Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H.

BackgroundOnly Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.MethodsWe therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay.ResultsAnti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody.ConclusionsThis 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism

Identification and characterisation of toxin-antitoxin systems (TA) in Burkholderia pseudomallei

The aim of this study was to identify and characterise type II toxin-antitoxin (TA) systems in Burkholderia pseudomallei, the causative agent of the human disease melioidosis. 8 putative TA systems were identified within the genome of B. pseudomallei K96243. 5 of these were located witihn genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. HicA also caused growth arrest in B. pseudomallei K96243 ΔhicAB. These toxin induced phenotypes were enhanced by an <3kDa extracellular factor that accumulated in the spent medium during growth. Expression of the cognate antitoxins could restore growth and culturability of cells. Expression of hicA in E. coli gave an increased number of persister cells in response to ciprofloxacin or ceftazidime. Site directed mutagenesis studies identified two key residues within the HicA toxin that were essential for both the reduced culturability and increased persistence phenotypes. Deletion of hicAB from B. pseudomallei K96243 did not affect persister cell or survival frequencies compared to the wild type following treatment with a variety of stress conditions. Deletion of the ΔhipBA locus from B. pseudomallei K96243 also had no affect on bacterial persistence or survival under the conditions tested

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties

YoeB toxin is activated during thermal stress.

Type II toxin-antitoxin (TA) modules are thought to mediate stress-responses by temporarily suppressing protein synthesis while cells redirect transcription to adapt to environmental change. Here, we show that YoeB, a ribosome-dependent mRNase toxin, is activated in Escherichia coli cells grown at elevated temperatures. YoeB activation is dependent on Lon protease, suggesting that thermal stress promotes increased degradation of the YefM antitoxin. Though YefM is efficiently degraded in response to Lon overproduction, we find that Lon antigen levels do not increase during heat shock, indicating that another mechanism accounts for temperature-induced YefM proteolysis. These observations suggest that YefM/YoeB functions in adaptation to temperature stress. However, this response is distinct from previously described models of TA function. First, YoeB mRNase activity is maintained over several hours of culture at 42°C, indicating that thermal activation is not transient. Moreover, heat-activated YoeB does not induce growth arrest nor does it suppress global protein synthesis. In fact, E. coli cells proliferate more rapidly at elevated temperatures and instantaneously accelerate their growth rate in response to acute heat shock. We propose that heat-activated YoeB may serve a quality control function, facilitating the recycling of stalled translation complexes through ribosome rescue pathways

Studies on the denaturation of antibody. IV. The influence of pH and certain other factors on the rate of inactivation of Staphylococcus antitoxin in urea solutions

In previous work on the denaturation of antibody, studies have been made of some of the factors influencing the inactivation of diphtheria antitoxin in urea solutions (1, 2). A quantitative formulation of a simple kinetic theory was found to fit satisfactorily the experimental data and to offer a reasonable explanation of the deviation of the inactivation from simple first order behavior (2). In the present work we have studied the inactivation of Staphylococcus antitoxin, investigating the influence of certain new factors on the rate and course of the over-all reaction, with a view toward gaining further insight into the mechanism of the reactions, and reinvestigating the influence of pH, a factor studied previously with diphtheria antitoxin, in order to test the applicability of the proposed kinetic mechanism to different antibodies

Type II and type IV toxin–antitoxin systems show different evolutionary patterns in the global Klebsiella pneumoniae population

The Klebsiella pneumoniae species complex includes important opportunistic pathogens which have become public health priorities linked to major hospital outbreaks and the recent emergence of multidrug-resistant hypervirulent strains. Bacterial virulence and the spread of multidrug resistance have previously been linked to toxin-antitoxin (TA) systems. TA systems encode a toxin that disrupts essential cellular processes, and a cognate antitoxin which counteracts this activity. Whilst associated with the maintenance of plasmids, they also act in bacterial immunity and antibiotic tolerance. However, the evolutionary dynamics and distribution of TA systems in clinical pathogens are not well understood. Here we present a comprehensive survey and description of the diversity of TA systems in 259 clinically relevant genomes of K. pneumoniae. We show that TA systems are highly prevalent with a median of 20 loci per strain. Importantly, these toxins differ substantially in their distribution patterns and in their range of cognate antitoxins. Classification along these properties suggests different roles of TA systems and highlights the association and co-evolution of toxins and antitoxins

Diversity of bacterial type II toxin–antitoxin systems: a comprehensive search and functional analysis of novel families

Type II toxin–antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as ‘addiction’, and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the ‘guilt by association’ principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes

Structure, Evolution, and Functions of Bacterial Type III Toxin-Antitoxin Systems.

Toxin-antitoxin (TA) systems are small genetic modules that encode a toxin (that targets an essential cellular process) and an antitoxin that neutralises or suppresses the deleterious effect of the toxin. Based on the molecular nature of the toxin and antitoxin components, TA systems are categorised into different types. Type III TA systems, the focus of this review, are composed of a toxic endoribonuclease neutralised by a non-coding RNA antitoxin in a pseudoknotted configuration. Bioinformatic analysis shows that the Type III systems can be classified into subtypes. These TA systems were originally discovered through a phage resistance phenotype arising due to a process akin to an altruistic suicide; the phenomenon of abortive infection. Some Type III TA systems are bifunctional and can stabilise plasmids during vegetative growth and sporulation. Features particular to Type III systems are explored here, emphasising some of the characteristics of the RNA antitoxin and how these may affect the co-evolutionary relationship between toxins and cognate antitoxins in their quaternary structures. Finally, an updated analysis of the distribution and diversity of these systems are presented and discussed.Work in the Salmond lab is supported by the BBSRC, UK; N.G. was supported by the Fonds National de la Recherche Luxembourg (9118191); B.C. was supported by a Cambridge International Scholarship from the Cambridge Commonwealth, European & International Trust; and A.D. was supported by a BBSRC -DTP studentship.This is the final version of the article. It first appeared from Molecular Diversity Preservation International via https://doi.org/10.3390/toxins810028