Briana M. Young

NOD1 and NOD2 signalling links ER stress with inflammation

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

The structure of xylem vessels in grapevine (Vitaceae) and a possible passive mechanism for the systemic spread of bacterial disease

Xylem-dwelling pathogens become systemic, suggesting that microorganisms move efficiently in the xylem. To better understand xylem pathways and how bacteria move within the xylem, vessel connectivity between stems and leaves of Vitis vinifera cv. Chardonnay and Muscadinia rotundifolia cv. Cowart was studied. Three methods were used: (1) the light-producing bacterium, Yersinia enterocolitica, (Ye) strain GY5232 was loaded into petioles and followed using X-ray film, (2) fluorescent beads were loaded and followed by microscopy, and (3) low-pressure air was pumped into leaves and extruded bubbles from cuts in submerged leaves were followed. Bacteria, beads, and air moved through long and branched xylem vessels from the petiole into the veins in leaves of both varieties. From the stem, bacteria and air traveled into primary and secondary veins of leaves one, two, and three nodes above the loading point of the bacteria or air. Particles and air could move unimpeded through single xylem vessels or multiple vessels (conduits) connected possibly through broken pit membranes from within the stem axis into leaf blades. Bacteria were also able to move long distances within minutes from stem to leaf passively without having to cross pit membranes. Such complex, open xylem conduits have not been well documented before; these findings will help elucidate mechanisms involved in the systemic spread of pathogens.

A Mechanism for Pituitary-Resistance to Thyroid Hormone (PRTH) Syndrome: a Loss in Cooperative Coactivator Contacts by Thyroid Hormone Receptor (TR)β2

Thyroid hormone receptors (TR) are hormone-modulated transcription factors that regulate overall metabolic rate, lipid utilization, heart rate, and development. TR are expressed as a mix of interrelated receptor isoforms. The TRβ2 isoform is expressed in the hypothalamus and pituitary, where it plays an important role in the feedback regulation of thyroid hormone levels. TRβ2 exhibits unique transcriptional properties that parallel the ability of this isoform to bind to certain coactivators cooperatively through multiple contact surfaces. The more peripherally expressed TRβ1 isoform, in contrast, appears to recruit these coactivators through a single contact mechanism. We report here that clusters of charged amino acids in the TR hormone-binding domain are required for this enhanced mode of coactivator recruitment and that mutations in these charge clusters, by disrupting TRβ2 coactivator binding, are a molecular basis for pituitary resistance to thyroid hormone, a disease characterized by inappropriate thyroid hormone feedback regulation. We propose that the charge clusters allow wild-type TRβ2 to assume a conformation compatible with its mode of multiple contact coactivator recruitment, whereas disruption of these charge clusters disrupts normal T(3) homeostasis by reducing TRβ2 to a TRβ1-like, single contact mode of coactivator binding.

The p160 coactivator PAS-B motif stabilizes nuclear receptor binding and contributes to isoform-specific regulation by thyroid hormone receptors.

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that play multiple roles in vertebrate endocrinology and development. TRs are expressed as a series of distinct receptor isoforms that mediate different biological functions. The TRβ2 isoform is expressed primarily in the hypothalamus, pituitary, cochlea, and retina, and displays an enhanced response to hormone agonist relative to the other TR isoforms. We report here that the unusual transcriptional properties of TRβ2 parallel the ability of this isoform to bind p160 coactivators cooperatively through multiple contact surfaces; the more broadly expressed TRβ1 isoform, in contrast, utilizes a single contact mechanism. Intriguingly, the PAS-B domain in the p160 N terminus plays a previously unanticipated role in permitting TRβ2 to recruit coactivator at limiting triiodothyronine concentrations. The PAS-B sequences also play an important role in coactivator binding by estrogen receptor-α. We propose that the PAS-B domain of the p160 coactivators is an important modulator of coactivator recruitment for a specific subset of nuclear receptors, permitting stronger transcriptional activation at lower hormone concentrations than would otherwise occur, and allowing isoform-specific mRNA splicing to customize the hormone response in different tissues.

Construction of a reporter system to study Burkholderia mallei type III secretion and identification of the BopA effector protein function in intracellular survival

Burkholderia mallei, the aetiological agent of glanders disease, is a Gram-negative facultative intracellular bacterium. Despite numerous studies, the detailed mechanism of its pathogenesis is almost unknown. The presence of a type III secretion system (TTSS) is one of the known mechanisms associated with virulence. An intact TTSS indicates that B. mallei is able to secrete proteins in response to different environmental conditions, which could play an important role in pathogenesis. Therefore, characterization of the TTSS and identification of the secreted proteins associated with bacterial pathogenesis could provide crucial information for the development of a candidate vaccine. In the current study, we used an enzymatic reporter system to establish some of the conditions enabling TTS. Construction of the TTSS bopA mutant revealed that BopA is important for B. mallei invasion and intracellular survival. Overall, our study elucidates how BopA can aid in the optimization of TTS and defines the function of TTS effectors in bacterial intracellular survival and invasion.

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.

Alteration of NCoR corepressor splicing in mice causes increased body weight and hepatosteatosis without glucose intolerance

ABSTRACT Alternative mRNA splicing is an important means of diversifying function in higher eukaryotes. Notably, both NCoR and SMRT corepressors are subject to alternative mRNA splicing, yielding a series of distinct corepressor variants with highly divergent functions. Normal adipogenesis is associated with a switch in corepressor splicing from NCoRω to NCoRδ, which appears to help regulate this differentiation process. We report here that mimicking this development switch in mice by a splice-specific whole-animal ablation of NCoRω is very different from a whole-animal or tissue-specific total NCoR knockout and produces significantly enhanced weight gain on a high-fat diet. Surprisingly, NCoRω−/− mice are protected against diet-induced glucose intolerance despite enhanced adiposity and the presence of multiple additional, prodiabetic phenotypic changes. Our results indicate that the change in NCoR splicing during normal development both helps drive normal adipocyte differentiation and plays a key role in determining a metabolically appropriate storage of excess calories. We also conclude that whole-gene “knockouts” fail to reveal how important gene products are customized, tailored, and adapted through alternative mRNA splicing and thus do not reveal all the functions of the protein products of that gene.

Phospholipase A1 Modulates the Cell Envelope Phospholipid Content of Brucella melitensis, Contributing to Polymyxin Resistance and Pathogenicity

ABSTRACT A subset of bacterial pathogens, including the zoonotic Brucella species, are highly resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed primarily to the modification of lipopolysaccharide; however, it is unknown what additional mechanisms mediate high-level resistance against this class of drugs. This work identified a role for the Brucella melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated that it is a phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, lipidomic analysis of B. melitensis revealed an excess of PE lipids in the bacterial membranes isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell envelope, BveA increases the resistance of B. melitensis to polymyxin B. BveA was required for survival and replication of B. melitensis in macrophages and for persistent infection in mice. BveA family esterases are encoded in the genomes of the alphaproteobacterial species that coexist with the polymyxin-producing bacteria in the rhizosphere, suggesting that maintenance of a low PE content in the bacterial cell envelope may be a shared persistence strategy for association with plant and mammalian hosts.

Utilization of Host Polyamines in Alternatively Activated Macrophages Promotes Chronic Infection by Brucella abortus

ABSTRACT Treatment of intracellular bacterial pathogens with antibiotic therapy often requires a long course of multiple drugs. A barrier to developing strategies that enhance antibiotic efficacy against these pathogens is our poor understanding of the intracellular nutritional environment that maintains bacterial persistence. The intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAMs); however, knowledge of the metabolic adaptations promoting exploitation of this niche is limited. Here we show that one mechanism promoting enhanced survival in AAMs is a shift in macrophage arginine utilization from production of nitric oxide (NO) to biosynthesis of polyamines, induced by interleukin 4 (IL-4)/IL-13 treatment. Production of polyamines by infected AAMs promoted both intracellular survival of B. abortus and chronic infection in mice, as inhibition of macrophage polyamine synthesis or inactivation of the putative putrescine transporter encoded by potIHGF reduced both intracellular survival in AAMs and persistence in mice. These results demonstrate that increased intracellular availability of polyamines induced by arginase-1 expression in IL-4/IL-13-induced AAMs promotes chronic persistence of B. abortus within this niche and suggest that targeting of this pathway may aid in eradicating chronic infection.