Studying Salmonellae and Yersiniae host-pathogen interactions using integrated 'omics and modeling.

Salmonella and Yersinia are two distantly related genera containing species with wide host-range specificity and pathogenic capacity. The metabolic complexity of these organisms facilitates robust lifestyles both outside of and within animal hosts. Using a pathogen-centric systems biology approach, we are combining a multi-omics (transcriptomics, proteomics, metabolomics) strategy to define properties of these pathogens under a variety of conditions including those that mimic the environments encountered during pathogenesis. These high-dimensional omics datasets are being integrated in selected ways to improve genome annotations, discover novel virulence-related factors, and model growth under infectious states. We will review the evolving technological approaches toward understanding complex microbial life through multi-omic measurements and integration, while highlighting some of our most recent successes in this area.

Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life.

Interaction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not only in vitro but also in vivo during infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

Biogenesis of bacterial membrane vesicles.

Membrane vesicle (MV) release remains undefined, despite its conservation among replicating Gram-negative bacteria both in vitro and in vivo. Proteins identified in Salmonella MVs, derived from the envelope, control MV production via specific defined domains that promote outer membrane protein-peptidoglycan (OM-PG) and OM protein-inner membrane protein (OM-PG-IM) interactions within the envelope structure. Modulation of OM-PG and OM-PG-IM interactions along the cell body and at division septa, respectively, maintains membrane integrity while co-ordinating localized release of MVs with distinct size distribution and protein content. These data support a model of MV biogenesis, wherein bacterial growth and division invoke temporary, localized reductions in the density of OM-PG and OM-PG-IM associations within the envelope structure, thus releasing OM as MVs.

A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions.

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling.

Adaptive Immune Responses during Salmonella Infection.

The interaction betweenSalmonella and its host is complex and dynamic: the host mounts an immune defense against the pathogen, which in turn acts to reduce, evade, or exploit these responses to successfully colonize the host. Although the exact mechanisms mediating protective immunity are poorly understood, it is known that T cells are a critical component of immunity to Salmonella infection, and a robust T-cell response is required for both clearance of primary infection and resistance to subsequent challenge. B-cell functions, including but not limited to antibody production, are also required for generation of protective immunity. Additionally, interactions among host cells are essential. For example, antigen-presenting cells (including B cells) express cytokines that participate in CD4+ T cell activation and differentiation. Differentiated CD4+ T cells secrete cytokines that have both autocrine and paracrine functions, including recruitment and activation of phagocytes, and stimulation of B cell isotype class switching and affinity maturation. Multiple bacterium-directed mechanisms, including altered antigen expression and bioavailability and interference with antigen-presenting cell activation and function, combine to modify Salmonella's "pathogenic signature" in order to minimize its susceptibility to host immune surveillance. Therefore, a more complete understanding of adaptive immune responses may provide insights into pathogenic bacterial functions. Continued identification of adaptive immune targets will guide rational vaccine development, provide insights into host functions required to resist Salmonella infection, and correspondingly provide valuable reagents for defining the critical pathogenic capabilities of Salmonella that contribute to their success in causing acute and chronic infections.

Membrane vesicles are immunogenic facsimiles of Salmonella typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo.

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-alpha, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4(+) T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4(+) T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.