In vitro and in vivo protective efficacies of antibodies that neutralize the RNA N-glycosidase activity of Shiga toxin 2

<p>Abstract</p> <p>Backgound</p> <p>Shiga toxin 2 (Stx2), one of two Stx liberated by Stx-producing <it>Escherichia coli</it>, is composed of an A subunit monomer and a B subunit pentamer, and is directly linked with hemolytic uremic syndrome in children. The pentameric B subunit binds to its cell surface receptor Gb₃for toxin internalization, and the A subunit follows intracellular retrograde transport to the cytosol where its RNA <it>N</it>-glycosidase activity (RNA-NGA) shuts down the protein synthesis, and leads to cell death. The present study investigated the ability of 19 Stx2 A subunit-specific human monoclonal antibodies (HuMAbs) to neutralize the RNA-NGA, and the association this neutralizing activity with protection of HeLa cells and mice against Stx2-induced death.</p> <p>Results</p> <p>The HuMAbs that were stronger inhibitors of RNA-NGA were also better at neutralizing Stx2 mediated HeLa cell death, and those that were weaker inhibitors of RNA-NGA activity were also weaker in protecting HeLa cells. These results suggest that the ability of an A subunit-specific antibody to block the RNA-NGA of the toxin is directly related to its ability to neutralize Stx2-mediated HeLa cell death. However, with the exception of the best RNA-NGA blocking antibodies 5C12 and 2F10, the efficacies of antibody neutralization of RNA-NGA of Stx2 did not correlate with their <it>in vivo </it>protective efficacies. The HuMAb 6C3, which neutralized RNA N-glycosidase activity of Stx2 less effectively than the HuMAbs 6D8 and 6B7, protected 100% of the mice against Stx2 challenge at 50 μg/mouse dose. In contrast, the HuMAbs 6D8 and 6B7, which neutralized RNA N-glycosidase activity of Stx2 more effectively than 6C3, protected 20% and 0% mice at that dose, respectively.</p> <p>Conclusions</p> <p>The neutralization efficiency of the RNA-NGA of Stx2 by A subunit-specific antibodies correlate strongly with their abilities to protect HeLa cells against Stx2-mediated toxicity but only the strongest RNA-NGA-neutralizing antibodies correlate very well with both protecting HeLa cells and mice against Stx2 challenge.</p

Effects of Surotomycin on Clostridium difficile Viability and Toxin Production In Vitro

The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

<p>Abstract</p> <p>Background</p> <p><it>Shigella dysenteriae </it>serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of <it>Shigella dysenteriae </it>serotype 1 (SD1) <it>in vitro </it>(derived from LB cell cultures) and <it>in vivo </it>(derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology.</p> <p>Results</p> <p>Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from <it>in vitro </it>and <it>in vivo </it>samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of <it>in silico </it>predicted SD1 membrane proteome) contributed to the most extensive survey of the <it>Shigella </it>membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under <it>in vivo </it>conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High <it>in vivo </it>abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while β-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers <it>in vivo</it>. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased <it>in vivo</it>.</p> <p>Conclusions</p> <p>Global proteomic profiling of SD1 comparing <it>in vivo vs. in vitro </it>proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under <it>in vivo </it>conditions, lending credence to their candidacy as potential vaccine targets.</p

A tetraspecific VHH-based neutralizing antibody modifies disease outcome in three animal models of Clostridium difficile infection

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd

The reduced genome of the parasitic microsporidian Enterocytozoon bieneusi lacks genes for core carbon metabolism

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial 2.5 License. The definitive version was published in Genome Biology and Evolution 2 (2010): 304, doi:10.1093/gbe/evq022.Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.This work was supported by grants from the Canadian Institutes for Health Research (MOP-84265), the National Institutes of Health (NIH AI31788, R21 AI52792, and R21 AI064118), and the National Science Foundation (MCB- 0135272). N.C. is a Scholar of the Canadian Institute for Advanced Research and is supported by a fellowship from the Swiss National Science Foundation (NSF) (PA00P3- 124166). D.E. is supported by the Swiss NSF. P.J.K. is a Fellow of the Canadian Institute for Advanced Research and a Senior Scholar of the Michael Smith Foundation for Health Research

Enhanced Actin Pedestal Formation by Enterohemorrhagic Escherichia coli O157:H7 Adapted to the Mammalian Host

Upon intestinal colonization, enterohemorrhagic Escherichia coli (EHEC) induces epithelial cells to generate actin “pedestals” beneath bound bacteria, lesions that promote colonization. To induce pedestals, EHEC utilizes a type III secretion system to translocate into the mammalian cell bacterial effectors such as translocated intimin receptor (Tir), which localizes in the mammalian cell membrane and functions as a receptor for the bacterial outer membrane protein intimin. Whereas EHEC triggers efficient pedestal formation during mammalian infection, EHEC cultured in vitro induces pedestals on cell monolayers with relatively low efficiency. To determine whether growth within the mammalian host enhances EHEC pedestal formation, we compared in vitro-cultivated bacteria with EHEC directly isolated from infected piglets. Mammalian adaptation by EHEC was associated with a dramatic increase in the efficiency of cell attachment and pedestal formation. The amounts of intimin and Tir were significantly higher in host-adapted than in in vitro-cultivated bacteria, but increasing intimin or Tir expression, or artificially increasing the level of bacterial attachment to mammalian cells, did not enhance pedestal formation by in vitro-cultivated EHEC. Instead, a functional assay suggested that host-adapted EHEC translocate Tir much more efficiently than does in vitro-cultivated bacteria. These data suggest that adaptation of EHEC to the mammalian intestine enhances bacterial cell attachment, expression of intimin and Tir, and translocation of effectors that promote actin signaling

Self-Regulation of Candida albicans Population Size during GI Colonization

Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth

Microsporidiosis and Malnutrition in Children with Persistent Diarrhea, Uganda

We show that the microsporidian fungus Enterocytozoon bieneusi is associated with lower rates of weight gain in children in Uganda with persistent diarrhea. This relationship remained after controlling for HIV and concurrent cryptosporidiosis. Children with microsporidiosis were predicted to weigh 1.3 kg less than children without microsporidiosis at 5 years of age

Adenovirus vector expressing Stx1/Stx2-neutralizing agent protects piglets infected with Escherichia coli O157: H7 against fatal systemic intoxication

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only V(H) domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant ‘targeting agent’ that binds a toxin at two unique sites and a ‘clearing Ab’ that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit