Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a "holy grail" in microbiology. This work describes a highly sensitive, broadly applicable, and costeffective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097-1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities. ambient mass spectrometry | microbial ecology | natural products M icrobes use secreted factors to interact, communicate with, and manipulate their local environment and neighboring cell populations in a process known as metabolic exchange (1-5). By using a wide breadth of molecules ranging from signaling compounds to defensive metabolites, metabolic exchange dictates not only basic microbial behavior, such as biofilm formation, sporulation, and motility, but also social interactions, such as syntrophy and quorum sensing, which enables microbes to establish communities (1-5). Despite these secreted factors, also known as the parvome, having a major impact on the phenotypic development of microbial populations, there is a lack of tools that enable scientists to probe the chemistry of microbial colonies in a direct manner. Currently, the chemistry of microbes is usually studied by monitoring individual molecular species and requires a significant time and monetary investment. Our laboratories are interested in the development of tools that make this process more efficient as well as making it easier for nonchemists to study the chemistry of microbes and nonmicrobe cell populations. Ideally, these tools should be easy to implement, compatible with existing infrastructure, and easily inco...
A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogs, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogs.
During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC 50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.etabolic exchange describes the process of exchanging signals or nutrients between cells or populations and is a common feature of all living systems. Bacteria produce a wide array of signaling molecules to control metabolic as well as morphological and developmental changes in either an interspecies or intraspecies manner (1). Bacillus subtilis, for example, has a complex life cycle and thrives in diverse living conditions ranging from soil, contaminated wounds, and the intestinal tract (2-4). To accommodate this, B. subtilis dedicates ∼10% of its genome to the production of specific molecules involved in intra-and interspecies metabolic exchange (5). Two of these molecules are sporulation delaying protein (SDP) and sporulation killing factor (SKF), which, based on genetic experiments, are proposed to lyse a subpopulation of B. subtilis cells to provide nutrients for the remaining cells, a process referred to as bacterial cannibalism (6-10). This behavior is dependent on Spo0A, a master transcriptional regulator that also controls biofilm formation and sporulation (6-13).We set out to characterize these cannibalistic compounds to establish their roles in the B. subtilis life cycle and to understand their structure ...
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.
The lifespan and survival of dendritic cells (DC) in vivo are potentially critical to the expansion of T cell immune responses. We have previously reported that DC loaded with specific antigen are rapidly eliminated by cytotoxic T lymphocytes (CTL) in vivo, but the site, mechanism, and consequences of DC elimination were not defined. In this article we show that DC elimination in vivo occurs in a perforin-dependent manner and does not require IFN-␥ or the presence of CD4 ؉ CD25 ؉ regulatory T cells. Most importantly, failure to eliminate DC had profound consequences on the CTL immune response. Perforin-deficient mice showed a progressive increase in the numbers of antigen-specific CD8 ؉ T cells after repeated immunizations with DC. In contrast, in control mice the number of antigen-specific CD8 ؉ T cells did not notably increase with repeated immunizations. Lastly, we also show that CTLmediated elimination of DC occurs in peripheral tissues but not in the lymph node. Our data suggest that CTL act as ''gatekeepers'' that control access of antigen-loaded DC into the lymph node, thereby preventing continued expansion of antigen-specific T cells.cytotoxic T cells ͉ immunoregulation ͉ killing D endritic cells (DC) are powerful antigen-presenting cells that are critical for the initiation of CD4 ϩ and CD8 ϩ T cell responses. DC reside in peripheral tissues where they take up antigens from the external environment, then migrate to the lymph nodes where they interact with antigen-specific T cells and induce their activation to proliferation and effector function (1, 2). The lifespan of DC once in the lymph node is thought to be relatively brief, but experimental estimates have not yielded a consistent figure (3-5).The mechanism and regulation of DC survival and death (6, 7) are likely to be important in maintaining the homeostatic balance of the immune system. A few reports have linked extended survival of DC to enhanced or dysregulated T cell immune responses and lymphoproliferative disease (8-10). In contrast, reduced survival of DC has been associated with impaired immune responses (7). It is presently unclear whether T cells also influence the lifespan of DC in an antigen-specific fashion. In lymph nodes of mice adoptively transferred with CD4 ϩ T cell receptor (TCR) transgenic T cells, DC presenting specific antigen disappear more rapidly than DC not presenting antigen (11), suggesting that T cells may have a role in regulating DC numbers and survival. Experiments using infection with Listeria or malaria also suggest that the number of antigen-presenting cells becomes limiting during the early phases of CD8 ϩ immune responses and prevents the continued expansion of antigen-specific T cells (12, 13). Together, these experiments suggest the attractive hypothesis that T cells may be able to regulate their own responses in a feedback fashion, by affecting the survival of antigen-presenting DC.We have previously reported that DC loaded with antigen and injected s.c. into immune mice are rapidly eliminated by CD8 ϩ T cel...
Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometrybased analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycopro-
Molecules that alter the normal dynamics of microtubule assembly and disassembly include many anticancer drugs in clinical use. So far all such therapeutics target β-tubulin, and structural biology has explained the basis of their action and permitted design of new drugs. However, by shifting the profile of β-tubulin isoforms, cancer cells become resistant to treatment. Compounds that bind to α-tubulin are less well characterized and unexploited. The natural product pironetin is known to bind to α-tubulin and is a potent inhibitor of microtubule polymerization. Previous reports had identified that pironetin reacts with lysine-352 residue however analogues designed on this model had much lower potency, which was difficult to explain, hindering further development. We report crystallographic and mass spectrometric data that reveal that pironetin forms a covalent bond to cysteine-316 in α-tubulin via a Michael addition reaction. These data provide a basis for the rational design of α-tubulin targeting chemotherapeutics.
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