Jonathan K. Hilmer

Characterization of the Archaeal Thermophile Sulfolobus Turreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life

ABSTRACT Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse MGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and k cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.

The Nucleotide Exchange Factor Ric-8A Is a Chaperone for the Conformationally Dynamic Nucleotide-Free State of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [15N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [15N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.

Proteomic Analysis of Sulfolobus solfataricus During Sulfolobus Turreted Icosahedral Virus Infection

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Demonstration of protein-based human identification using the hair shaft proteome

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.

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.

Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids

Chondrocytes are the sole cell type found in articular cartilage and are repeatedly subjected to mechanical loading in vivo. We hypothesized that physiological dynamic compression results in changes in energy metabolism to produce proteins for maintenance of the pericellular and extracellular matrices. The objective of this study was to develop an in-depth understanding for the short term (<30min) chondrocyte response to sub-injurious, physiological compression by analyzing metabolomic profiles for human chondrocytes harvested from femoral heads of osteoarthritic donors. Cell-seeded agarose constructs were randomly assigned to experimental groups, and dynamic compression was applied for 0, 15, or 30min. Following dynamic compression, metabolites were extracted and detected by HPLC-MS. Untargeted analyzes examined changes in global metabolomics profiles and targeted analysis examined the expression of specific metabolites related to central energy metabolism. We identified hundreds of metabolites that were regulated by applied compression, and we report the detection of 16 molecules not found in existing metabolite databases. We observed patient-specific mechanotransduction with aging dependence. Targeted studies found a transient increase in the ratio of NADP+ to NADPH and an initial decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. By characterizing metabolomics profiles of primary chondrocytes in response to applied dynamic compression, this study provides insight into how OA chondrocytes respond to mechanical load. These results are consistent with increases in glycolytic energy utilization by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

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.

Selective trapping of SNO-BSA and GSNO by benzenesulfinic acid sodium salt: mechanistic study of thiosulfonate formation and feasibility as a protein S-nitrosothiol detection strategy

The conversion of S-nitrosothiols to thiosulphonates by reaction with the sodium salt of benzenesulfinic acid (PhSO2Na) has been examined in detail with the exemplary substrates S-nitrosoglutathione (GSNO) and S-nitrosylated bovine serum albumin (SNO-BSA). The reaction stoichiometry (2:1, PhSO2Na:RSNO) and the rate law (first order in both PhSO2Na and RSNO) have been determined under mild acidic conditions (pH 4.0). The products have been identified as the corresponding thiosulphonates (GSSO2Ph and BSA-SSO2Ph) along with PhSO2NHOH obtained in a 1:1 ratio. GSH, GSSG, and BSA were unreactive to PhSO2Na.