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Squalamine was efficiently prepared in five steps utilizing a latent spermidine reagent. This reagent was shown to be most appropriate, as it is stable to reductive amination conditions. It is converted to spermidine under weakly acidic conditions. The soybean-based sterol, stigmasterol, which is used as the starting material to prepare the key intermediate, is both inexpensive and readily available. This stereoselective synthesis of squalamine from steroid has provided supplies for phase I clinical trials.

Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines requires testing of additional antigens, optimization of the vaccine formulation and a better understanding of the protective immune response. [ABSTRACT FROM AUTHOR]

Identification of specific antigens responsible for the ability of complex immunogens to induce protection is a major goal in development of bacterial vaccines. Much of the investigation has focused on highly abundant and highly immunodominant outer membrane proteins. Recently however, genomic and proteomic approaches have facilitated identification of minor components of the bacterial outer membrane that have previously been missed or ignored in immunological analyses. Immunization with Anaplasma marginale outer membranes or a cross-linked surface complex induces protection against bacteremia, however the components responsible for protection within these complex immunogens are unknown. Using outer membrane protein AM779 as a model, we demonstrated that this highly conserved but minor component of the A. marginale surface was immunologically sub-dominant in the context of the outer membrane or surface complex vaccines. Immunologic sub-dominance could be overcome by targeted vaccination with AM779 for T lymphocyte responses but not for antibody responses, suggesting that both abundance and intrinsic immunogenicity determine relative dominance. Importantly, immunization with AM779 supports that once priming is achieved by specific targeting, recall upon infectious challenge is achieved. While immunization with AM779 alone was not sufficient to induce protection, the ability of targeted immunization to prime the immune response to highly conserved but low abundance proteins supports continued investigation into the role of sub-dominant antigens, individually and collectively, in vaccine development for A. marginale and related bacterial pathogens. [ABSTRACT FROM AUTHOR]

Background: To decipher the complexity and improve the understanding of host-pathogen interactions, biologists must adopt new system level approaches in which the hierarchy of biological interactions and dynamics can be studied. This paper presents the application of systems biology for the cross-comparative analysis and interactome modeling of three different infectious agents, leading to the identification of novel, unique and common molecular host responses (biosignatures). Methods: A computational systems biology method was utilized to create interactome models of the host responses to Brucella melitensis (BMEL), Salmonella enterica Typhimurium (STM) and Mycobacterium avium paratuberculosis (MAP). A bovine ligated ileal loop biological model was employed to capture the host gene expression response at four time points post infection. New methods based on Dynamic Bayesian Network (DBN) machine learning were employed to conduct a systematic comparative analysis of pathway and Gene Ontology category perturbations. Results: A cross-comparative assessment of 219 pathways and 1620 gene ontology (GO) categories was performed on each pathogen-host condition. Both unique and common pathway and GO perturbations indicated remarkable temporal differences in pathogen-host response profiles. Highly discriminatory pathways were selected from each pathogen condition to create a common system level interactome model comprised of 622 genes. This model was trained with data from each pathogen condition to capture unique and common gene expression features and relationships leading to the identification of candidate host-pathogen points of interactions and discriminatory biosignatures. Conclusions: Our results provide deeper understanding of the overall complexity of host defensive and pathogen invasion processes as well as the identification of novel host-pathogen interactions. The application of advanced computational methods for developing interactome models based on DBN has proven to be instrumental in conducting multi-conditional cross-comparative analyses. Further, this approach generates a fully simulateable [ABSTRACT FROM AUTHOR]

Global protein identification through current proteomics methods typically depends on the availability of sequenced genomes. In spite of increasingly high throughput sequencing technologies, this information is not available for every microorganism and rarely available for entire microbial communities. Nevertheless, the protein-level homology that exists between related bacteria makes it possible to extract biological information from the proteome of an organism or microbial community by using the genomic sequences of a near neighbor organism. Here, we demonstrate a trans-organism search strategy for determining the extent to which near-neighbor genome sequences can be applied to identify proteins in unsequenced environmental isolates. In proof of concept testing, we found that within a CLUSTAL W distance of 0.089, near-neighbor genomes successfully identified a high percentage of proteins within an organism. Application of this strategy to characterize environmental bacterial isolates lacking sequenced genomes, but having 16S rDNA sequence similarity to Shewanella resulted in the identification of 300-500 proteins in each strain. The majority of identified pathways mapped to core processes, as well as to processes unique to the Shewanellae, in particular to the presence of c-type cytochromes. Examples of core functional categories include energy metabolism, protein and nucleotide synthesis and cofactor biosynthesis, allowing classification of bacteria by observation of conserved processes. Additionally, within these core functionalities, we observed proteins involved in the alternative lactate utilization pathway, recently described in Shewanella. [ABSTRACT FROM AUTHOR]

Abstract: Smooth Brucella abortus S2308 is virulent while rough derivatives are attenuated. Intracellular killing is often blamed for these differences. In the studies described, uptake kinetics and interaction of S2308 and S2308 manBA∷Tn5 (CA180) rough mutants with macrophages were investigated. The results revealed that smooth B. abortus was rapidly internalized, achieving a maximum level in less than 5min without additional uptake over the next 30min. In contrast, continued uptake of the rough mutant was observed and only achieves a maximum level after 30min. The results were confirmed by the differences in F-actin polymerization, lipid raft staining, early endosome colocalization and electron microscopic observations after smooth and rough Brucella infection. We also demonstrated for the first time that uptake of S2308, but not rough mutant CA180 was PI3-kinase and toll-like receptor 4 (TLR4) dependent. Differences in uptake were associated with differences in macrophage activation with regard to NF-κB translocation and cytokine production. These results provide evidence that the presence of B. abortus OPS dictates the interactions between Brucella and specific cell surface receptors minimizing macrophage activation and enhancing Brucella survival and/or persistence. [Copyright &y& Elsevier]

While comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry, experimental validation of the existence of this core genome requires extensive measurement and is typically not undertaken. Enabled by an extensive proteome database developed over six years, we have experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. Although genomic studies can establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits. [ABSTRACT FROM AUTHOR]

Dermacentor andersoni , known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified. [ABSTRACT FROM AUTHOR]

Abstract: The aim of research on infectious diseases is their prevention, and brucellosis and salmonellosis as such are classic examples of worldwide zoonoses for application of a systems biology approach for enhanced rational vaccine development. When used optimally, vaccines prevent disease manifestations, reduce transmission of disease, decrease the need for pharmaceutical intervention, and improve the health and welfare of animals, as well as indirectly protecting against zoonotic diseases of people. Advances in the last decade or so using comprehensive systems biology approaches linking genomics, proteomics, bioinformatics, and biotechnology with immunology, pathogenesis and vaccine formulation and delivery are expected to enable enhanced approaches to vaccine development. The goal of this paper is to evaluate the role of computational systems biology analysis of host:pathogen interactions (the interactome) as a tool for enhanced rational design of vaccines. Systems biology is bringing a new, more robust approach to veterinary vaccine design based upon a deeper understanding of the host–pathogen interactions and its impact on the host''s molecular network of the immune system. A computational systems biology method was utilized to create interactome models of the host responses to Brucella melitensis (BMEL), Mycobacterium avium paratuberculosis (MAP), Salmonella enterica Typhimurium (STM), and a Salmonella mutant (isogenic ΔsipA, sopABDE2) and linked to the basis for rational development of vaccines for brucellosis and salmonellosis as reviewed by Adams et al. and Ficht et al. . A bovine ligated ileal loop biological model was established to capture the host gene expression response at multiple time points post infection. New methods based on Dynamic Bayesian Network (DBN) machine learning were employed to conduct a comparative pathogenicity analysis of 219 signaling and metabolic pathways and 1620 gene ontology (GO) categories that defined the host''s biosignatures to each infectious condition. Through this DBN computational approach, the method identified significantly perturbed pathways and GO category groups of genes that define the pathogenicity signatures of the infectious agent. Our preliminary results provide deeper understanding of the overall complexity of host innate immune response as well as the identification of host gene perturbations that defines a unique host temporal biosignature response to each pathogen. The application of advanced computational methods for developing interactome models based on DBNs has proven to be instrumental in elucidating novel host responses and improved functional biological insight into the host defensive mechanisms. Evaluating the unique differences in pathway and GO perturbations across pathogen conditions allowed the identification of plausible host–pathogen interaction mechanisms. Accordingly, a systems biology approach to study molecular pathway gene expression profiles of host cellular responses to microbial pathogens holds great promise as a methodology to identify, model and predict the overall dynamics of the host–pathogen interactome. Thus, we propose that such an approach has immediate application to the rational design of brucellosis and salmonellosis vaccines. [Copyright &y& Elsevier]