Activation of the cannabinoid receptor CB₂ has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB₂ plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB₂ agonists, I set out to examine whether CB₂ modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB₂ had any effect on CB₂ agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB₁/CB₂, G_i/o-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&5, CYSLTR1&2 and GPER1, were not responsible for CB₂ agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB₂ agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB₂. I found that CB₂^-/- mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB₂ specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB₂ acts to regulate neutrophil recruitment during the acute inflammatory response.

When, at the beginning of 1972, my scientific attention was first drawn to the subject of cellular participation in the inflanunatory response, I was struck by the need for an understanding of the cellular and molecular mechanisms whereby blood leucocytes are attracted to an area of injury. The literature at that time contained good technical accounts of methods available for studying chemotaxis in vitro and many reports of diverse compounds of biological origin with attractant, or chemotactic, properties for motile, phagocytic cells. In general, these reports tended to substantiate the belief that chemical substances generated at an inflannnatory source attracted cells to that source in a teleologically appropriate way and they justified, by the consistent correlation observed, the relevance of in vitro procedures for studying the phenomenon. In other words, answers were available to the question "What substances attract?"; very few were available to the question, "How do they attract?".

The flagellated soil-dwelling bacterium Sinorhizobium meliloti is known for its symbiotic relationship with several leguminous genera. The symbiosis between the bacterium and its host plants facilitates fixation of atmospheric nitrogen and ultimately replenishment of nitrogen to the soil. However, before nitrogen fixation can occur, the bacterial cells must actively travel to the plant's roots and successfully induce formation of a plant organ called a root nodule. To initiate the nodulation process, the bacterium needs to be in direct contact with the root hairs. This requires free living bacterial cells within the soil to sense the presence of their host plant and travel to its roots. S. meliloti is able to do this through a process called chemotaxis. Chemotaxis is the ability to respond to chemical gradients within the environment by directed movement. It is facilitated by Methyl-accepting Chemotaxis Proteins (MCPs) as part of a two-component signal transduction system. These receptor proteins are able to bind ligands and influence the state of the signal transduction system, ultimately controlling flagellar behavior. The chemotaxis system of Escherichia coli has been well characterized and serves as a useful point of comparison to that of S. melilot throughout this work. Within this work we have determined the stoichiometry of all chemotaxis proteins of S. meliloti by means of quantitative immunoblotting. Chapter 2 addresses the stoichiometry of MCPs and the histidine kinase CheA. The eight MCPs were grouped by total abundance within the cell, in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY, McpZ and McpT). The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 22.3:1, highly similar to the 23:1 ratio known for Bacilius subtiltis. Chapter 3 continues the investigation of the protein stoichiometry, expanding to all chemotaxis proteins. We compare ratios of S. meliloti chemotaxis proteins to those of E. coli and B. subtilis. We address the possible reasons for the high ratio of CheR / CheB to the total amount of receptors. Proteins again can be grouped by abundance: CheD, CheY1, and CheY2 are the most abundant. CheR and CheB appear in lower amounts, CheS and CheT appear to be auxiliary proteins, and finally CheW1 and CheW2, which directly interact with the receptors and CheA. Chapter 4 focuses on altered receptor abundance in S. meliloti due to the fusion of common epitope tags to the C-terminus. The fusion of these tags promotes greater cellular abundance of many receptors including McpU. The fusion of charged residues to the C-terminus promotes a greater increase in McpU abundance thanthe addition of single amino acid residues. Truncations of McpU were made to investigate the presence of a protease recognition site near the C-terminus. These truncations resulted in an increase in abundance similar to those resulting from epitope tag fusions. As epitope tags are widely used in protein studies to help determine protein stoichiometry, this study obviates a potential stumbling block for future experimenters. The function of CheT, a small protein (13.4 kDa) encoded by the last gene in S. meliloti's major chemotaxis operon, is the subject of chapter 5. A cheT deletion strain is chemotactically deficient compared to the S. meliloti wild-type strain. Through two separate experiments (a glutaraldehyde cross-linking assay and co-purification) we demonstrated that CheT interacts with the methyltransferase CheR. We also investigate its possible role in CheR's methylation activity through a series of methylation assays. This work contributes to our understanding of Sinorhizobium meliloti's chemotaxis signal transduction system. We have discovered evidence for new a protein-protein interaction within our system and have revealed the abundance of all chemotaxis proteins within the cell. We also showed that fusions of epitope tags to various chemotaxis proteins can dramatically influence their abundance. We shed light on the possible function of a previously uncharacterized protein, although more work is required to determine its exact role. / Doctor of Philosophy / Sinorhizboium meliloti is a bacterium that lives in the soil and forms a symbiotic relationship with many plants including alfalfa, a commonly grown cover crop. This symbiotic relationship is important because it allows for nitrogen to be replenished into the soil without the use of artificial fertilizer. However, to form this relationship the bacterial cells in the soil must be able to colonize the plant roots. The soil is a complex environment with many different kinds of chemical molecules and sources of nutrients. Like many other types of bacteria, S. meliloti uses flagella (long helical structures that rotate much like a propeller) to move through the soil. Control of the flagella falls to what is known as a chemotaxis signal transduction system, which can be thought of as a navigation system for each bacterial cell. The system has proteins that act as receptors to sense different chemical molecules. The bacterial cells can sense signals for the plant and move towards their host. This work shows the abundance of each type of receptor and other components within the cell. It also examines the function of a previously unknown protein, CheT, within the chemotaxis system.

Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses.

Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility. Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle. A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation. The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.

Thesis (M.S.)--University of Michigan, 1972.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 108-136).

Thesis (M.S.)--University of Michigan, 1972.

Sinorhizobium meliloti (Ensifer meliloti) is a bacterium that will exist saprotrophically in the soil and rhizosphere or as a differentiated bacteroid inside root nodules of the legume genera Medicago, Melilotus, and Trigonella. It exists in symbiosis when inside a host plant and will fix gaseous N2 into ammonium for the plant. In return, a population of the bacteria is harbored inside the plant where it can proliferate beyond what would be possible in the rhizosphere or bulk soil. This symbiosis is a defining feature of the Fabaceae (legume) family, a clade that diverged approximately 60 million years ago and is now the 5th largest plant family by species count. Each legume species pairs with one or several strains of bacteria, referred to broadly as rhizobia. The rhizobia identify their proper host plant by a cocktail of secondary metabolites called flavonoids released from specific parts of the roots. Initiation of the symbiosis may only occur at the tips of young root hairs. Therefore, the means rhizobia take to localize themselves to these sites must be the inceptive step in the symbiotic interaction. The studies here examine the mechanisms and priorities rhizobia use to achieve this goal. Movement of bacteria is referred to as motility and is achieved via (in rhizobia, multiple) rotating flagella, proteinaceous extracellular appendages that propel the cell through liquid environments. On their own, flagella may only move but not guide the cell. Navigation is achieved through sensors that detect chemical attractant or repellent cues in the environment and an intracellular signaling system that relays information to appropriately control locomotion. This sensing is called chemotaxis. A research focus is directed on the sensing aspect of chemotaxis to understand which chemical compounds are the preferred attractants for S. meliloti. An emphasis is placed on those compounds released from germinating host seeds. Chapter 2 spearheads our research goals by examining the chemotactic potential of host-derived flavonoids, the compounds that induce the symbiotic signaling in the rhizobial symbiont. While a logical place to start, this study reveals that our strain of rhizobia is not attracted to flavonoids. We determined that the best chemoattractants are hydrophilic in nature and that hydrophobic compounds, such as flavonoids, are not effective chemoattractants. In addition, we discuss the nature of chemotactic agents and symbiosis inducers to fortify our understanding of how classes of compounds contribute to the rhizobia-plant interaction. In chapter 3, we characterize the sensor protein, McpV, and its ligand profile for carboxylates. The protein is first screened using a high-throughput assay to test numerous possible ligands simultaneously. We confirm positive reactions using direct binding studies and quantify dissociation constants. Then, the phenotypic response to these ligands is measured using capillary chemotaxis assays, and the role mcpV plays in this response is confirmed using deletion mutants. Last, the symbiotic context is addressed by quantifying these ligands in exudates of the host alfalfa. These experiments show that McpV is a chemotactic sensor dedicated to detecting 2 – 4 C monocarboxylates. Only one of the compounds found in the ligand profile, glycolate, was detected in seed exudates, so the contribution of McpV to host sensing is yet to be expounded. Chapter 4 follows the model of chapter 2 but is complicated when the ligand screen used previously gives ambiguous results. Using direct binding studies, we were able to confirm the true ligand amidst numerous false positives. Analytical gel filtration suggests that McpT exists as a dimer regardless of ligand binding. Capillary chemotaxis assays quantified the responses mediated by McpT to di- and tri-carboxylates, which were slightly weaker, but still on-par with the responses to McpV ligands. Strains with mcpT deletions showed strongly reduced, but in some cases, not abolished, chemotaxis to carboxylates. Chapter 5 examines McpX – the chemoreceptor already known to be a sensor of quaternary ammonium compounds. This is a structural investigation into the binding of McpX to its ligands. A crystal structure of the ligand binding region of the protein is resolved to understand how ligands fit into the binding pocket of McpX and what determines its structurally diverse ligand profile. The contribution of certain residues to ligand binding are further probed using direct binding studies on single point variants of McpX. The analysis of chemoreceptor functions hint at what kinds of molecules are most important to bacterial survival and reproduction. Knowing what the bacterium is tuned to seek out grants understanding of what niches they prefer, and how they thrive in those niches. For S. meliloti and other rhizobia, the preeminent niche is one in symbiosis with a host plant. The sum of this knowledge we have accrued with S. meliloti lends itself to agricultural goals of soil enrichment, legume inoculation, nutrient cycling, and environmentally safe and efficient crop fertilization. / Doctor of Philosophy / Sinorhizobium meliloti and other soil-dwelling bacteria termed rhizobia are crucial to the cultivation of leguminous crops such as alfalfa, soy, pea, lentil, peanut, and many more. The bacterium can be internalized by the plant host's roots where it will supply the plant with nitrogen. This is a great boon to crops when they need to accumulate more protein in seed stores, or for plants that survive in nutrient depleted soils. The bacterium must begin seeking out the host plant by sensing chemical cues. It can navigate to the proper location by using a process called chemotaxis. This process is centered around chemoreceptors that can be likened to the nose of the bacterium. Using these chemoreceptors, the bacterium will seek out compounds that benefits it – these are usually food sources. Identifying what each individual chemoreceptor senses allows us to understand what the bacterium needs to seek out to survive. We correlate this information with compounds that the plant secretes and find that many chemoreceptors have evolved to sense signals that will lead the bacterium to a plant root. This interaction is a key part of how the symbiosis is propagated and ultimately benefits the agriculture of leguminous plants.

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