Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation.

Systemic blood coagulation accompanies inflammation during severe infection like sepsis and COVID. We've previously established a link between pyroptosis, a vital defense mechanism against infection, and coagulopathy. During pyroptosis, the formation of gasdermin-D (GSDMD) pores on the plasma membrane leads to the release of tissue factor (TF)-positive microvesicles (MVs) that are procoagulant. Mice lacking GSDMD release fewer TF MVs. However, the specific mechanisms leading from activation of GSDMD to MV release remain unclear. Plasma membrane rupture (PMR) in pyroptosis was recently reported to be actively mediated by the transmembrane protein Ninjurin-1 (NINJ1). Here we show that NINJ1 promotes procoagulant MV release during pyroptosis. Haploinsuffciency or glycine inhibition of NINJ1 limited the release of procoagulant MVs and inflammatory cytokines and protected against blood coagulation and lethality triggered by bacterial flagellin. Our findings suggest a crucial role for NINJ1-dependent PMR in inflammasome-induced blood coagulation and inflammation.

Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection.

Inflammasome activation is a critical defense mechanism against bacterial infection. Previous studies suggest that inflammasome activation protects against Salmonella oral infection. Here we find inflammasome activation plays a critical role in the pathogenesis of Salmonella systemic infection. We show that in a systemic infection model by i.p. injection of Salmonella, deficiency of caspase-1 or gasdermin-D prolonged survival time, reduced plasma concentrations of the proinflammatory cytokines IL-1ß, IL-6 and TNFα. These deficiencies also protected against coagulopathy during Salmonella infection as evidenced by diminished prolongation of prothrombin time and increase in plasma thrombin-antithrombin complex concentrations in the caspase-1 or gasdermin-D deficient mice. Activation of the NAIP/NLRC4 inflammasome by flagellin and/or the components of the SPI1 type 3 secretion system played a critical role in Salmonella-induced coagulopathy. In the absence of flagellin and SPI1, the Salmonella mutant strain still triggered coagulopathy through the caspase-11/NLRP3 pathway. Our results reveal a previously undisclosed role of the inflammasomes and pyroptosis in the pathogenesis of Salmonella systemic infection.

A monoadduct generating Ru(ii) complex induces ribosome biogenesis stress and is a molecular mimic of phenanthriplatin.

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

Emergence of two AcrB substitutions conferring multidrug resistance to Salmonella spp.

AcrAB-TolC is a major tripartite multidrug efflux pump conferring resistance to a wide variety of compounds in Gram-negative pathogens. Many AcrB mutants have been constructed through site-directed mutagenesis to probe the mechanism of AcrB function in antibiotic resistance. However, much less is known about the actual drug resistance related mutants that naturally occur in clinically isolated pathogens. Here, we report two novel AcrB substitutions, M78I and P319L, in clinically isolated Salmonella strains with high-level ciprofloxacin resistance. Plasmids expressing the detected acrB mutations were constructed and introduced into SL1344△acrB Antimicrobial susceptibility assay showed that all AcrB M78I, AcrB P319L and AcrB M78I/319L conferred reduced susceptibilities to multiple substrates, including fluoroquinolones, erythromycin, tetracyclines, bile salts and dyes. Site-directed mutagenesis and MIC results revealed that increased hydrophobicity of M78I was one of the reasons why AcrB M78I had lower susceptibility to fluoroquinolones. Fluorescence labeling experiments suggested that the AcrB M78I substitution enhanced the binding of substrates to certain amino acid sites in the efflux pathway (e.g., site Q89, E673 and F617) and weakened the binding to other amino acids (e.g., S134 and N274). Structural modeling disclosed the increased flexibility of Leu was favorable for the functional rotation of AcrB compared to the original Pro. AcrA 319L makes the functional rotation of AcrB more flexible, this enables substrate efflux more efficiently. In order to understand the mechanism of AcrAB-TolC drug efflux well, interaction between AcrA and AcrB in the role of substrate efflux of AcrAB-TolC should be further investigated.

Aerosol capture and coronavirus spike protein deactivation by enzyme functionalized antiviral membranes.

The airborne nature of coronavirus transmission makes it critical to develop new barrier technologies that can simultaneously reduce aerosol and viral spread. Here, we report nanostructured membranes with tunable thickness and porosity for filtering coronavirus-sized aerosols, combined with antiviral enzyme functionalization that can denature spike glycoproteins of the SARS-CoV-2 virus in low-hydration environments. Thin, asymmetric membranes with subtilisin enzyme and methacrylic functionalization show more than 98.90% filtration efficiency for 100-nm unfunctionalized and protein-functionalized polystyrene latex aerosol particles. Unfunctionalized membranes provided a protection factor of 540 ± 380 for coronavirus-sized particle, above the Occupational Safety and Health Administration's standard of 10 for N95 masks. SARS-CoV-2 spike glycoprotein on the surface of coronavirus-sized particles was denatured in 30 s by subtilisin enzyme-functionalized membranes with 0.02-0.2% water content on the membrane surface.

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

Transport Across Two Membrane Bilayers in E. coli by Efflux Pumps of Different Dimensions.

AcrAB-TolC and CusBAC are two of the most well-studied Resistance-Nodulation-Division (RND) family tripartite efflux pumps in E. coli. AcrAB-TolC is a multidrug efflux system, while CusBAC transports Cu(I), Cu(II) and Ag(I). The RND pump complexes span both the inner membrane (IM) and the outer membrane (OM). The long axis dimension of the fully assembled AcrAB-TolC is ∼3 nm longer than that of CusBAC. To probe if these two efflux systems with different dimensions affect each other when they need to work simultaneously in the same cell, two real-time assays were used to monitor the efflux activities of these two pumps and their impact on each other. The results showed that the presence of AcrAB-TolC substrates accelerated the accumulation of Cu(I) in BW25113 but not in BW25113ΔcusBA or BW25113ΔtolC strains. Similarly, the presence of Ag(I) slowed down the Nile red efflux in the parent strain more significantly than in the CusBA deficient mutant. To further investigate the impact of the OM/IM distance on the function of these tripartite complexes, we experimented with strains lacking the lipoprotein Lpp or containing Lpp mutant of different lengths. Data from efflux/accumulation assays and susceptibility tests revealed that mutation of Lpp resulted in functional deficiency of both AcrAB-TolC and CusBAC. In conclusion, this study demonstrated that when AcrAB-TolC and CusBAC functioned simultaneously, it took the cell a few minutes to adjust. Furthermore, the presence of Lpp of proper length is important to support full efflux activity of transporters spanning both membrane layers in E. coli.

Pyroptosis-Induced Inflammation and Tissue Damage.

Programmed cell deaths are pathways involving cells playing an active role in their own destruction. Depending on the signaling system of the process, programmed cell death can be divided into two categories, pro-inflammatory and non-inflammatory. Pyroptosis is a pro-inflammatory form of programmed cell death. Upon cell death, a plethora of cytokines are released and trigger a cascade of responses from the neighboring cells. The pyroptosis process is a double-edged sword, could be both beneficial and detrimental in various inflammatory disorders and disease conditions. A physiological outcome of these responses is tissue damage, and sometimes death of the host. In this review, we focus on the inflammatory response triggered by pyroptosis, and resulting tissue damage in selected organs.

Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria.

Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the "Trojan horse strategy". In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.

Insight into the AcrAB-TolC Complex Assembly Process Learned from Competition Studies.

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called "dominant negative" effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.

Donnan Potential across the Outer Membrane of Gram-Negative Bacteria and Its Effect on the Permeability of Antibiotics.

The cell envelope structure of Gram-negative bacteria is unique, composed of two lipid bilayer membranes and an aqueous periplasmic space sandwiched in between. The outer membrane constitutes an extra barrier to limit the exchange of molecules between the cells and the exterior environment. Donnan potential is a membrane potential across the outer membrane, resulted from the selective permeability of the membrane, which plays a pivotal role in the permeability of many antibiotics. In this review, we discussed factors that affect the intensity of the Donnan potential, including the osmotic strength and pH of the external media, the osmoregulated periplasmic glucans trapped in the periplasmic space, and the displacement of cell surface charges. The focus of our discussion is the impact of Donnan potential on the cellular permeability of selected antibiotics including fluoroquinolones, tetracyclines, ß-lactams, and trimethoprim.

Inflammasome activation promotes venous thrombosis through pyroptosis.

Crosstalk between coagulation and innate immunity contributes to the progression of many diseases, including infection and cardiovascular disease. Venous thromboembolism (VTE), including pulmonary embolism and deep vein thrombosis (DVT), is among the most common causes of cardiovascular death. Here, we show that inflammasome activation and subsequent pyroptosis play an important role in the development of venous thrombosis. Using a flow restriction-induced mouse venous thrombosis model in the inferior vena cava (IVC), we show that deficiency of caspase-1, but not caspase-11, protected against flow restriction-induced thrombosis. Interleukin-1ß expression increased in the IVC following ligation, indicating that inflammasome is activated during injury. Deficiency of gasdermin D (GSDMD), an essential mediator of pyroptosis, protected against restriction-induced venous thrombosis. After induction of venous thrombosis, fibrin was deposited in the veins of wild-type mice, as detected using immunoblotting with a monoclonal antibody that specifically recognizes mouse fibrin, but not in the caspase-1-deficient or GSDMD-deficient mice. Depletion of macrophages by gadolinium chloride or deficiency of tissue factor also protected against venous thrombosis. Our data reveal that tissue factor released from pyroptotic monocytes and macrophages following inflammasome activation triggers thrombosis.

Synthesis and biological evaluation of stilbene-based peptoid mimics against the phytopathogenic bacterium Xanthomonas citri pv. citri.

BACKGROUND: The emergence of drug-resistant phytopathogenic bacteria and the need for new types of biological disease-control agents have accelerated efforts toward searching for alternative candidates with a low propensity for resistance development. In this study, a new series of stilbene-based peptoid mimics were synthesized, and their biological activities were evaluated against citrus pathogenic bacteria in vitro and in vivo. RESULTS: Antibacterial bioassay results showed that the dicationic peptoid mimics 9a and 9b displayed excellent bioactivity against Xanthomonas citri pv. citri, with the minimum inhibitory concentration values of 25 µM, which were superior to those of commercial copper biocides Delite (200 µM) and Kasumin Bordeaux (100 µM). In vivo bioassay further confirmed their control efficacy against plant bacterial diseases. In addition, the antibacterial mechanism of action elucidated their membrane-disruption effects resulting in the leakage of the bacterial membranes, which was similar to that of antimicrobial peptides. Moreover, the inhibition effect on biofilm formation of peptoid mimics has also been demonstrated. CONCLUSION: Stilbene-based peptoid mimics synthesized in this study showed promising antibacterial activity with a potent membrane-disruptive mechanism. The results suggested that stilbene-based peptoid mimics have the potential as a candidate new type of bactericide for citrus disease protection.

Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli.

The double-layered cell envelope of Gram-negative bacteria and active drug efflux present a formidable barrier for antimicrobial compounds to penetrate. Fluoroquinolones are among the few classes of antimicrobials that are clinically useful in the treatment of Gram-negative bacterial infection. Previous studies on fluoroquinolone accumulation measured total bacteria associated compounds, rather than the cytoplasmic accumulation. Fluoroquinolones target the type II topoisomerases in the cytoplasm. Thus, the cytoplasmic accumulation is expected to be more relevant to the potency of the drugs. Here, we fractionated and measured the concentration of nine fluoroquinolone compounds in the periplasm and the cytoplasm of two strains of E. coli cells, a parent strain and its isogenic efflux-deficient tolC knockout strain. The potency of the drugs was determined using the minimum inhibitory concentration (MIC) assay. We found that all fluoroquinolones tested accumulated at much higher concentrations in the periplasm than in the cytoplasm. The periplasmic concentrations were 2-15 folds higher than the cytoplasmic concentration, while the actual distribution ratio varies drastically among the compounds tested. Good correlation between the MIC and the cytoplasmic accumulation, but not whole cell accumulation, was observed using a pair of isogenic wild type and drug-efflux deficient strains.

Probing the Dynamics of AcrB Through Disulfide Bond Formation.

The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in Escherichia coli. AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an acrB knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in E. coli.

Periplasmic Targets for the Development of Effective Antimicrobials against Gram-Negative Bacteria.

Antibiotic resistance has emerged as a serious threat to global public health in recent years. Lack of novel antimicrobials, especially new classes of compounds, further aggravates the situation. For Gram-negative bacteria, their double layered cell envelope and an array of efflux pumps act as formidable barriers for antimicrobials to penetrate. While cytoplasmic targets are hard to reach, proteins in the periplasm are clearly more accessible, as the drug only needs to breach the outer membrane. In this review, we summarized recent efforts on the validation and testing of periplasmic proteins as potential antimicrobial targets and the development of related inhibitors that either inhibit the growth of a bacterial pathogen or reduce its virulence during interaction with host cells. We conclude that the periplasm contains a promising pool of novel antimicrobial targets that should be scrutinized more closely for the development of effective treatment against multidrug-resistant Gram-negative bacteria.

Application of Fluorescence in Studying Therapeutic Enzymes.

Fluorescence spectroscopy is one of the most important techniques in the study of therapeutic enzymes. The fluorescence phenomenon has been discovered and exploited for centuries, while therapeutic enzymes have been used in treatment of disease for only decades. This chapter provides a brief summary of the current applications of fluorescence methods in studying therapeutic enzymes to provide some insights on the selection of proper method tailored to the goal. First a brief introduction about therapeutic enzymes and history of fluorescence were provided, followed by discussions on how fluorescence was applied in the studies. Four popular fluorescence methods are discussed: fluorescence tracing, fluorescence resonance energy transfer (FRET), fluorescence quenching and fluorescence polarization. Selected application of the fluorescence methods in studying therapeutic enzymes are listed, and discussed in details in the following paragraphs.

Automatic segmentation of arterial tree from 3D computed tomographic pulmonary angiography (CTPA) scans.

Pulmonary embolism (PE) and other pulmonary vascular diseases, have been found associated with the changes in arterial morphology. To detect arterial changes, we propose a novel, fully automatic method that can extract pulmonary arterial tree in computed tomographic pulmonary angiography (CTPA) images. The approach is based on the fuzzy connectedness framework, combined with 3D vessel enhancement and Harris Corner detection to achieve accurate segmentation. The effectiveness and robustness of the method is validated in clinical datasets consisting of 10 CT angiography scans (6 without PE and 4 with PE). The performance of our method is compared with manual classification and machine learning method based on random forest. Our method achieves a mean accuracy of 92% when compared to manual reference, which is higher than the 89% accuracy achieved by machine learning. This performance of the segmentation for pulmonary arteries may provide a basis for the CAD application of PE.

Increasing Salt Rejection of Polybenzimidazole Nanofiltration Membranes via the Addition of Immobilized and Aligned Aquaporins.

Aquaporins are water channel proteins in cell membrane, highly specific for water molecules while restricting the passage of contaminants and small molecules, such as urea and boric acid. Cysteine functional groups were installed on aquaporin Z for covalent attachment to the polymer membrane matrix so that the proteins could be immobilized to the membranes and aligned in the direction of the flow. Depth profiling using x-ray photoelectron spectrometer (XPS) analysis showed the presence of functional groups corresponding to aquaporin Z modified with cysteine (Aqp-SH). Aqp-SH modified membranes showed a higher salt rejection as compared to unmodified membranes. For 2 M NaCl and CaCl2 solutions, the rejection obtained from Aqp-SH membranes was 49.3 ± 7.5% and 59.1 ± 5.1%. On the other hand, the rejections obtained for 2 M NaCl and CaCl2 solutions from unmodified membranes were 0.8 ± 0.4% and 1.3 ± 0.2% respectively. Furthermore, Aqp-SH membranes did not show a significant decrease in salt rejection with increasing feed concentrations, as was observed with other membranes. Through simulation studies, it was determined that there was approximately 24% capping of membrane pores by dispersed aquaporins.

Gasdermin D (GSDMD) as a new target for the treatment of infection.

The discovery of a previously unknown protein, gasdermin D (GSDMD), as the key effector that leads to pyroptosis and NETosis has created much excitement. Since its initial report in Oct. 2015, more than 200 papers have been published on studies of the structure and mechanism of GSDMD and its homologues. The clear connection between infection and inflammasome activation made GSDMD a promising target for the development of anti-infection treatment. In this mini review, we discuss first the current understanding of the structure and mechanism of GSDMD, focusing on its potential as a druggable target, and then recent efforts in the development of inhibitors to interfere with the pore-forming function of GSDMD and thus alleviate the detrimental effects due to pyroptotic cell death.