A novel Shiga toxin 2a neutralizing antibody therapeutic with low immunogenicity and high efficacy.

Shiga toxin-producing Escherichia coli infections are difficult to treat due to the risk of antibiotic-induced stress upregulating the production of toxins, medical treatment is consequently limited to supportive care to prevent the development of hemolytic uremic syndrome (HUS). Here, we introduce a potentially therapeutic humanized mouse monoclonal antibody (Hu-mAb 2-5) targeting Stx2a, the most common Shiga toxin subtype identified from outbreaks. We demonstrate that Hu-mAb 2-5 has low immunogenicity in healthy adults ex vivo and high neutralizing efficacy in vivo, protecting mice from mortality and HUS-related tissue damage.

SwrA-mediated Multimerization of DegU and an Upstream Activation Sequence Enhance Flagellar Gene Expression in Bacillus subtilis.

The earliest genes in bacterial flagellar assembly are activated by narrowly-conserved proteins called master regulators that often act as heteromeric complexes. A complex of SwrA and the response-regulator transcription factor DegU is thought to form the master flagellar regulator in Bacillus subtilis but how the two proteins co-operate to activate gene expression is poorly-understood. Here we find using ChIP-Seq that SwrA interacts with a subset of DegU binding sites in the chromosome and does so in a DegU-dependent manner. Using this information, we identify a DegU-specific inverted repeat DNA sequence in the Pflache promoter region and show that SwrA synergizes with DegU phosphorylation to increase binding affinity. We further demonstrate that the SwrA/DegU footprint extends from the DegU binding site towards the promoter, likely through SwrA-induced DegU multimerization. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription by disrupting a previously-unrecognized upstream activation sequence (UAS). Thus, the SwrA-DegU heteromeric complex likely enables both remote binding and interaction between the activator and RNA polymerase. Small co-activator proteins like SwrA may allow selective activation of subsets of genes where activator multimerization is needed. Why some promoters require activator multimerization and some require UAS sequences is unknown.

SwrA extends DegU over an UP element to activate flagellar gene expression in Bacillus subtilis.

SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and elevate flagellar density to enable swarming over solid surfaces. Here we use ChIP-seq to show that SwrA interacts with many sites on the chromosome in a manner that depends on the response regulator DegU. We identify a DegU-specific inverted repeat DNA sequence and show that SwrA synergizes with phosphorylation to increase DegU DNA binding affinity. We further show that SwrA increases the size of the DegU footprint expanding the region bound by DegU towards the promoter. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription more that could be explained by deactivation. We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and interaction between the activator and RNA polymerase in the context of an interceding UP element. We speculate that multimeric activators that resolve cis-element spatial conflicts are common in bacteria and likely act on flagellar biosynthesis loci and other long operons of other multi-subunit complexes. IMPORTANCE: In Bacteria, the sigma subunit of RNA polymerase recognizes specific DNA sequences called promoters that determine where gene transcription begins. Some promoters also have sequences immediately upstream called an UP element that is bound by the alpha subunit of RNA polymerase and is often necessary for transcription. Finally, promoters may be activated by transcription factors that bind DNA specific sequences and help recruit RNA polymerase to weak promoter elements. Here we show that the promoter for the 32 gene long flagellar operon in Bacillus subtilis requires an UP element and is activated by a heteromeric transcription factor of DegU and SwrA. Our evidence suggests that SwrA oligomerizes DegU over the DNA to allow RNA polymerase to interact with DegU and the UP element simultaneously. Heteromeric activator complexes are known but poorly-understood in bacteria and we speculate they may be needed to resolve spatial conflicts in the DNA sequence.

Development of Thermally Stable Nanobodies for Detection and Neutralization of Staphylococcal Enterotoxin B.

In this study, sixteen unique staphylococcal enterotoxin B (SEB)-reactive nanobodies (nbs), including ten monovalent and six bivalent nbs, were developed. All characterized nbs were highly specific for SEB and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb). The lowest limit of detection (LOD) reached 50 pg/mL in PBS. When applied to an ELISA to detect SEB-spiked milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the sixteen nbs, with a subset of nbs, SEB-5, SEB-9, and SEB-62, retaining activity even after exposure to 95 °C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat-labile properties. Several nbs demonstrated a long shelf-life, with one nb (SEB-9) retaining 93% of its activity after two weeks of storage at room temperature. In addition to their usage in toxin detection, eleven out of fifteen nbs were capable of neutralizing SEB's super-antigenic activity, demonstrated by their inhibition on IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs are relatively small, thermally stable, and easy to produce, making them useful in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products.

Validation of a Cell-Based Assay for Detection of Active Shiga Toxins Produced by Escherichia coli in Water.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the EPA's current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. In this study, a HeLa cell-based assay is validated for the detection of metabolically active Stxs produced by STEC in water, including tap, bottled, and pond water. Active Stxs are detected even when the number of Stx-producing bacteria is less than 0.4 CFU/mL and the assay performance is not affected by background flora or chlorine in the water. This assay is not only as simple and affordable as cell-free assays but also detects active holotoxins without the use of live animals. In addition, the assay is designed for use in multi-well formats, making it ideal for high-throughput screening of water samples and therefore useful for environmental public health surveillance programs to reduce human risk of infection with STEC.

Structural and Functional Characterization of Stx2k, a New Subtype of Shiga Toxin 2.

Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs.

In Vivo Evidence for ATPase-Dependent DNA Translocation by the Bacillus subtilis SMC Condensin Complex.

Structural maintenance of chromosomes (SMC) complexes shape the genomes of virtually all organisms, but how they function remains incompletely understood. Recent studies in bacteria and eukaryotes have led to a unifying model in which these ring-shaped ATPases act along contiguous DNA segments, processively enlarging DNA loops. In support of this model, single-molecule imaging experiments indicate that Saccharomyces cerevisiae condensin complexes can extrude DNA loops in an ATP-hydrolysis-dependent manner in vitro. Here, using time-resolved high-throughput chromosome conformation capture (Hi-C), we investigate the interplay between ATPase activity of the Bacillus subtilis SMC complex and loop formation in vivo. We show that point mutants in the SMC nucleotide-binding domain that impair but do not eliminate ATPase activity not only exhibit delays in de novo loop formation but also have reduced rates of processive loop enlargement. These data provide in vivo evidence that SMC complexes function as loop extruders.

The C-Terminal Region of Bacillus subtilis SwrA Is Required for Activity and Adaptor-Dependent LonA Proteolysis.

SwrA is the master activator of flagellar biosynthesis in Bacillus subtilis, and SwrA activity is restricted by regulatory proteolysis in liquid environments. SwrA is proteolyzed by the LonA protease but requires a proteolytic adaptor protein, SmiA. Here, we show that SwrA and SmiA interact directly. To better understand SwrA activity, SwrA was randomly mutagenized and loss-of-function and gain-of-function mutants were localized primarily to the predicted unstructured C-terminal region. The loss-of-function mutations impaired swarming motility and activation from the Pfla-che promoter. The gain-of-function mutations increased protein stability but did not abolish SmiA binding, suggesting that SmiA association was a precursor to, but not sufficient for, LonA-dependent proteolysis. Finally, one allele abolished simultaneously SwrA activity and regulatory proteolysis, suggesting that the two functions may be in steric competition.IMPORTANCE SwrA is the master activator of flagellar biosynthesis in Bacillus subtilis, and its mechanism of activation is poorly understood. Moreover, SwrA levels are restricted by SmiA, the first adaptor protein reported for the Lon family of proteases. Here, we show that the C-terminal region of SwrA is important for both transcriptional activation and regulatory proteolysis. Competition between the two processes at this region may be critical for responding to cell contact with a solid surface and the initiation of swarming motility.

MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

Cure of prediabetic mice by viral infections involves lymphocyte recruitment along an IP-10 gradient.

Viruses can cause but can also prevent autoimmune disease. This dualism has certainly hampered attempts to establish a causal relationship between viral infections and type 1 diabetes (T1D). To develop a better mechanistic understanding of how viruses can influence the development of autoimmune disease, we exposed prediabetic mice to various viral infections. We used the well-established NOD and transgenic RIP-LCMV models of autoimmune diabetes. In both cases, infection with the lymphocytic choriomeningitis virus (LCMV) completely abrogated the diabetic process. Interestingly, such therapeutic viral infections resulted in a rapid recruitment of T lymphocytes from the islet infiltrate to the pancreatic draining lymph node, where increased apoptosis was occurring. In both models this was associated with a selective and extensive expression of the chemokine IP-10 (CXCL10), which predominantly attracts activated T lymphocytes, in the pancreatic draining lymph node, and in RIP-LCMV mice it depended on the viral antigenic load. In RIP-LCMV mice, blockade of TNF-alpha or IFN-gamma in vivo abolished the prevention of T1D. Thus, virally induced proinflammatory cytokines and chemokines can influence the ongoing autoaggressive process beneficially at the preclinical stage, if produced at the correct location, time, and levels.