Steven J. Bark

A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotic-tolerant bacterial infections.IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.

Enhancing MALDI Time-Of-Flight Mass Spectrometer Performance through Spectrum Averaging

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometers are simple and robust mass spectrometers used for analysis of biologically relevant molecules in diverse fields including pathogen identification, imaging mass spectrometry, and natural products chemistry. Despite high nominal resolution and accuracy, we have observed significant variability where 30–50% of individual replicate measurements have errors in excess of 5 parts-per-million, even when using 5-point internal calibration. Increasing the number of laser shots for each spectrum did not resolve this observed variability. What is responsible for our observed variation? Using a modern MALDI-TOF/TOF instrument, we evaluated contributions to variability. Our data suggest a major component of variability is binning of the raw flight time data by the electronics and clock speed of the analog-to-digital (AD) detection system, which requires interpolation by automated peak fitting algorithms and impacts both calibration and the observed mass spectrum. Importantly, the variation observed is predominantly normal in distribution, which implies multiple components contribute to the observed variation and suggests a method to mitigate this variability through spectrum averaging. Restarting the acquisition impacts each spectrum within the electronic error of the AD detector system and defines a new calibration function. Therefore, averaging multiple independent spectra and not a larger number of laser shots leverages this inherent binning error to mitigate variability in accurate MALDI-TOF mass measurements.

Insights into the anti-angiogenic properties of phosphaplatins

Phosphaplatins are platinum-based antitumor compounds that, unlike other clinically utilized platinum drugs (i.e. cisplatin, carboplatin, and oxaliplatin), appear to target proteins rather than DNA. Because of their unique mode of action, phosphaplatins are promising drug candidates for cisplatin-resistant cancers. In this study, we discovered that Pt(II) and Pt(IV) phosphaplatins possess diverse antitumor properties. In addition to targeting apoptosis antigen (FAS) and proapoptotic gene products as described previously, phosphaplatins also target angiogenesis. We demonstrate that phosphaplatins inhibit human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and suppress tumor angiogenesis and growth in immunodeficient mice that were inoculated with A2780 ovarian cancer cells in vivo. To provide insight into this novel antitumor mechanism, phosphaplatin-treated HUVECs were found to exhibit lower gene expression levels of vascular endothelial growth factors (VEGFs) and the VEGFR-2 receptor compared to untreated cells. Kinase inhibition studies suggest that phosphaplatins are inhibitors of VEGFR-2. In ligand exchange experiments using both Pt atomic absorption and 31P NMR spectroscopies, we show that phosphaplatins most likely bind to VEGFR-2 through metal-ligand coordination rather than electrostatic interactions. These studies enhance our understanding of the diverse and novel mechanisms of action of the phosphaplatin antitumor agents, which could potentially be used as chemotherapeutic agents against cisplatin-resistant cancers.

Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein

Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.

Formaldehyde scavengers function as novel antigen retrieval agents.

Antigen retrieval agents improve the detection of formaldehyde-fixed proteins, but how they work is not well understood. We demonstrate that formaldehyde scavenging represents a key characteristic associated with effective antigen retrieval; under controlled temperature and pH conditions, scavenging improves the typical antigen retrieval process through reversal of formaldehyde-protein adduct formation. This approach provides a rational framework for the identification and development of more effective antigen retrieval agents.

Twenty-Five Years Of Investigating The Universal Stress Protein: Function, Structure, And Applications

Since the initial discovery of universal stress protein A (UspA) 25 years ago, remarkable advances in molecular and biochemical technologies have revolutionized our understanding of biology. Many studies using these technologies have focused on characterization of the uspA gene and Usp-type proteins. These studies have identified the conservation of Usp-like proteins across bacteria, archaea, plants, and even some invertebrate animals. Regulation of these proteins under diverse stresses has been associated with different stress-response genes including spoT and relA in the stringent response and the dosR two-component signaling pathways. These and other foundational studies suggest Usps serve regulatory and protective roles to enable adaptation and survival under external stresses. Despite these foundational studies, many bacterial species have multiple paralogs of genes encoding these proteins and ablation of the genes does not provide a distinct phenotype. This outcome has limited our understanding of the biochemical functions of these proteins. Here, we summarize the current knowledge of Usps in general and UspA in particular across different genera as well as conclusions about their functions from seminal studies in diverse organisms. Our objective has been to organize the foundational studies in this field to identify the significant impediments to further understanding of Usp functions at the molecular level. We propose ideas and experimental approaches that may overcome these impediments and drive future development of molecular approaches to understand and target Usps as central regulators of stress adaptation and survival. Despite the fact that the full functions of Usps are still not known, creative many applications have already been proposed, tested, and used. The complementary approaches of basic research and applications, along with new technology and analytic tools, may yield the elusive yet critical functions of universal stress proteins in diverse systems.