The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis.

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.

Design of Optical-Imaging Probes by Screening of Diverse Substrate Libraries Directly in Disease-Tissue Extracts.

Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection, and surgical guidance. These probes are often designed to target specific enzymes associated with diseases by direct optimization using single purified enzymes. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino-acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any a priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.

Optimization of a Protease Activated Probe for Optical Surgical Navigation.

Molecularly targeted optical contrast agents have the potential to enable surgeons to visualize specific molecular markers that can help improve surgical precision and thus outcomes. Fluorescently quenched substrates can be used to highlight tumor lesions by targeting proteases that are highly abundant in the tumor microenvironment. However, the majority of these and other molecularly targeted optical contrast agents are labeled with reporter dyes that are not ideally matched to the properties of clinical camera systems, which are typically optimized for detection of indocyanine-green (ICG). While a wide range of near-infrared (NIR) dyes are suitable for use with highly sensitive and highly tunable research-focused small animal imaging systems, most have not been evaluated for use with commonly used clinical imaging systems. Here we report the optimization of a small molecule fluorescently quenched protease substrate probe 6QC-ICG, which uses the indocyanine green (ICG) dye as its optical reporter. We evaluated dosing and kinetic parameters of this molecule in tumor-bearing mice and observed optimal tumor over background signals in as little as 90 min with a dose of 2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal in tumors did not linearly scale with dose, suggesting the importance of detailed dosing studies. Furthermore, when imaged using the FDA approved da Vinci Si surgical system with Firefly detection, signals were significantly higher for the ICG probe compared to a corresponding probe containing a dye with similar quantum yield but with a slightly shifted excitation and emission profile. The increased signal intensity generated by the optimal dye and dose of the ICG labeled probe enabled detection of small, flat lesions that were less than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe that performs optimally with clinical imaging systems and has great potential for applications in optical surgical navigation.

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini.

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies.

Stress-resistant Translation of Cathepsin L mRNA in Breast Cancer Progression.

The cysteine protease cathepsin L (CTSL) is often thought to act as a tumor promoter by enhancing tumor progression and metastasis. This goes along with increased CTSL activity in various tumor entities; however, the mechanisms leading to high CTSL levels are incompletely understood. With the help of the polyoma middle T oncogene driven breast cancer mouse model expressing a human CTSL genomic transgene, we show that CTSL indeed promotes breast cancer metastasis to the lung. During tumor formation and progression high expression levels of CTSL are maintained by enduring translation of CTSL mRNA. Interestingly, human breast cancer specimens expressed the same pattern of 5' untranslated region (UTR) splice variants as the transgenic mice and the human cancer cell line MDA-MB 321. By polyribosome profiling of tumor tissues and human breast cancer cells, we observe an intrinsic resistance of CTSL to stress-induced shutdown of translation. This ability can be attributed to all 5' UTR variants of CTSL and is not dependent on a previously described internal ribosomal entry site motif. In conclusion, we provide in vivo functional evidence for overexpressed CTSL as a promoter of lung metastasis, whereas high CTSL levels are maintained during tumor progression due to stress-resistant mRNA translation.

Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

Low-level lysosomal membrane permeabilization for limited release and sublethal functions of cathepsin proteases in the cytosol and nucleus.

For a long time, lysosomes were purely seen as organelles in charge of garbage disposal within the cell. They destroy any cargo delivered into their lumen with a plethora of highly potent hydrolytic enzymes, including various proteases. In case of damage to their limiting membranes, the lysosomes release their soluble content with detrimental outcomes for the cell. In recent years, however, this view of the lysosome changed towards acknowledging it as a platform for integration of manifold intracellular and extracellular signals. Even impaired lysosomal membrane integrity is no longer considered to be a one-way street to cell death. Increasing evidence suggests that lysosomal enzymes, mainly cathepsin proteases, can be released in a spatially and temporarily restricted manner that is compatible with cellular survival. This way, cathepsins can act in the cytosol and the nucleus, where they affect important cellular processes such as cell division. Here, we review this evidence and discuss the routes and molecular mechanisms by which the cathepsins may reach their unusual destination.

AND-gate contrast agents for enhanced fluorescence-guided surgery.

Surgical resection of tumours requires precisely locating and defining the margins between lesions and normal tissue. However, this is made difficult by irregular margin borders. Although molecularly targeted optical contrast agents can be used to define tumour margins during surgery in real time, the selectivity of the contrast agents is often limited by the target being expressed in both healthy and tumour tissues. Here, we show that AND-gate optical imaging probes that require the processing of two substrates by multiple tumour-specific enzymes produce a fluorescent signal with significantly improved specificity and sensitivity to tumour tissue. We evaluated the performance of the probes in mouse models of mammary tumours and of metastatic lung cancer, as well as during fluorescence-guided robotic surgery. Imaging probes that rely on multivariate activation to selectively target complex patterns of enzymatic activity should be useful in disease detection, treatment and monitoring.

Methods for analysis of near-infrared (NIR) quenched-fluorescent contrast agents in mouse models of cancer.

Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.

The Clinical Drug Ebselen Attenuates Inflammation and Promotes Microbiome Recovery in Mice after Antibiotic Treatment for CDI.

Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We reported that the safe-in-human clinical drug ebselen (ClinicalTrials.gov: NCT03013400, NCT01452607, NCT00762671, and NCT02603081) has biochemical, cell-based, and in vivo efficacy against the toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster model of relapsing CDI. Furthermore, ebselen treatment does not alter microbiome diversity and promotes recovery back to that of healthy controls after antibiotic-induced dysbiosis in healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers, suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

Short-Wave Infrared Fluorescence Chemical Sensor for Detection of Otitis Media.

Otitis media (OM) or middle ear infection is one of the most common diseases in young children around the world. The diagnosis of OM is currently performed using an otoscope to detect middle ear fluid and inflammatory changes manifested in the tympanic membrane. However, conventional otoscopy cannot visualize across the tympanic membrane or sample middle ear fluid. This can lead to low diagnostic certainty and overdiagnoses of OM. To improve the diagnosis of OM, we have developed a short-wave infrared (SWIR) otoscope in combination with a protease-cleavable biosensor, 6QC-ICG, which can facilitate the detection of inflammatory proteases in the middle ear with an increase in contrast. 6QC-ICG is a fluorescently quenched probe, which is activated in the presence of cysteine cathepsin proteases that are up-regulated in inflammatory immune cells. Using a preclinical model and custom-built SWIR otomicroscope in this proof-of-concept study, we successfully demonstrated the feasibility of robustly distinguishing inflamed ears from controls (p = 0.0006). The inflamed ears showed an overall signal-to-background ratio of 2.0 with a mean fluorescence of 81 ± 17 AU, while the control ear exhibited a mean fluorescence of 41 ± 11 AU. We envision that these fluorescently quenched probes in conjunction with SWIR imaging tools have the potential to be used as an alternate/adjunct tool for objective diagnosis of OM.

Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models.

Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFß) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.

Proteomic analysis of lung metastases in a murine breast cancer model reveals divergent influence of CTSB and CTSL overexpression.

Studies in the MMTV-PyMT (PyMT) breast cancer mouse model have shown a strong influence of the lysosomal cysteine cathepsins B or L on lung metastasis formation. Transgenic expression of human CTSB (tgCTSB) or CTSL (tgCTSL) both led to similar metastatic phenotypes with increased metastatic burden in the PyMT mice. However, recent studies in other tumor models proved marked differences in effects of either cathepsin on the proteome composition. We sought to analyze and compare proteome changes in the metastatic proteome of PyMT mice expressing either tgCTSB or tgCTSL to evaluate similarities and differences in those models. Performing an explorative, quantitative proteome comparison based on LC-MS/MS, we identified up to 3,000 proteins from murine lung metastases in three independent biological replicates per genotype. In both cases, when compared to wild-type (WT) mice, we noticed a pronounced impact of transgene cathepsin expression on the metastasis proteome. Highlights include increased moesin, integrin beta 1 and vinexin levels in the tgCTSB dataset and increased saposin and granulin levels in the tgCTSL dataset. Importantly, non-supervised hierarchical clustering clearly separated tgCTSB vs. tgCTSL induced proteome changes. In summary, tgCTSB and tgCTSL both display a strong and distinct impact on proteome composition of lung macrometastases in the PyMT model. Our observations suggest that they impact malignant behavior in distinct ways, thus further emphasizing interest into their tumor-contextual functionality.

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo.

The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.