Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location-specific mitochondrial targeting signals and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, targeting signals of multi-pass Tims are less defined. Here, we report the characterization of the targeting signals of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy, we located at least two internal targeting signals (ITS): (i) within TM1 (31-50 AAs) and (ii) TM4 + loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the ITS for a multipass inner membrane protein in a divergent eukaryote, like T. brucei.IMPORTANCEAfrican trypanosomiasis (AT) is a deadly disease in human and domestic animals, caused by the parasitic protozoan Trypanosoma brucei. Therefore, AT is not only a concern for human health but also for economic development in the vast area of sub-Saharan Africa. T. brucei possesses a single mitochondrion per cell that imports hundreds of nuclear-encoded mitochondrial proteins for its functions. T. brucei Tim17 (TbTim17), an essential component of the TbTIM17 complex, is a nuclear-encoded protein; thus, it is necessary to be imported from the cytosol to form the TbTIM17 complex. Here, we demonstrated that the internal targeting signals within the transmembrane 1 (TM1) and TM4 with loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block TbTim17 function and parasite growth.

Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location specific mitochondrial targeting signals (MTSs) and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, MTSs of multi-pass Tims are less defined. Here we report the characterization of the MTSs of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy we located at least two internal signals, 1) within TM1 (31-50 AAs) and 2) TM4 + Loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K 122 ) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the internal mitochondrial targeting signals (ITS) for a multipass inner membrane protein in a divergent eukaryote, like T. brucei . Summary: Internal targeting signals within the TM1, TM4 with Loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block parasite growth.

Effects of Lactobacillus reuteri and Streptomyces coelicolor on Growth Performance of Broiler Chickens.

There are well documented complications associated with the continuous use of antibiotics in the poultry industry. Over the past few decades, probiotics have emerged as viable alternatives to antibiotics; however, most of these candidate probiotic microorganisms have not been fully evaluated for their effectiveness as potential probiotics for poultry. Recent evaluation of a metagenome of broiler chickens in our laboratory revealed a prevalence of Lactobacillus reuteri (L. reuteri) and Actinobacteria class of bacteria in their gastrointestinal tract. In this study Lactobacillus reuteri and Streptomyces coelicolor (S. coelicolor) were selected as probiotic bacteria, encapsulated, and added into broiler feed at a concentration of 100 mg/kg of feed. In an 8-week study, 240 one day-old chicks were randomly assigned to four dietary treatments. Three dietary treatments contained two probiotic bacteria in three different proportions (L. reuteri and S. coelicolor individually at 100 ppm, and mixture of L. reuteri and S. coelicolor at 50 ppm each). The fourth treatment had no probiotic bacteria and it functioned as the control diet. L. reuteri and S. coelicolor were added to the feed by using wheat middlings as a carrier at a concentration of 100 ppm (100 mg/kg). Chickens fed diets containing L. reuteri and S. coelicolor mixture showed 2% improvement in body weight gain, 7% decrease in feed consumption, and 6-7% decrease in feed conversion ratios. This research suggests that L. reuteri and S. coelicolor have the potential to constitute probiotics in chickens combined or separately, depending on the desired selection of performance index.

Expression of Potential Regulatory Genes in Abdominal Adipose Tissue of Broiler Chickens during Early Development.

The identities of genes that underlie population variation in adipose tissue development in farm animals are poorly understood. Previous studies in our laboratory have suggested that increased fat tissue involves the expression modulation of an array of genes in broiler chickens. Of special interest are eight genes, FGFR3, EPHB2, IGFBP2, GREM1, TNC, COL3A1, ACBD7, and SCD. To understand their expression regulation and response to dietary manipulation, we investigated their mRNA levels after dietary manipulation during early development. Chickens were fed either a recommended standard or a high caloric diet from hatch to eight weeks of age (WOA). The high caloric diet markedly affected bodyweight of the broiler birds. mRNA levels of the eight genes in the abdominal adipose tissue were assayed at 2, 4, 6, and 8 WOA using RT-qPCR. Results indicate that (1) FGFR3 mRNA level was affected significantly by diet, age, and diet:age interaction; (2) COL3A mRNA level was repressed by high caloric diet; (3) mRNA levels of EPHB2, ACBD7, and SCD were affected by age; (4) mRNA level of TNC was modulated by age:diet interaction; (5) changes in GREM1 and IGFBP2 mRNA levels were not statistically different.