mTORC1 Inactivation Promotes Colitis-Induced Colorectal Cancer but Protects from APC Loss-Dependent Tumorigenesis

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Identification and preliminary structure-activity relationship studies of novel pyridyl sulfonamides as potential Chagas disease therapeutic agents

Chagas disease is a neglected pathology responsible for about 12,000 deaths every year across Latin America. Although six million people are infected by the Trypanosoma cruzi, current therapeutic options are limited, highlighting the need for new drugs. Here we report the preliminary structure activity relationships of a small library of 17 novel pyridyl sulfonamide derivatives. Analogues 4 and 15 displayed significant potency against intracellular amastigotes with EC₅₀ of 5.4?µM and 8.6?µM. In cytotoxicity assays using mice fibroblast L929 cell lines, both compounds indicated low toxicity with decent selectivity indices (SI) >36 and?>23 respectively. Hence these compounds represent good starting points for further lead optimization.     

Antibodies Are Generated during Infection to Coxiella burnetii Macrophage Infectivity Potentiator Protein (Cb-Mip)

Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C. burnetii macrophage infectivity potentiator protein (Cb-Mip) cloned in Escherichia coli. Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb-Mip by immunoblot analysis. In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb-Mip. This study indicates that Cb-Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip-like proteins of intracellular bacteria in human sera. Cb-Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.     

Isolates of Arthrobacter from the soils of Schirmacher Oasis,Antarctica

Summary Thirteen isolates of bacteria from the soils of Schirmacher Oasis, Antarctica have been identified as members of the genus Arthrobacter. All the isolates exhibited a rod-coccus cycle during growth; were gram positive, catalase positive, non-motile and non-fermentative; did not form endospores; and contained MK-8 (H₂) as the major menaquinone. The mole %G + C in DNA of the isolates ranged from 58% to 72%. Based on their morphology, physiology, nutritional requirements, biochemical characteristics, and mole %G + C of their DNA, the isolates were identified as A. globiformis, A. pascens and A. protophormiae. However, unlike the mesophilic isolates the Antarctic Arthrobacter could be considered to be unique as they were psychrotrophic, contained glucose and lysine in the cell wall, and did not hydrolyze starch.     

In vivo determination of Neisseria meningitidis serogroup A capsular polysaccharide by whole cell high-resolution magic angle spinning NMR spectroscopy

High resolution-magic angle spinning (HRMAS) NMR spectroscopy was applied to serogroup A Neisseria meningitidis (NMA) to determine precise structures of capsular polysaccharide (CPS) expressed on the meningococcal surface. Both the O-acetylated (OAc) NMA parent and a mynC::aphA3 OAc- mutant demonstrated characteristic CPS-derived NMR signals indicating cell-surface expression of CPS, but only the parent expressed O-3 and O-4 acetylation signals. A capsule-defective strain showed no NMR signals for CPS. The (1)H NMR HRMAS spectral patterns correlated with the purified CPS (1)H NMR profiles. HRMAS NMR can distinguish detailed complex carbohydrate structures expressed on bacteria. NMA express both O-3 and O-4 acetylated polymers but not in equimolar ratio amounts in vivo.     

Inactivation of the fibronectin-binding adhesin gene bbk32 significantly attenuates the infectivity potential of Borrelia burgdorferi

Borrelia burgdorferi, the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surface-exposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi. Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.     

A limited role for antibody in protective immunity induced by rCPAF and CpG vaccination against primary genital Chlamydia muridarum challenge

Mice deficient in B cells (μmT mice) were used to evaluate the role of antibody in enhanced chlamydial clearance and reduction of pathology afforded by vaccination with recombinant chlamydial protease-like activity factor (rCPAF). Enhanced, but comparable, chlamydial clearance was observed in μmT and wild-type (WT) mice after rCPAF+CpG vaccination. Chlamydia -induced pathology was present in mock-immunized animals, but at significantly greater levels in μmT than WT mice, whereas vaccinated μmT and WT mice exhibited similar reductions in pathology. Thus, antibodies may play a role in protection against chlamydial pathology after primary infection, but were largely dispensable in rCPAF+CpG-induced chlamydial clearance and reduction in pathology.     

A bacterial two-hybrid system that utilizes Gateway cloning for rapid screening of protein-protein interactions

Comprehensive clone sets representing the entire genome now exist for a large number of organisms. The Gateway entry clone sets are a particularly useful means to study gene function, given the ease of introduction into any Gateway-suitable destination vector. We have adapted a bacterial two-hybrid system for use with Gateway entry clone sets, such that potential interactions between proteins encoded within these clone sets can be determined by new destination vectors. We show that utilizing the Gateway clone sets for Francisella tularensis and Vibrio cholerae, known interactions between F. tularensis IglA and IglB and V. cholerae VipA and VipB could be confirmed with these destination vectors. Moreover, the introduction of unique tags into each vector allowed for visualization of the expressed hybrid proteins via Western immunoblot. This Gateway-suitable bacterial two-hybrid system provides a new tool for rapid screening of protein-protein interactions.