Enzyme-functionalized alginate microparticles enable anaerobic culture under ambient oxygen.

Methods for culturing oxygen-sensitive cells and organisms under anaerobic conditions are vital to biotechnology research. Here, we report a biomaterial-based platform for anaerobic culture that consists of glucose oxidase (GOX) functionalized alginate microparticles (ALG-GOX), which are designed to deplete dissolved [O2 ] through enzymatic activity. ALG-GOX microparticles were synthesized via a water-in-oil emulsion and had a size of 132.0 ± 51.4 µm. Despite having a low storage modulus, the microparticles remained stable under aqueous conditions due to covalent crosslinking through amide bonds. Enzyme activity was tunable based on the loaded GOX concentration, with a maximum activity of 3.6 ± 0.3 units/mg of microparticles being achieved at an initial loading concentration of 5 mg/mL of GOX in alginate precursor solution. High enzyme activity in ALG-GOX microparticles resulted in rapid oxygen depletion, producing a suitable environment for anaerobic culture. Microparticles loaded with both GOX and catalase (ALG-GOX-CAT) to reduce H2 O2 buildup exhibited sustained activity for potential long-term anaerobic culture. ALG-GOX-CAT microparticles were highly effective for the anaerobic culture of Bacteroides thetaiotaomicron, with 10 mg/mL of ALG-GOX-CAT microparticles supporting the same level of growth in an aerobic environment compared to an anaerobic chamber after 16 h (8.70 ± 0.96 and 10.03 ± 1.03 million CFU, respectively; N.S. p = 0.07). These microparticles could be a valuable tool for research and development in biotechnology.

Cryptosporidium parvum Elongation Factor 1α Participates in the Formation of Base Structure at the Infection Site During Invasion.

BACKGROUND: Cryptosporidium is a genus of apicomplexan parasites, the causative agents of cryptosporidiosis in humans and/or animals. Although most apicomplexans parasitize within the host cell cytosols, Cryptosporidium resides on top of host cells, but it is embraced by a double-layer parasitophorous vacuole membrane derived from host cell. There is an electron-dense band to separate the parasite from host cell cytoplasm, making it as an intracellular but extracytoplasmic parasite. However, little is known on the molecular machinery at the host cell-parasite interface. METHODS: Cryptosporidium parvum at various developmental stages were obtained by infecting HCT-8 cells cultured in vitro. Immunofluorescence assay was used to detect CpEF1α with a polyclonal antibody and host cell F-actin with rhodamine-phalloidin. Recombinant CpEF1α protein was used to evaluate its effect on the invasion by the parasite. RESULTS: We discovered that a C parvum translation elongation factor 1α (CpEF1α) was discharged from the invading sporozoites into host cells, forming a crescent-shaped patch that fully resembles the electron-dense band. At the same time, host cell F-actin aggregated to form a globular-shaped plug beneath the CpEF1α patch. The CpEF1α patch remained for most of the time but became weakened and dissolved upon the completion of the invasion process. In addition, recombinant CpEF1α protein could effectively interfere the invasion of sporozoites into host cells. CONCLUSIONS: CpEF1α plays a role in the parasite invasion by participating in the formation of electron-dense band at the base of the parasite infection site.

Novel Antiparasitic Activity of the Antifungal Lead Occidiofungin.

Novel antiparasitic activity was observed for the antifungal occidiofungin. It efficaciously and irreversibly inhibited the zoonotic enteric parasite Cryptosporidium parvumin vitro with limited cytotoxicity (50% effective concentration [EC50] = 120 nM versus 50% cytotoxic concentration [TC50] = 988 nM), and its application disrupted the parasite morphology. This study expands the spectrum of activity of a glycolipopeptide named occidiofungin. Occidiofungin has poor gastrointestinal tract absorption properties, supporting future investigations into its potential activities on other enteric parasites.

The Action of the Hexokinase Inhibitor 2-deoxy-d-glucose on Cryptosporidium parvum and the Discovery of Activities against the Parasite Hexokinase from Marketed Drugs.

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2-deoxy-d-glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG-6-phosphate (2DG6P), the anti-cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose-6-phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti-cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC50  = 0.53, 1.77, 8.1 and 165 µM, respectively). The anti-CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase.

The Existing Drug Vorinostat as a New Lead Against Cryptosporidiosis by Targeting the Parasite Histone Deacetylases.

Background: Cryptosporidiosis affects all human populations, but can be much more severe or life-threatening in children and individuals with weak or weakened immune systems. However, current options to treat cryptosporidiosis are limited. Methods: An in vitro phenotypic screening assay was employed to screen 1200 existing drugs for their anticryptosporidial activity and to determine the inhibitory kinetics of top hits. Selected top hits were further evaluated in mice. The action of the lead compound vorinostat on the parasite histone deacetylase (HDAC) was biochemically validated. Results: Fifteen compounds exhibited anticryptosporidial activity at nanomolar level in vitro. Among them, the histone deacetylase (HDAC) inhibitor vorinostat retained outstanding efficacy in vitro (half maximal effective concentration, EC50 = 203 nM) and in an interleukin 12 knockout mouse model (50% inhibition dose = 7.5 mg/kg). Vorinostat was effective on various parasite developmental stages and could irreversibly kill the parasite. Vorinostat was highly effective against the parasite native HDAC enzymes (half maximal inhibitory concentration, IC50 = 90.0 nM) and a recombinant Cryptosporidium parvum HDAC (the inhibitor constant, Ki = 123.0 nM). Conclusions: These findings suggest the potential for repurposing of vorinostat to treat cryptosporidiosis, and imply that the parasite HDAC can be explored for developing more selective anticryptosporidial therapeutics.

Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics.

The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly-if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 µM and 55.6 µM, respectively), as well as parasite growth in vitro (IC50 = 11.8 µM and 39.5 µM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available.

Multiyear Survey of Coccidia, Cryptosporidia, Microsporidia, Histomona, and Hematozoa in Wild Quail in the Rolling Plains Ecoregion of Texas and Oklahoma, USA.

We developed nested PCR protocols and performed a multiyear survey on the prevalence of several protozoan parasites in wild northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) in the Rolling Plains ecoregion of Texas and Oklahoma (i.e. fecal pellets, bird intestines and blood smears collected between 2010 and 2013). Coccidia, cryptosporidia, and microsporidia were detected in 46.2%, 11.7%, and 44.0% of the samples (n = 687), whereas histomona and hematozoa were undetected. Coccidia consisted of one major and two minor Eimeria species. Cryptosporidia were represented by a major unknown Cryptosporidium species and Cryptosporidium baileyi. Detected microsporidia species were highly diverse, in which only 11% were native avian parasites including Encephalitozoon hellem and Encephalitozoon cuniculi, whereas 33% were closely related to species from insects (e.g. Antonospora, Liebermannia, and Sporanauta). This survey suggests that coccidia infections are a significant risk factor in the health of wild quail while cryptosporidia and microsporidia may be much less significant than coccidiosis. In addition, the presence of E. hellem and E. cuniculi (known to cause opportunistic infections in humans) suggests that wild quail could serve as a reservoir for human microsporidian pathogens, and individuals with compromised or weakened immunity should probably take precautions while directly handling wild quail.

Seroprevalence of five parasitic pathogens in pregnant women in ten Caribbean countries.

To date, published epidemiological studies of parasitic infections in humans in the Caribbean region are very limited. Here, we report the seroprevalence of five parasitic pathogens, including Ascaris lumbricoides, Entamoeba histolytica, Giardia lamblia, Schistosoma mansoni, and Toxocara canis in 435 serum samples collected between 2008 and 2011 from pregnant women in ten Caribbean islands. We tested the serum samples for IgG antibodies against the five parasites by enzyme-linked immunosorbent assay (ELISA). Among them, 66.2 % were serologically positive for at least one parasite. The most prevalent parasite was G. lamblia (40.5 %), followed by A. lumbricoides (37.9 %), T. canis (14.5 %), E. histolytica (6.7 %), and S. mansoni (3.0 %). Evidence of infections of G. lamblia and A. lumbricoides were detected in all ten Caribbean countries. Seroprevalence estimates significantly differed between countries for A. lumbricoides, E. histolytica, and T. canis (p values <0.001). For S. mansoni, significance was observed by Fisher's exact test (p = 0.013) but not by multiple comparisons. The prevalence of G. lamblia was not significantly different between countries (p = 0.089). A significant negative correlation between the gross domestic product (GDP) per capita and overall seroprevalence by country was also observed (Pearson's r = -0.9202, p = 0.0002). The data strongly indicates that neglected parasitic infections remain a significant health burden on people in these countries. Thus, justification has been provided to regional health planners to enhance existing public health surveillance programs on parasitic diseases and to heighten the public's awareness through education and outreach programs on how they can minimize the occurrence of parasitic infections.

Differential Gene Expression and Protein Localization of Cryptosporidium parvum Fatty Acyl-CoA Synthetase Isoforms.

Cryptosporidium parvum is unable to synthesize fatty acids de novo, but possesses three long-chain fatty acyl-CoA synthetase (CpACS) isoforms for activating fatty acids. We have recently shown that these enzymes could be targeted to kill the parasite in vitro and in vivo. Here, we demonstrated that the CpACS genes were differentially expressed during the parasite life cycle, and their proteins were localized to different subcellular structures by immunofluorescence and immuno-electron microscopies. Among them, CpACS1 displayed as an apical protein in sporozoites and merozoites, but no or little presence during the intracellular merogony until the release of merozoites, suggesting that CpACS1 probably functioned mainly during the parasite invasion and/or early stage of intracellular development. Both CpACS2 and CpACS3 proteins were present in all parasite life cycle stages, in which CpACS2 was present in the parasite and the parasitophorous vacuole membranes (PVM), whereas CpACS3 was mainly present in the parasite plasma membranes with little presence in the PVM. These observations suggest that CpACS2 and CpACS3 may participate in scavenging and transport of fatty acids across the PVM and the parasite cytoplasmic membranes, respectively.

Amelioration of Cryptosporidium parvum infection in vitro and in vivo by targeting parasite fatty acyl-coenzyme A synthetases.

BACKGROUND: Cryptosporidium is emerging as 1 of the 4 leading diarrheal pathogens in children in developing countries. Its infections in patients with AIDS can be fatal, whereas fully effective treatments are unavailable. The major goal of this study is to explore parasite fatty acyl-coenzyme A synthetase (ACS) as a novel drug target. METHODS: A colorimetric assay was developed to evaluate biochemical features and inhibitory kinetics of Cryptosporidium parvum ACSs using recombinant proteins. Anticryptosporidial efficacies of the ACS inhibitor triacsin C were evaluated both in vitro and in vivo. RESULTS: Cryptosporidium ACSs displayed substrate preference toward long-chain fatty acids. The activity of parasite ACSs could be specifically inhibited by triacsin C with the inhibition constant Ki in the nanomolar range. Triacsin C was highly effective against C. parvum growth in vitro (median inhibitory concentration, 136 nmol/L). Most importantly, triacsin C effectively reduced parasite oocyst production up to 88.1% with no apparent toxicity when administered to Cryptosporidium-infected interleukin 12 knockout mice at 8-15 mg/kg/d for 1 week. CONCLUSIONS: The findings of this study not only validated Cryptosporidium ACS (and related acyl-[acyl-carrier-protein]-ligases) as pharmacological targets but also indicate that triacsin C and analogues can be explored as potential new therapeutics against the virtually untreatable cryptosporidial infection in immunocompromised patients.

Molecular cloning and differential expression in tissues of a tyrosinase gene in the Pacific oyster Crassostrea gigas.

Tyrosinases are a group of type-3 copper proteins that catalyze the first two reactions in the melanin biosynthesis in organisms ranging from bacteria, fungi, plants to animals. Tyrosinases are not only involved in pigmentation, but also play an important role in the innate immunity in invertebrates. Additionally, tyrosinases are also known to be involved in the biogenesis and pigmentation of shells. The recently published Crassostrea gigas genome sequences revealed that the Pacific oyster possesses at least 26 tyrosinase isoforms. However, their molecular features are largely understudied. In fact, the full-length mRNA sequence was determined for one of the tyrosinase genes (i.e., CgTry1; aka cgi-tyr1). Here we report the full-length transcript of a second C. gigas tyrosinase (CgTyr2) sequence and the determination of its sequence features characteristic to the tyrosinase family proteins. We also showed that CgTyr2 gene was differentially expressed with the highest level of expression in mantle edges, suggesting its potential role in the formation of periostracum/pigmentation. Our comprehensive phylogenetic reconstructions supported that hemocyanins possibly evolved from a tyrosinase by an ancient gene duplication followed by functional differentiation, and the current large number of tyrosinase isoforms in C. gigas and other mollusks were originated from multiple gene duplication events that took places before and after mollusk species were established.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

Gene discovery, evolutionary affinity and molecular detection of Oxyspirura petrowi, an eye worm parasite of game birds.

BACKGROUND: Oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact Northern Bobwhite quail individuals and populations within Texas and other regions of the United States. Despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of O. petrowi. To fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18S rRNA and the intergenic region between the 18S and 28S rRNA genes, studied its phylogenetic affinity, and developed a PCR protocol for the detection of this eye worm. RESULTS: We have generated ~240 kb of genome sequence data derived from 348 clones by a random genome survey of an O. petrowi genomic library. The eye worm genome is AT-rich (i.e., 62.2% AT-content), and contains a high number of microsatellite sequences. The discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. Phylogenetic analysis based on 18S rRNA sequences indicate that the Spiruroidea is paraphyletic, in which Oxyspirura and its closely related species are sisters to the filarial nematodes. We have also developed a PCR protocol based on the ITS2 sequence that allows sensitive and specific detection of eye worm DNA in feces. Using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from Northern Bobwhites and Scaled Quail in Texas in the spring of 2013 are O. petrowi positive. CONCLUSIONS: The O. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. This eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as Loa loa would be referential in developing treatments for the Thelazoidea parasites. Our qPCR-based survey has confirmed that O. petrowi infection is of potential concern to quail managers in Texas.

Engineering live attenuated vaccines: Old dogs learning new tricks.

Autoimmune diseases such as rheumatoid arthritis and type 1 diabetes are increasingly common global problems. Concerns about increases in the prevalence of such diseases and the limited efficacy of conventional treatment regimens necessitates new therapies to address these challenges. Autoimmune disease severity and dysbiosis are interconnected. Although probiotics have been established as a therapy to rebalance the microbiome and suppress autoimmune symptoms, these microbes tend to lack a number of advantageous qualities found in non-commensal bacteria. Through attenuation and genetic manipulation, these non-commensal bacteria have been engineered into recombinant forms that offer malleable platforms capable of addressing the immune imbalances found in RA and T1D. Such bacteria have been engineered to express valuable gene products known to suppress autoimmunity such as anti-inflammatory cytokines, autoantigens, and enzymes synthesizing microbial metabolites. This review will highlight current and emerging trends in the field and discuss how they may be used to prevent and control autoimmune diseases.

An automated system for interrogating the evolution of microbial endosymbiosis.

Inter-kingdom endosymbiotic interactions between bacteria and eukaryotic cells are critical to human health and disease. However, the molecular mechanisms that drive the emergence of endosymbiosis remain obscure. Here, we describe the development of a microfluidic system, named SEER (S̲ystem for the E̲volution of E̲ndosymbiotic R̲elationships), that automates the evolutionary selection of bacteria with enhanced intracellular survival and persistence within host cells, hallmarks of endosymbiosis. Using this system, we show that a laboratory strain of Escherichia coli that initially possessed limited abilities to survive within host cells, when subjected to SEER selection, rapidly evolved to display a 55-fold enhancement in intracellular survival. Notably, molecular dissection of the evolved strains revealed that a single-point mutation in a flexible loop of CpxR, a gene regulator that controls bacterial stress responses, substantially contributed to this intracellular survival. Taken together, these results establish SEER as the first microfluidic system for investigating the evolution of endosymbiosis, show the importance of CpxR in endosymbiosis, and set the stage for evolving bespoke inter-kingdom endosymbiotic systems with novel or emergent properties.

Involvement of host cell integrin α2 in Cryptosporidium parvum infection.

Cryptosporidium parvum is an opportunistic pathogen in AIDS patients. It is an intracellular but extracytoplasmic parasite residing in a host cell-derived parasitophorous vacuole. It is still poorly understood how this parasite interacts with host cells. We observed that expression of the integrin α2 (ITGA2) gene in host cells was significantly upregulated upon C. parvum infection, and a higher level of ITGA2 protein was present in the parasite infection sites. The infection could be reduced by the treatment of antibodies against ITGA2 and integrin ß1 (ITGB1) subunits, as well as by type I collagen (an integrin α2ß1 ligand). We also generated stable knockdown of ITGA2 gene expression in HCT-8 cells and observed consistent reduction of parasite infection in these knockdown cells. Collectively, our evidence indicates that host cell ITGA2 might be involved in interacting with Cryptosporidium during infection, probably acting as part of the regulatory elements upstream of the reported recruiting and reorganization of F actin at the infection sites.

Transcriptome analysis reveals unique metabolic features in the Cryptosporidium parvum Oocysts associated with environmental survival and stresses.

BACKGROUND: Cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. Little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. Here we report the development of the first Agilent microarray for C. parvum (CpArray15K) that covers all predicted ORFs in the parasite genome. Global transcriptome analysis using CpArray15K coupled with real-time qRT-PCR uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite. RESULTS: Oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. The proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. Energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase (LDH) gene. We also studied parasite responses to UV-irradiation, and observed complex and dynamic regulations of gene expression. Notable changes included increased transcript levels of genes involved in DNA repair and intracellular trafficking. Among the stress-related genes, TCP-1 family members and some thioredoxin-associated genes appear to play more important roles in the recovery of UV-induced damages in the oocysts. Our observations also suggest that UV irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking. CONCLUSIONS: CpArray15K is the first microarray chip developed for C. parvum, which provides the Cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. CpArray15K has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to UV-irradiation. These observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as UV irradiation.

3-(4-Bromo-phen-yl)-1-(4-chloro-benz-yl)-1H-pyrazole-5-carbaldehyde.

The title compound, C(17)H(12)BrClN(2)O, was synthesized by oxidation of [3-(4-bromo-phen-yl)-1-(4-chloro-benz-yl)-1H-pyrazol-5-yl]methanol under mild conditions. The pyrazole ring makes dihedral angles of 3.29 (9) and 74.91 (4)°, respectively, with the bromo-phenyl and chloro-phenyl rings.

Live attenuated bacterium limits cancer resistance to CAR-T therapy by remodeling the tumor microenvironment.

The tumor microenvironment (TME) is characterized by the activation of immune checkpoints, which limit the ability of immune cells to attack the growing cancer. To overcome immune suppression in the clinic, antigen-expressing viruses and bacteria have been developed to induce antitumor immunity. However, the safety and targeting specificity are the main concerns of using bacteria in clinical practice as antitumor agents. In our previous studies, we have developed an attenuated bacterial strain (Brucella melitensis 16M ∆vjbR, henceforth Bm∆vjbR) for clinical use, which is safe in all tested animal models and has been removed from the select agent list by the Centers for Disease Control and Prevention. In this study, we demonstrated that Bm∆vjbR homed to tumor tissue and improved the TME in a murine model of solid cancer. In addition, live Bm∆vjbR promoted proinflammatory M1 polarization of tumor macrophages and increased the number and activity of CD8+ T cells in the tumor. In a murine colon adenocarcinoma model, when combined with adoptive transfer of tumor-specific carcinoembryonic antigen chimeric antigen receptor CD8+ T cells, tumor cell growth and proliferation was almost completely abrogated, and host survival was 100%. Taken together, these findings demonstrate that the live attenuated bacterial treatment can defeat cancer resistance to chimeric antigen receptor T-cell therapy by remodeling the TME to promote macrophage and T cell-mediated antitumor immunity.

A metabolically engineered bacterium controls autoimmunity and inflammation by remodeling the pro-inflammatory microenvironment.

Immunotherapy has led to impressive advances in the treatment of autoimmune and pro-inflammatory disorders; yet, its clinical outcomes remain limited by a variety of factors including the pro-inflammatory microenvironment (IME). Discovering effective immunomodulatory agents, and the mechanisms by which they control disease, will lead to innovative strategies for enhancing the effectiveness of current immunotherapeutic approaches. We have metabolically engineered an attenuated bacterial strain (i.e., Brucella melitensis 16M ∆vjbR, Bm∆vjbR::tnaA) to produce indole, a tryptophan metabolite that controls the fate and function of regulatory T (Treg) cells. We demonstrated that treatment with Bm∆vjbR::tnaA polarized macrophages (Mφ) which produced anti-inflammatory cytokines (e.g., IL-10) and promoted Treg function; moreover, when combined with adoptive cell transfer (ACT) of Treg cells, a single treatment with our engineered bacterial strain dramatically reduced the incidence and score of autoimmune arthritis and decreased joint damage. These findings show how a metabolically engineered bacterium can constitute a powerful vehicle for improving the efficacy of immunotherapy, defeating autoimmunity, and reducing inflammation by remodeling the IME and augmenting Treg cell function.