Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.     

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.     

Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes

Fatty acid translocase (FAT)/CD36 is one of several putative plasma membrane long-chain fatty acid (LCFA) transport proteins; however, its role in intestinal absorption of LCFA is unknown. We hypothesized that FAT/CD36 would be differentially expressed along the longitudinal axis of the gut and during intestinal development, suggesting specificity of function. We found that intestinal mucosal FAT/CD36 mRNA levels varied by anatomic location along the longitudinal gut axis: stomach 45 +/- 7, duodenum 173 +/- 29, jejunum 238 +/- 17, ileum 117 +/- 14, and colon 9 +/- 1% (means +/- SE with 18S mRNA as control). FAT/CD36 protein levels were also higher in proximal compared with distal intestinal mucosa. Mucosal FAT/CD36 mRNA was also regulated during intestinal maturation, with a fourfold increase from neonatal to adult animals. In addition, FAT/CD36 mRNA levels and enterocyte LCFA uptake were rapidly downregulated by intraduodenal oleate infusion. These findings suggest that FAT/CD36 plays a role in the uptake of LCFA by small intestinal enterocytes. This may have important implications in understanding fatty acid absorption in human physiological and pathophysiological conditions.     

Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin.     

Microfluidics-based diagnostics of infectious diseases in the developing world

One of the great challenges in science and engineering today is to develop technologies to improve the health of people in the poorest regions of the world. Here we integrated new procedures for manufacturing, fluid handling and signal detection in microfluidics into a single, easy-to-use point-of-care (POC) assay that faithfully replicates all steps of ELISA, at a lower total material cost. We performed this 'mChip' assay in Rwanda on hundreds of locally collected human samples. The chip had excellent performance in the diagnosis of HIV using only 1 μl of unprocessed whole blood and an ability to simultaneously diagnose HIV and syphilis with sensitivities and specificities that rival those of reference benchtop assays. Unlike most current rapid tests, the mChip test does not require user interpretation of the signal. Overall, we demonstrate an integrated strategy for miniaturizing complex laboratory assays using microfluidics and nanoparticles to enable POC diagnostics and early detection of infectious diseases in remote settings.     

The adipocyte as an important target cell for Trypanosoma cruzi infection

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.