Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.

Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

IgM-associated gut bacteria in obesity and type 2 diabetes in C57BL/6 mice and humans.

AIMS/HYPOTHESIS: IgM is the primary antibody produced by B cells and we hypothesise that IgM antibodies to gut microbiota may play a role in immunometabolism in obesity and type 2 diabetes. To test our hypothesis, we used B6 mice deficient in activation-induced cytidine deaminase (Aid-/- [also known as Aicda-/-]) which secrete only IgM antibodies, and human faecal samples. METHODS: We studied the immunometabolic effects and gut microbial changes in high-fat-diet-induced obesity (HFDIO) in Aid-/- B6 mice compared with wild-type mice. To determine similarities between mice and humans, human stool samples were collected from children and adolescents who were obese with normal glucose tolerance (NGT), obese with glucose intolerance (IGT), or obese and newly diagnosed with type 2 diabetes, for faecal microbiota transplant (FMT) into germ-free (GF) B6 mice and we assessed IgM-bound bacteria and immune responses. RESULTS: Compared with wild-type mice, Aid-/- B6 mice developed exacerbated HFDIO due to abundant levels of IgM. FMT from Aid-/- B6 to GF B6 mice promoted greater weight gain in recipient mice compared with FMT using wild-type mouse faecal microbiota. Obese youth with type 2 diabetes had more IgM-bound gut bacteria. Using the stools from the obese youth with type 2 diabetes for FMT to GF B6 mice, we observed that the gut microbiota promoted body weight gain and impaired glucose tolerance in the recipient GF B6 mice. Importantly, some clinical features of these obese young individuals were mirrored in the GF B6 mice following FMT. CONCLUSIONS/INTERPRETATION: Our results suggest that IgM-bound gut microbiota may play an important role in the immuno-pathogenesis of obesity and type 2 diabetes, and provide a novel link between IgM in obesity and type 2 diabetes in both mice and humans. DATA AVAILABILITY: The 16s rRNA sequencing datasets supporting the current study have been deposited in the NCBI SRA public repository ( https://www.ncbi.nlm.nih.gov/sra ; accession no. SAMN18796639).

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9.

BACKGROUND & AIM: Ammonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model. METHODS: Ammonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50µg/mouse) was evaluated. RESULTS: Following NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells. CONCLUSION: Ammonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.

Toll-like receptor 9 negatively regulates pancreatic islet beta cell growth and function in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Innate immune effectors interact with the environment to contribute to the pathogenesis of the autoimmune disease, type 1 diabetes. Although recent studies have suggested that innate immune Toll-like receptors (TLRs) are involved in tissue development, little is known about the role of TLRs in tissue development, compared with autoimmunity. We aimed to fill the knowledge gap by investigating the role of TLR9 in the development and function of islet beta cells in type 1 diabetes, using NOD mice. METHODS: We generated Tlr9-/- NOD mice and examined them for type 1 diabetes development and beta cell function, including insulin secretion and glucose tolerance. We assessed islet and beta cell number and characterised CD140a expression on beta cells by flow cytometry. We also tested beta cell function in Tlr9-/- C57BL/6 mice. Finally, we used TLR9 antagonists to block TLR9 signalling in wild-type NOD mice to verify the role of TLR9 in beta cell development and function. RESULTS: TLR9 deficiency promoted pancreatic islet development and beta cell differentiation, leading to enhanced glucose tolerance, improved insulin sensitivity and enhanced first-phase insulin secretory response. This was, in part, mediated by upregulation of CD140a (also known as platelet-derived growth factor receptor-α [PDGFRα]). In the absence of TLR9, induced by either genetic targeting or treatment with TLR9 antagonists, which had similar effects on ontogenesis and function of beta cells, NOD mice were protected from diabetes. CONCLUSIONS/INTERPRETATION: Our study links TLR9 and the CD140a pathway in regulating islet beta cell development and function and indicates a potential therapeutic target for diabetes prevention and/or treatment.

Maternal Antibiotic Treatment Protects Offspring from Diabetes Development in Nonobese Diabetic Mice by Generation of Tolerogenic APCs.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the slow, progressive destruction of islet ß cells and loss of insulin production, as a result of interaction with environmental factors, in genetically susceptible individuals. The gut microbiome is established very early in life. Commensal microbiota establish mutualism with the host and form an important part of the environment to which individuals are exposed in the gut, providing nutrients and shaping immune responses. In this study, we studied the impact of targeting most Gram-negative bacteria in the gut of NOD mice at different time points in their life, using a combination of three antibiotics--neomycin, polymyxin B, and streptomycin--on diabetes development. We found that the prenatal period is a critical time for shaping the immune tolerance in the progeny, influencing development of autoimmune diabetes. Prenatal neomycin, polymyxin B, and streptomycin treatment protected NOD mice from diabetes development through alterations in the gut microbiota, as well as induction of tolerogenic APCs, which led to reduced activation of diabetogenic CD8 T cells. Most importantly, we found that the protective effect was age dependent, and the most profound protection was found when the mice were treated before birth. This indicates the importance of the prenatal environment and early exposure to commensal bacteria in shaping the host immune system and health.

NLRP3 deficiency protects from type 1 diabetes through the regulation of chemotaxis into the pancreatic islets.

Studies in animal models and human subjects have shown that both innate and adaptive immunity contribute to the pathogenesis of type 1 diabetes (T1D). Whereas the role of TLR signaling pathways in T1D has been extensively studied, the contribution of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 inflammasome pathway remains to be explored. In this study, we report that NLRP3 plays an important role in the development of T1D in the nonobese diabetic (NOD) mouse model. NLRP3 deficiency not only affected T-cell activation and Th1 differentiation, but also modulated pathogenic T-cell migration to the pancreatic islet. The presence of NLRP3 is critical for the expression of the chemokine receptors CCR5 and CXCR3 on T cells. More importantly, NLRP3 ablation reduced the expression of chemokine genes CCL5 and CXCL10 on pancreatic islet cells in an IRF-1-dependent manner. Our results suggest that molecules involved in chemotaxis, accompanied by the activation of the NLRP3 inflammasome, may be effective targets for the treatment of T1D.

Type 1 diabetes and gut microbiota: Friend or foe?

Type 1 diabetes is a T cell-mediated autoimmune disease. Environmental factors play an important role in the initiation of the disease in genetically predisposed individuals. With the improved control of infectious disease, the incidence of autoimmune diseases, particularly type 1 diabetes, has dramatically increased in developed countries. Increasing evidence suggests that gut microbiota are involved in the pathogenesis of type 1 diabetes. Here we focus on recent advances in this field and provide a rationale for novel therapeutic strategies targeting gut microbiota for the prevention of type 1 diabetes.

Combination treatment with anti-CD20 and oral anti-CD3 prevents and reverses autoimmune diabetes.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease, although B cells also play an important role in T1D development. Both T cell- and B cell-directed immunotherapies have shown efficacy in the prevention and reversal of T1D. However, whether the combined strategy of targeting both T and B cells could further improve therapeutic efficacy remains to be explored. We show that combined treatment with intravenous antihuman CD20 (hCD20) and oral anti-CD3 significantly delays diabetes development in prediabetic hCD20 transgenic NOD mice. More importantly, the combined treatment reverses diabetes in >60% of mice newly diagnosed with diabetes. Further mechanistic studies demonstrated that the addition of oral anti-CD3 to the B-cell depletion therapy synergistically enhances the suppressive function of regulatory T cells. Of note, the oral anti-CD3 treatment induced a fraction of interleukin (IL)-10-producing CD4 T cells in the small intestine through IL-10- and IL-27-producing dendritic cells. Thus, the findings demonstrate that combining anti-CD20 and oral anti-CD3 is superior to anti-CD20 monotherapy for restoring normoglycemia in diabetic NOD mice, providing important preclinical evidence for the optimization of B cell-directed therapy for T1D.

The dual effects of B cell depletion on antigen-specific T cells in BDC2.5NOD mice.

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet ß cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (T(reg)) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in T(reg) cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d(-) fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and T(reg) cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.

The role of Gr1+ cells after anti-CD20 treatment in type 1 diabetes in nonobese diabetic mice.

Studies suggest that Gr1(+)CD11b(+) cells have immunoregulatory function, and these cells may play an important role in autoimmune diseases. In this study, we investigated the regulatory role of Gr1(+)CD11b(+) cells in protecting against type 1 diabetes in NOD mice. In this study, we showed that temporary B cell depletion induced the expansion of Gr1(+)CD11b(+) cells. Gr1(+)CD11b(+) cells not only directly suppress diabetogenic T cell function but also can induce regulatory T cell differentiation in a TGF-ß-dependent manner. Furthermore, we found that Gr1(+)CD11b(+) cells could suppress diabetogenic CD4 and CD8 T cell function in an IL-10-, NO-, and cell contact-dependent manner. Interestingly, single anti-Gr1 mAb treatment can also induce a transient expansion of Gr1(+)CD11b(+) cells that delayed diabetes development in NOD mice. Our data suggest that Gr1(+)CD11b(+) cells contribute to the establishment of immune tolerance to pancreatic islet autoimmunity. Manipulation of Gr1(+)CD11b(+) cells could be considered as a novel immunotherapy for the prevention of type 1 diabetes.

To B or not to B--pathogenic and regulatory B cells in autoimmune diabetes.

B cells have a vitally important function to produce antibodies which are directly pathogenic in some autoimmune diseases. However, it is clear that a number of other B cell functions are also critical in the pathogenesis of organ-specific autoimmune diseases that were previously thought to be mainly T cell mediated. Therapeutic agents that target B cells and their functions may therefore be of considerable importance in these autoimmune diseases. In this review, we will focus on B cell characteristics and functions that contribute to type 1 diabetes (T1D) and discuss why anti-B cell treatment may be effective in T1D, a disease that was previously considered to be primarily T cell mediated.

Innate immunity and intestinal microbiota in the development of Type 1 diabetes.

Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.

Infections with immunogenic trypanosomes reduce tsetse reproductive fitness: potential impact of different parasite strains on vector population structure.

The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.

The role of Toll-like receptors 3 and 9 in the development of autoimmune diabetes in NOD mice.

Innate immunity is mediated, at least in part, through a number of receptors known as Toll-like receptors (TLRs), which are activated by different microbial stimuli. Adaptive immunity, including autoimmunity, follows the innate response in a more specific manner. To investigate the roles of TLR3 and TLR9 in the development of type 1 diabetes, we generated NOD mice that are deficient in TLR3 and 9, respectively. There was no obvious difference in the incidence of spontaneous diabetes between TLR3-deficient mice and TLR3 heterozygous mice. However, TLR9-deficient mice were markedly protected from the disease compared to TLR9 heterozygous mice. Our results suggest that different TLRs play a varying role in autoimmune diseases.

Anti-CD20 treatment prolongs syngeneic islet graft survival and delays the onset of recurrent autoimmune diabetes.

Type 1 diabetes is an autoimmune disease characterized by T cell-mediated destruction of pancreatic islet beta cells. Pancreatic islet transplantation with long-term immunosuppressive drug treatment is an accepted therapeutic option for patients with type 1 diabetes suffering from disabling hypoglycemia on insulin treatment. Here we investigated the replacement of immunosuppressive drug treatment with immune tolerance establishment induced by temporary B cell-depletion therapy for islet transplantation. The result suggested that the combined therapy of B cell depletion and syngeneic islet transplantation may reverse the disease in hCD20/NOD mice.

Treatment with CD20-specific antibody prevents and reverses autoimmune diabetes in mice.

The precise roles of B cells in promoting the pathogenesis of type 1 diabetes remain undefined. Here, we demonstrate that B cell depletion in mice can prevent or delay diabetes, reverse diabetes after frank hyperglycemia, and lead to the development of cells that suppress disease. To determine the efficacy and potential mechanism of therapeutic B cell depletion, we generated a transgenic NOD mouse expressing human CD20 (hCD20) on B cells. A single cycle of treatment with an antibody specific for hCD20 temporarily depleted B cells and significantly delayed and/or reduced the onset of diabetes. Furthermore, disease established to the point of clinical hyperglycemia could be reversed in over one-third of diabetic mice. Why B cell depletion is therapeutic for a variety of autoimmune diseases is unclear, although effects on antibodies, cytokines, and antigen presentation to T cells are thought to be important. In B cell-depleted NOD mice, we identified what we believe is a novel mechanism by which B cell depletion may lead to long-term remission through expansion of Tregs and regulatory B cells. Our results demonstrate clinical efficacy even in established disease and identify mechanisms for therapeutic action that will guide design and evaluation of parallel studies in patients.

Replication of flock house virus in three genera of medically important insects.

Flock House Virus (family Nodaviridae, genus Alphanodavirus, FHV) was originally isolated from grass grubs Costelytra zealandica (White) (Coleoptera: Scarabaeidae) in New Zealand and belongs to a family of divided genome, plus-sense RNA insect viruses. FHV replicates in insects, a nematode, plants, and yeast. We previously reported replication of FHV in four genera of mosquitoes and expression of green fluorescent protein in Aedes aegypti (L.) produced by an FHV-based vector. We report here that FHV multiplies vigorously in vivo in the malaria vectors Anopheles gambiae Giles and An. stephensi Liston and in vitro in a cell line derived from An. gambiae. In addition, FHV multiplies extensively in two other medically important insects, the tsetse fly, Glossina morsitans morsitans Westwood, and the reduviid bug Rhodnius prolixus Stal, extending its host range to four orders of insects (Coleoptera, Lepidoptera, Diptera, and Hemiptera). The virus disseminates in all the major tissues of the insects studied. Anopheles and Glossina show mortality when FHV is injected at a dose above 10(4) plaque-forming units (pfu) or the virus accumulates to titer above 10(8) pfu. A lower dose (10(3) pfu) promotes more extensive virus multiplication and reduces mortality to < 10%. No adverse effects are observed in Ae. aegypti, Culex pipiens pipiens L., and Armigeres subalbatus (Coquillett), when injected with a dose of up to 10(7) pfu. Mosquitoes orally fed with FHV exhibited slower virus growth rate with lower mortality. Our results indicate that FHV has uniquely broad insect host range and that the virus can be used to study virus host interactions in a variety of medically important insects.

Innate immune responses regulate trypanosome parasite infection of the tsetse fly Glossina morsitans morsitans.

Tsetse flies transmit the protozoan parasite African trypanosomes, the agents of human sleeping sickness in sub-Saharan Africa. Parasite transmission in the insect is restricted by a natural resistance phenomenon (refractoriness). Understanding the mechanism of parasite resistance is important as strengthening fly's response(s) via transgenic approaches can prevent parasite transmission and lead to the development of novel vector control strategies. Here, we investigated the role of one of the two major pathways regulating innate immunity in invertebrates, the immunodeficiency (Imd) pathway, for Glossina morsitans morsitans's natural defence against Trypanosoma brucei spp. infections. We determined the molecular structure of the Imd pathway transcriptional activator Relish (GmmRel), which shows high amino acid identity and structural similarity to its Drosophila homologue. Through a double-stranded RNA-based interference approach, we showed that the pathogen-induced expression profile of the antimicrobial peptides (AMPs) attacin and cecropin is under the regulation of GmmRel. Unexpectedly, the AMP diptericin appears to be constitutively expressed in tsetse independent of the presence of the Rel factor. Through GmmRel knock-down, we could successfully block the induction of attacin and cecropin expression in the immune responsive tissues fat body and proventriculus (cardia) following microbial challenge. The midgut and salivary gland trypanosome infection prevalence, as well as the intensity of midgut parasite infections were found to be significantly higher in flies when attacin and relish expression were knocked down. Our results provide the first direct evidence for the involvement of antimicrobial peptides in trypanosome transmission in tsetse.

Requirement of the N-terminus for dimer formation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate synthase AroG of Escherichia coli.

The first regulatory step in the synthesis of aromatic amino acids is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). In Escherichia coli, the allosteric DAHPS exists as three isozymes, AroG, AroF and AroH, each independently feedback-inhibited by corresponding end product amino acids, phenylalanine, tyrosine and typtophan. Structural biological evidences have suggested that the N-terminus of AroG is involved in the formation of a putative inhibitor-binding site and feedback inhibition signal transmission. Our previous work showed that a single amino acid residue replacement Ile10Ala or deletion of 15 N-terminal amino acids could lead to a dramatic loss of AroG enzymatic activity (Hu et al. 2003). Here we demonstrate that the deletion of N-terminus prevents the enzyme from forming a dimeric structure, indicating that the N-terminus of AroG plays a critical role in the formation of the essential tight dimeric structure.