Down regulation of Tim50 in Trypanosoma brucei increases tolerance to oxidative stress.

Trypanosoma brucei, the causative agent for African trypanosomiasis, possesses a single mitochondrion that imports hundreds of proteins from the cytosol. However, the parasite only possesses a few homologs of the canonical protein translocases found in fungi and animals. We recently characterized a homolog of the translocase of the mitochondrial inner membrane, Tim50, in T. brucei. TbTim50 knockdown (KD) moderately reduced cell growth, decreased the mitochondrial membrane potential, and inhibited import of proteins into mitochondria. In contrast to Tim50 KD, we show here that TbTim50 overexpression (OE) increased the mitochondrial membrane potential as well as increased the production of cellular reactive oxygen species (ROS). Therefore, TbTim50 OE also inhibits cell growth. In addition, TbTim50 OE and KD cells showed different responses upon treatment with H2O2. Surprisingly, TbTim50 KD cells showed a greater tolerance to oxidative stress. Further analysis revealed that TbTim50 KD inhibits transition of cells from an early to late apoptotic stage upon exposure to increasing concentrations of H2O2. On the other hand TbTim50 OE caused cells to be in a pro-apoptotic stage and thus they underwent increased cell death upon H2O2 treatment. However, externally added H2O2 similarly increased the levels of cellular ROS and decreased the mitochondrial membrane potential in both cell types, indicating that tolerance to ROS is mediated through induction of the stress-response pathway due to TbTim50 KD. Together, these results suggest that TbTim50 acts as a stress sensor and that down regulation of Tim50 could be a survival mechanism for T. brucei exposed to oxidative stress.

Tim50 in Trypanosoma brucei possesses a dual specificity phosphatase activity and is critical for mitochondrial protein import.

In eukaryotes, proteins are imported into mitochondria via multiprotein translocases of the mitochondrial outer and inner membranes, TOM and TIM, respectively. Trypanosoma brucei, a hemoflagellated parasitic protozoan and the causative agent of African trypanosomiasis, imports about a thousand proteins into the mitochondrion; however, the mitochondrial protein import machinery in this organism is largely unidentified. Here, we characterized a homolog of Tim50 that is localized in the mitochondrial membrane in T. brucei. Similar to Tim50 proteins from fungi and mammals, Tim50 in T. brucei (TbTim50) possesses a mitochondrial targeting signal at its N terminus and a C-terminal domain phosphatase motif at its C terminus. Knockdown of TbTim50 reduced cell growth and inhibited import of proteins that contain N-terminal targeting signals. Co-immunoprecipitation analysis revealed that TbTim50 interacts with TbTim17. Unlike its fungal counterpart but similar to the human homolog of Tim50, recombinant TbTim50 possesses a dual specificity phosphatase activity with a greater affinity for protein tyrosine phosphate than for protein serine/threonine phosphate. Mutation of the aspartic acid residues to alanine in the C-terminal domain phosphatase motif (242)DXDX(V/T)(246) abolished activity for both type of substrates. TbTim50 knockdown increased and its overexpression decreased the level of voltage-dependent anion channel (VDAC). However, the VDAC level was unaltered when the phosphatase-inactive mutant of TbTim50 was overexpressed, suggesting that the phosphatase activity of TbTim50 plays a role in regulation of VDAC expression. In contrast, phosphatase activity of the TbTim50 is required neither for mitochondrial protein import nor for its interaction with TbTim17. Overall, our results show that TbTim50 plays additional roles in mitochondrial activities besides preprotein translocation.

Antimicrobial susceptibility testing of Shiga toxin-producing Escherichia coli from various samples by using a spiral gradient endpoint technique.

Shiga toxin-producing Escherichia coli (STEC) remains a major public health concern. Microbial resistance may be due to use of antimicrobial agents (AAs) as a growth promoter in food animals or overuse of AAs in humans. The objective of the current study was to determine the antimicrobial susceptibility patterns of STEC strains isolated from food, veterinary, and clinical sources against 14 AAs by using the spiral gradient endpoint method. One hundred ten isolates from three sources were characterized. Results of the current study showed that all strains were resistant to the folate pathway inhibiting AAs including tylosin tartrate (gradient minimum inhibitory concentration [GMIC] ranges from ≥180.00 to 256.00 µg/mL; end concentration [EC] ranges from ≥130.00 to 151.22 µg/mL; and tail-end concentration [TEC] ≥145.00 µg/mL). All the strains isolated from three sources were susceptible to the fluoroquinolone class of AAs (GMIC ranges from ≤1.00 to 64.30 µg/mL; EC ranges from ≤3.33 to 72.00 µg/mL; and TEC ranges from ≤12.13 to 45.00 µg/mL). Among the food isolates, less resistance was found within the aminoglycoside and amphenicol group (GMIC ≥256.00 µg/mL; EC=161.00 µg/mL). Eight strains were resistant to one to three, 44 strains were resistant to four to six, and two strains were resistant to seven or more AAs. All the clinical isolates (100%) were susceptible to the fluoroquinolones and gentamycin. Results also showed that antimicrobial resistance was observed between four and six AAs among the isolates. Some veterinary isolates were resistant to five AAs. Least AAs resistance was shown by 3.7% of isolates to gentamycin and 7.45% to chloramphenicol. This study showed an increasing trend of antimicrobial resistant strains of STEC, and we suggest that periodic surveillance of the antimicrobial susceptibility may be a useful measure to detect the antimicrobial resistant pathogens.

Determination of antimicrobial activity of sorrel (Hibiscus sabdariffa) on Escherichia coli O157:H7 isolated from food, veterinary, and clinical samples.

The use of medicinal plants as natural antimicrobial agents is gaining popularity. Sorrel (Hibiscus sabdariffa) is widely used for the treatment of diseases. The objective of this study was to investigate the antimicrobial activity of sorrel on Escherichia coli O157:H7 isolates from food, veterinary, and clinical samples. Phenolics of the calyces were extracted from 10 g of ground, freeze-dried samples using 100 mL of 80% aqueous methanol. Concentrations of 10%, 5%, and 2.5% methanol extract of sorrel were investigated for its antimicrobial activity. Inhibition zones were indicated by a lack of microbial growth due to inhibitory concentrations of sorrel diffused into semisolid culture medium beneath the sorrel-impregnated disk. The results of this experiment showed that the most potent sorrel concentration was 10%, then 5%, and finally 2.5%. The overall mean zone of inhibition for the sorrel extract was 12.66 mm for 10%, 10.75 mm for 5%, and 8.9 mm for 2.5%. The highest inhibition zones (11.16 mm) were observed in veterinary samples, and the lowest (10.57 mm) in the food samples. There were significant (P<.05) differences among mean zones of inhibition found in the food, veterinary, and clinical sources. Based on the source of samples and concentration of sorrel extract, the lowest mean inhibition was 7.00±0.04 mm from clinical samples, and the highest was 15.37±0.61 mm from a food source. These findings indicated that sorrel was effective at all levels in inhibiting E. coli O157:H7; thus it possesses antimicrobial activity and hold great promise as an antimicrobial agent.