compartments, and their contribution to HIV pathogenesis have great relevance for defining novel therapeutic strategies to achieve the goals of HIV cure. In this context, a recent study by Damouche and colleagues published in the September 2015 issue of PLoS Pathogens has extensively addressed the contribution of adipose tissue to maintenance of viral reservoirs and as a site of persistent