RTS,S/AS02A Malaria Vaccine-Induced IgG Responses Equally Recognize Native-Like Fucosylated and Nonfucosylated Plasmodium falciparum Circumsporozoite Proteins.

The RTS,S/AS02A malaria vaccine is based on the Plasmodium falciparum circumsporozoite protein (PfCSP), which is O-fucosylated on the sporozoite surface. We determined whether RTS,S/AS02A-induced immunoglobulin G (IgG) antibodies recognize vaccine-like nonfucosylated PfCSP better than native-like fucosylated PfCSP. Similar to previous vaccine trials, RTS,S/AS02A vaccination induced high anti-PfCSP IgG levels associated with malaria protection. IgG recognition of nonfucosylated and fucosylated PfCSP was equivalent, suggesting that PfCSP fucosylation does not affect antibody recognition. Clinical Trials Registration. NCT00197041.

Search for a Shared Genetic or Biochemical Basis for Biofilm Tolerance to Antibiotics across Bacterial Species.

Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for 3 days and then challenged with respective antibiotics (ciprofloxacin, daptomycin, and tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells, and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of individual microorganisms in biofilms and contribute to antibiotic tolerance.

Conceptual Model of Biofilm Antibiotic Tolerance That Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology.

Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip flow reactors on a medium composed to mimic the exudate from a chronic wound. After 4 days, the biofilm was 114 µm thick with 9.45 log10 CFU cm-2 These biofilms exhibited tolerance, relative to exponential-phase planktonic cells, to subsequent treatment with ciprofloxacin. The specific growth rate of the biofilm was estimated via elemental balances to be approximately 0.37 h-1 and with a reaction-diffusion model to be 0.32 h-1, or one-third of the maximum specific growth rate for planktonic cells. Global analysis of gene expression indicated lower transcription of ribosomal genes and genes for other anabolic functions in biofilms than in exponential-phase planktonic cells and revealed the induction of multiple stress responses in biofilm cells, including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing. Metabolic pathways for phenazine biosynthesis and denitrification were transcriptionally activated in biofilms. A customized reaction-diffusion model predicted that steep oxygen concentration gradients will form when these biofilms are thicker than about 40 µm. Mutant strains that were deficient in Psl polysaccharide synthesis, the stringent response, the stationary-phase response, and the membrane stress response exhibited increased ciprofloxacin susceptibility when cultured in biofilms. These results support a sequence of phenomena leading to biofilm antibiotic tolerance, involving oxygen limitation, electron acceptor starvation and growth arrest, induction of associated stress responses, and differentiation into protected cell states.IMPORTANCE Bacteria in biofilms are protected from killing by antibiotics, and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.

Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry.

Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.

Multi-omics analysis provides insight to the Ignicoccus hospitalis-Nanoarchaeum equitans association.

BACKGROUND: Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS: We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS: Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS: This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE: Our study applies statistical and visualization techniques to a mixed-source omics dataset to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.

Expanding the paradigm of thiol redox in the thermophilic root of life.

BACKGROUND: The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides. METHODS: Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition. RESULTS: Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain. CONCLUSIONS: In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability. GENERAL SIGNIFICANCE: Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.

Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR.

Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements.

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0-30min, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

Adipokinetic hormone signaling in the malaria vector Anopheles gambiae facilitates Plasmodium falciparum sporogony.

An obligatory step in the complex life cycle of the malaria parasite is sporogony, which occurs during the oocyst stage in adult female Anopheles mosquitoes. Sporogony is metabolically demanding, and successful oocyst maturation is dependent on host lipids. In insects, lipid energy reserves are mobilized by adipokinetic hormones (AKHs). We hypothesized that Plasmodium falciparum infection activates Anopheles gambiae AKH signaling and lipid mobilization. We profiled the expression patterns of AKH pathway genes and AgAkh1 peptide levels in An. gambiae during starvation, after blood feeding, and following infection and observed a significant time-dependent up-regulation of AKH pathway genes and peptide levels during infection. Depletion of AgAkh1 and AgAkhR by RNAi reduced salivary gland sporozoite production, while synthetic AgAkh1 peptide supplementation rescued sporozoite numbers. Inoculation of uninfected female mosquitoes with supernatant from P. falciparum-infected midguts activated AKH signaling. Clearly, identifying the parasite molecules mediating AKH signaling in P. falciparum sporogony is paramount.

Niclosamide as a chemical probe for analyzing SARS-CoV-2 modulation of host cell lipid metabolism.

Introduction: SARS-CoV-2 subverts host cell processes to facilitate rapid replication and dissemination, and this leads to pathological inflammation. Methods: We used niclosamide (NIC), a poorly soluble anti-helminth drug identified initially for repurposed treatment of COVID-19, which activates the cells' autophagic and lipophagic processes as a chemical probe to determine if it can modulate the host cell's total lipid profile that would otherwise be either amplified or reduced during SARS-CoV-2 infection. Results: Through parallel lipidomic and transcriptomic analyses we observed massive reorganization of lipid profiles of SARS-CoV-2 infected Vero E6 cells, especially with triglycerides, which were elevated early during virus replication, but decreased thereafter, as well as plasmalogens, which were elevated at later timepoints during virus replication, but were also elevated under normal cell growth. These findings suggested a complex interplay of lipid profile reorganization involving plasmalogen metabolism. We also observed that NIC treatment of both low and high viral loads does not affect virus entry. Instead, NIC treatment reduced the abundance of plasmalogens, diacylglycerides, and ceramides, which we found elevated during virus infection in the absence of NIC, resulting in a significant reduction in the production of infectious virions. Unexpectedly, at higher viral loads, NIC treatment also resulted in elevated triglyceride levels, and induced significant changes in phospholipid metabolism. Discussion: We posit that future screens of approved or new partner drugs should prioritize compounds that effectively counter SARS-CoV-2 subversion of lipid metabolism, thereby reducing virus replication, egress, and the subsequent regulation of key lipid mediators of pathological inflammation.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

NPC1161B, an 8-Aminoquinoline Analog, Is Metabolized in the Mosquito and Inhibits Plasmodium falciparum Oocyst Maturation.

Malaria is a major global health threat, with nearly half the world's population at risk of infection. Given the recently described delayed clearance of parasites by artemisinin-combined therapies, new antimalarials are needed to facilitate the global effort toward elimination and eradication. NPC1161 is an 8-aminoquinoline that is derived from primaquine with an improved therapeutic profile compared to the parent compound. The (R)-(-) enantiomer (NPC1161B) has a lower effective dose that results in decreased toxic side effects such as hemolysis compared to the (S)-(+)-enantiomer, making it a promising compound for consideration for clinical development. We explored the effect of NPC1161B on Plasmodium falciparum oocyst and sporozoite development to evaluate its potential transmission-blocking activity viz. its ability to cure mosquitoes of an ongoing infection. When mosquitoes were fed NPC1161B 4 days after P. falciparum infection, we observed that total oocyst numbers were not affected by NPC1161B treatment. However, the sporozoite production capacity of the oocysts was impaired, and salivary gland sporozoite infections were completely blocked, rendering the mosquitoes non-infectious. Importantly, NPC1161B did not require prior liver metabolism for its efficacy as is required in mammalian systems, suggesting that an alternative metabolite is produced in the mosquito that is active against the parasite. We performed liquid chromatography-mass spectrometry (LC-MS)/MS analysis of methanol extracts from the midguts of mosquitoes fed on an NPC1161B (434.15 m/z)-treated blood meal and identified a compound with a mass of 520.2 m/z, likely a conjugate of NPC1161B or an oxidized metabolite. These findings establish NPC1161B, and potentially its metabolites, as transmission-blocking candidates for the treatment of P. falciparum.

Critical Size Limit of Biodegradable Nanoparticles for Enhanced Lymph Node Trafficking and Paracortex Penetration.

Lymph node (LN) targeting through interstitial drainage of nanoparticles (NPs) is an attractive strategy to stimulate a potent immune response, as LNs are the primary site for lymphocyte priming by antigen presenting cells (APCs) and triggering of an adaptive immune response. NP size has been shown to influence the efficiency of LN-targeting and retention after subcutaneous injection. For clinical translation, biodegradable NPs are preferred as carrier for vaccine delivery. However, the selective "size gate" for effective LN-drainage, particularly the kinetics of LN trafficking, is less well defined. This is partly due to the challenge in generating size-controlled NPs from biodegradable polymers in the sub-100-nm range. Here, we report the preparation of three sets of poly(lactic-co-glycolic)-b-poly(ethylene-glycol) (PLGA-b-PEG) NPs with number average diameters of 20-, 40-, and 100-nm and narrow size distributions using flash nanoprecipitation. Using NPs labeled with a near-infrared dye, we showed that 20-nm NPs drain rapidly across proximal and distal LNs following subcutaneous inoculation in mice and are retained in LNs more effectively than NPs with a number average diameter of 40-nm. The drainage of 100-nm NPs was negligible. Furthermore, the 20-nm NPs showed the highest degree of penetration around the paracortex region and had enhanced access to dendritic cells in the LNs. Together, these data confirmed that small, size-controlled PLGA-b-PEG NPs at the lower threshold of about 30-nm are most effective for LN trafficking, retention, and APC uptake after s.c. administration. This report could inform the design of LN-targeted NP carrier for the delivery of therapeutic or prophylactic vaccines.

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development.

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.

The Selection of a Hepatocyte Cell Line Susceptible to Plasmodium falciparum Sporozoite Invasion That Is Associated With Expression of Glypican-3.

In vitro studies of liver stage (LS) development of the human malaria parasite Plasmodium falciparum are technically challenging; therefore, fundamental questions about hepatocyte receptors for invasion that can be targeted to prevent infection remain unanswered. To identify novel receptors and to further understand human hepatocyte susceptibility to P. falciparum sporozoite invasion, we created an optimized in vitro system by mimicking in vivo liver conditions and using the subcloned HC-04.J7 cell line that supports mean infection rates of 3-5% and early development of P. falciparum exoerythrocytic forms-a 3- to 5-fold improvement on current in vitro hepatocarcinoma models for P. falciparum invasion. We juxtaposed this invasion-susceptible cell line with an invasion-resistant cell line (HepG2) and performed comparative proteomics and RNA-seq analyses to identify host cell surface molecules and pathways important for sporozoite invasion of host cells. We identified and investigated a hepatocyte cell surface heparan sulfate proteoglycan, glypican-3, as a putative mediator of sporozoite invasion. We also noted the involvement of pathways that implicate the importance of the metabolic state of the hepatocyte in supporting LS development. Our study highlights important features of hepatocyte biology, and specifically the potential role of glypican-3, in mediating P. falciparum sporozoite invasion. Additionally, it establishes a simple in vitro system to study the LS with improved invasion efficiency. This work paves the way for the greater malaria and liver biology communities to explore fundamental questions of hepatocyte-pathogen interactions and extend the system to other human malaria parasite species, like P. vivax.

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.

Investigations into the Use of a Protein Sensor Assay for Metabolite Analysis.

Rapid and definitive classification of biological samples has application in industrial, agricultural, and clinical settings. Considerable effort has been given to analytical methods to address such applications over the past 50 years, with the majority of successful solutions focusing on a single molecular target. However, in many cases, a single or even a few features are insufficient for accurate characterization or classification. Serum albumin (SA) proteins are a class of cargo-carrying proteins in blood that have evolved to transport a wide variety of metabolites and peptides in mammals. These proteins have up to seven binding sites which communicate allosterically to orchestrate a complex pick-up and delivery system involving a large number of different molecules at any time. The ability of SA proteins to bind multiple molecular species in a sophisticated manner inspired the development of assays to differentiate complex biological solutions. The combination of SA and high-resolution liquid chromatography mass spectrometry (LC-MS) is showing exciting promise as a protein sensor assay (PSA) for classification of complex biological samples. In this study, the PSA has been applied to cells undergoing and recovering from mild oxidative stress. Analysis using traditional LC-MS-based metabolomics failed to differentiate samples into treatment or temporal groups, whereas samples first treated with the PSA were cleanly classified into both correct treatment and temporal groups. The success of the PSA could be attributed to selective binding of metabolites, leading to a reduction in sample complexity and a general reduction in chemical noise. Metabolites important to successful sample classification were often enriched by 100-fold or more yet displayed a wide range of affinities for SA. The end result of PSA treatment is better classification of samples with a reduction in the number of features seen overall. Together, these results demonstrate how the use of a protein-based assay before LC-MS analysis can greatly improve separation and lead to more accurate and successful tracking of the metabolic state in an organism, suggesting potential application in a wide range of fields.

Untargeted metabolomics studies employing NMR and LC-MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis.

Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans, including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans's consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans. Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites.

Bovine serum albumin as a molecular sensor for the discrimination of complex metabolite samples.

The potential for using serum albumin (SA) as a broadly applicable molecular sensor was explored in an effort to develop a method for rapid analysis of complex metabolite samples. SA is a protein present at high concentration in blood, which transports a diverse set of compounds including fatty acids, hormones, and drugs. The effectiveness of the bovine ortholog (BSA) as a molecular sensor was tested by analyzing the pool of small molecules bound to the protein after a brief incubation with complex fluids of biological origin. As an initial test, three varietals of red wine were readily distinguished. Further analysis using four varietals of white wine also showed clear separation. In a second analysis using urine, animals in hemorrhagic shock were separated from a group of comparably treated controls. A time course analysis showed that recovery from injury could also be followed using the assay. This finding is significant as there currently is no method or biomarker for predicting the onset of shock. Comparison of samples was based on liquid chromatography mass spectrometry (LCMS) analysis of compounds selectively bound by BSA. Analysis of the samples after protein selection revealed a significant reduction in complexity and clear separation of groups by Principle Component Analysis (PCA). These results show the potential for using cargo-carrying proteins as molecular sensors for screening complex samples without the need for prior knowledge of sample composition or concentration and may streamline elucidation of biomarkers.