RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆2903 and ∆lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly. IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

Spacer Fidelity Assessments of Guide RNA by Top-Down Mass Spectrometry.

The advancement of CRISPR-based gene editing tools into biotherapeutics offers the potential for cures to genetic disorders and for new treatment paradigms for even common diseases. Arguably, the most important component of a CRISPR-based medicine is the guide RNA, which is generally large (>100-mer) synthetic RNA composed of a "tracr" and "spacer" region, the latter of which dictates the on-target editing site as well as potential undesired off-target edits. Aiming to advance contemporary capabilities for gRNA characterization to ensure the spacer region is of high fidelity, top-down mass spectrometry was herein implemented to provide direct and quantitative assessments of highly modified gRNA. In addition to sequencing the spacer region and pinpointing modifications, top-down mass spectra were utilized to quantify single-base spacer substitution impurities down to <1% and to decipher highly dissimilar spacers. To accomplish these results in an automated fashion, we devised custom software capable of sequencing and quantifying impurities in gRNA spacers. Notably, we developed automated tools that enabled the quantification of single-base substitutions, including advanced isotopic pattern matching for C > U and U > C substitutions, and created a de novo sequencing strategy to facilitate the identification and quantification of gRNA impurities with highly dissimilar spacer regions.

Mass Spectrometry Imaging Reveals Abnormalities in Cardiolipin Composition and Distribution in Astrocytoma Tumor Tissues.

Cardiolipin (CL) is a mitochondrial lipid with diverse roles in cellular respiration, signaling, and organelle membrane structure. CL content and composition are essential for proper mitochondrial function. Deranged mitochondrial energy production and signaling are key components of glial cell cancers and altered CL molecular species have been observed in mouse brain glial cell xenograft tumors. The objective of this study was to describe CL structural diversity trends in human astrocytoma tumors of varying grades and correlate these trends with histological regions within the heterogeneous astrocytoma microenvironment. To this aim, we applied desorption electrospray ionization coupled with high field asymmetric ion mobility mass spectrometry (DESI-FAIMS-MS) to map CL molecular species in human normal cortex (N = 29), lower-grade astrocytoma (N = 19), and glioblastoma (N = 28) tissues. With this platform, we detected 46 CL species and 12 monolysocardiolipin species from normal cortex samples. CL profiles detected from glioblastoma tissues lacked diversity and abundance of longer chain polyunsaturated fatty acid containing CL species when compared to CL detected from normal and lower-grade tumors. CL profiles correlated with trends in tumor viability and tumor infiltration. Structural characterization of the CL species by tandem MS experiments revealed differences in fatty acid and double bond isomer composition among astrocytoma tissues compared with normal cortex and glioblastoma tissues. The GlioVis platform was used to analyze astrocytoma gene expression data from the CGGA dataset. Decreased expression of several mitochondrial respiratory enzyme encoding-genes was observed for higher-grade versus lower-grade tumors, however no significant difference was observed for cardiolipin synthesis enzyme CRLS1.

Type III CRISPR-Cas complexes act as protein-assisted ribozymes during target RNA cleavage.

CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic6,7. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors8,9, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.

Mapping paratopes of nanobodies using native mass spectrometry and ultraviolet photodissociation.

Following immense growth and maturity of the field in the past decade, native mass spectrometry has garnered widespread adoption for the structural characterization of macromolecular complexes. Routine analysis of biotherapeutics by this technique has become commonplace to assist in the development and quality control of immunoglobulin antibodies. Concurrently, 193 nm ultraviolet photodissociation (UVPD) has been developed as a structurally sensitive ion activation technique capable of interrogating protein conformational changes. Here, UVPD was applied to probe the paratopes of nanobodies, a class of single-domain antibodies with an expansive set of applications spanning affinity reagents, molecular imaging, and biotherapeutics. Comparing UVPD sequence fragments for the free nanobodies versus nanobody·antigen complexes empowered assignment of nanobody paratopes and intermolecular salt-bridges, elevating the capabilities of UVPD as a new strategy for characterization of nanobodies.

Investigation of Product Ions Generated by 193 nm Ultraviolet Photodissociation of Peptides and Proteins Containing Disulfide Bonds.

Disulfide bridges are unique post-translational modifications (PTM) that contribute to protein architecture and modulate function. This PTM, however, challenges top-down mass spectrometry by cyclizing stretches of the protein sequence. In order to produce and release detectable product ions that contribute to the assignment of proteoforms, regions of a protein encapsulated by disulfide bonds require two fragmentation events: cleavage of the protein backbone and cleavage of the disulfide bond. Traditional collisional activation methods do not cleave disulfide bonds efficiently, often leading to low sequence coverage of proteins that incorporate this feature. To address this challenge, we have evaluated the fragmentation pathways enabled by 193 nm ultraviolet photodissociation (UVPD) and UVPD coupled to electron transfer dissociation for the characterization of protein structures incorporating disulfide bonds. Cleavage of disulfide bonds by either approach results in S-S and C-S dissociation products that result from a combination of homolytic cleavage and hydrogen-transfer processes. Characterization of these product ions elevates interpretation of complex top-down spectra of proteins that incorporate disulfide bonds.

Enhanced Characterization of Cardiolipins via Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Cardiolipins (CLs) constitute a structurally complex class of glycerophospholipids with a unique tetraacylated structure accompanied by distinctive functional roles. Aberrations in the composition of this lipid class have been associated with disease states, spurring interest in the development of new approaches to differentiate the structures of diverse CLs in complex mixtures. The structural characterization of these complex lipids using conventional methods, however, suffers from limited resolution and frequently proves unable to discern subtle yet biologically significant features such as unsaturation sites or acyl chain position assignments. Here, we describe the synergistic use of chemical derivatization and hybrid dissociation techniques to characterize CL from complex biological mixtures with both double bond and sn positional isomer resolution in a shotgun mass spectrometry strategy. Utilizing (trimethylsilyl)diazomethane (TMSD), CL phosphate groups were methylated to promote positive-mode ionization by the production of metal-cationized lipids, enabling structural interrogation via hybrid higher-energy collisional activation/ultraviolet photodissociation (HCD/UVPD). This combination of TMSD derivatization and HCD/UVPD fragmentation results in diagnostic product ions that permit distinction and relative quantitation of sn-stereoisomers and the localization of double bonds. Applying this strategy to a total lipid extract from a thyroid carcinoma revealed a previously unreported 18:2/18:1 motif, elucidating a structural feature unique to the lipid class.

Influence of Primary Structure on Fragmentation of Native-Like Proteins by Ultraviolet Photodissociation.

Analysis of native-like protein structures in the gas phase via native mass spectrometry and auxiliary techniques has become a powerful tool for structural biology applications. In combination with ultraviolet photodissociation (UVPD), native top-down mass spectrometry informs backbone flexibility, topology, hydrogen bonding networks, and conformational changes in protein structure. Although it is known that the primary structure affects dissociation of peptides and proteins in the gas phase, its effect on the types and locations of backbone cleavages promoted by UVPD and concomitant influence on structural characterization of native-like proteins is not well understood. Here, trends in the fragmentation of native-like proteins were evaluated by tracking the propensity of 10 fragment types (a, a+1, b, c, x, x+1, y, y-1, Y, and z) in relation to primary structure in a native-top down UVPD data set encompassing >9600 fragment ions. Differing fragmentation trends are reported for the production of distinct fragment types, attributed to a combination of both direct dissociation pathways from excited electronic states and those surmised to involve intramolecular vibrational energy redistribution after internal conversion. The latter pathways were systematically evaluated to evince the role of proton mobility in the generation of "CID-like" fragments through UVPD, providing pertinent insight into the characterization of native-like proteins. Fragmentation trends presented here are envisioned to enhance analysis of the protein higher-order structure or augment scoring algorithms in the high-throughput analysis of intact proteins.

Relative Quantitation of Unsaturated Phosphatidylcholines Using 193 nm Ultraviolet Photodissociation Parallel Reaction Monitoring Mass Spectrometry.

Structural characterization of glycerophospholipids beyond the fatty acid level has become a major endeavor in lipidomics, presenting an opportunity to advance the understanding of the intricate relationship between lipid metabolism and disease state. Distinguishing subtle lipid structural features, however, remains a major challenge for high-throughput workflows that implement traditional tandem mass spectrometry (MS/MS) techniques, stunting the molecular depth of quantitative strategies. Here, reversed phase liquid chromatography is coupled to parallel reaction mass spectrometry utilizing the double bond localization capabilities of ultraviolet photodissociation (UVPD) mass spectrometry to produce double bond isomer specific responses that are leveraged for relative quantitation. The strategy provides lipidomic characterization at the double bond level for phosphatidylcholine phospholipids from biological extracts. In addition to quantifying monounsaturated lipids, quantitation of phospholipids incorporating isomeric polyunsaturated fatty acids is also achieved. Using this technique, phosphatidylcholine isomer ratios are compared across human normal and tumor breast tissue to reveal significant structural alterations related to disease state.

Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae.

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Double Bond Characterization of Free Fatty Acids Directly from Biological Tissues by Ultraviolet Photodissociation.

Free fatty acids (FA) are a vital component of cells and are critical to cellular structure and function, so much so that alterations in FA are often associated with cell malfunction and disease. Analysis of FA from biological samples can be achieved by mass spectrometry (MS), but these analyses are often not capable of distinguishing the fine structural alterations within FA isomers and often limited to global profiling of lipids without spatial resolution. Here, we present the use of ultraviolet photodissociation (UVPD) for the characterization of double bond positional isomers of charge inverted dication·FA complexes and the subsequent implementation of this method for online desorption electrospray ionization (DESI) MS imaging of FA isomers from human tissue sections. This method allows relative quantification of FA isomers from heterogeneous biological tissue sections, yielding spatially resolved information about alterations in double bond isomers within these samples. Applying this method to the analysis of the monounsaturated FA 18:1 within breast cancer subtypes uncovered a correlation between double bond positional isomer abundance and the hormone receptor status of the tissue sample, an important factor in the prognosis and treatment of breast cancer patients. This result further validates similar studies that suggest FA synthase activity and FA isomer abundances are significantly altered within breast cancer tissue.

Localization of Double Bonds in Bacterial Glycerophospholipids Using 193 nm Ultraviolet Photodissociation in the Negative Mode.

The need for detailed structural characterization of glycerophospholipids (GPLs) for many types of biologically motivated applications has led to the development of novel mass spectrometry-based methodologies that utilize alternative ion activation methods. Ultraviolet photodissociation (UVPD) has shown great utility for localizing sites of unsaturation within acyl chains and to date has predominantly been used for positive mode analysis of GPLs. In the present work, UVPD is used to localize sites of unsaturation in GPL anions. Similar to UVPD mass spectra of GPL cations, UVPD of deprotonated or formate-adducted GPLs yields diagnostic fragment ions spaced 24 Da apart. This method was integrated into a liquid chromatography workflow and used to evaluate profiles of sites of unsaturation of lipids in Escherichia coli (E. coli) and Acinetobacter baumannii (A. baumannii). When assigning sites of unsaturation, E. coli was found to contain all unsaturation elements at the same position relative to the terminal methyl carbon of the acyl chain; the first carbon participating in a site of unsaturation was consistently seven carbons along the acyl chain when counting carbons from the terminal methyl carbon. GPLs from A. baumannii exhibited more variability in locations of unsaturation. For GPLs containing sites of unsaturation in both acyl chains, an MS3 method was devised to assign sites to specific acyl chains.

Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry Localizes Cardiolipin Unsaturations.

Developing alternative MS/MS strategies to distinguish isomeric lipids has become a high impact goal in shotgun lipidomics. Novel approaches have been developed to resolve structural features that are not discernible by traditional shotgun methods and have consequently promoted the discovery of new disease biomarkers. However, these methods have largely been limited to characterizing lipids with low structural complexity. Here, ultraviolet photodissociation (UVPD) strategies for phospholipid characterization are expanded for analysis of cardiolipins (CL), a class of phospholipids that exhibits a higher degree of structural complexity. A hybrid collision induced dissociation/193 nm UVPD (CID/UVPD) approach was implemented to pinpoint the location of both double bond and cyclopropyl unsaturations on the four acyl chains of CLs. This strategy was complemented with CID for the de novo elucidation of unknown CLs in biological extracts.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods.

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Attractiveness of food and avoidance from contamination as conflicting stimuli to habitat selection by fish.

Habitat selection by fish is the outcome of a choice between different stimuli. Typically, the presence of food tends to attract organisms, while contamination triggers an avoidance response to prevent toxic effects. Given that both food and contaminants are not homogeneously distributed in the environment and that food can be available in contaminated zones, a key question has been put forward in the present study: does a higher availability of food in contaminated areas interfere in the avoidance response to contaminants regardless of the contamination level? Tilapia fry (Oreochromis sp.; 2.5-3.0 cm and 0.5-0.8 g) were exposed to two different effluent samples, diluted along a free-choice, non-forced exposure system simulating a contamination gradient. Initially, avoidance to the effluents was checked during a one hour exposure. Afterwards, food was added to the system so that the availability of food increased with the increase in the level of contamination, and the avoidance response to contamination was checked during another hour. Results clearly showed a concentration-dependent avoidance response for both effluents during the first hour (i.e., with no food). However, in presence of the food, the avoidance pattern was altered: organisms were propelled to intermittently move towards contaminated areas where food availability was higher. The incursions were taken regardless of the potential risk linked to the toxic effects. In conclusion, even when the risk of toxicity was imminent, tilapia fry were more intensively stimulated by the attractiveness of the food than by repulsion to the contamination.

Predicting the effects of copper on local population decline of 2 marine organisms, cobia fish and whiteleg shrimp, based on avoidance response.

The present study focuses on avoidance response to predict population decline of the marine fish Rachycentron canadum (cobia) and larvae of the estuarine shrimp Litopenaeus vannamei (whiteleg shrimp). Avoidance of approximately 60% was recorded for the cobia fry exposed to 1.0 mg Cu/L, 1.60 mg Cu/L, and 1.80 mg Cu/L. For the shrimp larvae, avoidance was approximately 80% for all Cu concentrations. The population decline of cobia fry was conditioned by avoidance in lower concentrations. However, in higher concentrations mortality begins to play an important role. The displacement toward uncontaminated habitats might determine shrimp population decline. A Cu-contaminated environment can determine the habitat selection of both species and, therefore, their local population decline.

Heavy metals in yellowfin tuna (Thunnus albacares) and common dolphinfish (Coryphaena hippurus) landed on the Ecuadorian coast.

Heavy metals are contaminants of great environmental concern due to their multiple origins (natural and anthropogenic), the ability to accumulate in organs and tissues, and the deleterious effects they can cause in organisms. Studies on the accumulation of metals in seafood, such as fish, have increased in importance due to the risk for human health when consuming fish contaminated by metals. The present work was aimed at verifying the concentrations of cadmium (Cd), mercury (Hg) and lead (Pb) in the muscular tissue and liver of yellowfin tuna (Thunnus albacares) and common dolphinfish (Coryphaena hippurus) from the Eastern Pacific Ocean landed in Manta city, Ecuador. Samples were analyzed by inductively coupled plasma mass spectroscopy (ICP-MS). Around half of the muscle samples of both species presented levels of Cd and Hg above the limits considered safe for human consumption established by the European Union. For Pb,most of the muscle samples were considered acceptable for consumption. Results indicate that both species should be consumed with some caution. Considering the tolerable weekly intake recommended for adults by the World Health Organization, results indicate that Hg is the main metal that limits the consumption of yellowfin tuna and common dolphinfish, with a recommended maximum ingestion, respectively, of 191 and 178 g per week for an adult.c