POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.

Trypanosoma brucei causes human African trypanosomiasis and sequentially expresses distinct VSGs, its major surface antigen, to achieve host immune evasion. VSGs are monoallelically expressed from subtelomeric loci, and telomere proteins regulate VSG monoallelic expression and VSG switching. T. brucei telomerase is essential for telomere maintenance, but no regulators of telomerase have been identified. T. brucei appears to lack OB fold-containing telomere-specific ssDNA binding factors that are critical for coordinating telomere G- and C-strand syntheses in higher eukaryotes. We identify POLIE as a telomere protein essential for telomere integrity. POLIE-depleted cells have more frequent VSG gene conversion-mediated VSG switching and an increased amount of telomeric circles (T-circles), indicating that POLIE suppresses DNA recombination at the telomere/subtelomere. POLIE-depletion elongates telomere 3' overhangs dramatically, indicating that POLIE is essential for coordinating DNA syntheses of the two telomere strands. POLIE depletion increases the level of telomerase-dependent telomere G-strand extension, identifying POLIE as the first T. brucei telomere protein that suppresses telomerase. Furthermore, depletion of POLIE results in an elevated telomeric C-circle level, suggesting that the telomere C-strand experiences replication stress and that POLIE may promote telomere C-strand synthesis. Therefore, T. brucei uses a novel mechanism to coordinate the telomere G- and C-strand DNA syntheses.

Trypanosoma brucei RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions.

RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1's self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Detection of multidrug resistant (MDR) bacteria in untreated waste water disposals of hospitals in Dhaka City, Bangladesh.

BACKGROUND: Emergence of MDR bacteria is a global problem and a major burden for treatment of various infectious diseases. This study was performed to detect antibiotic resistant bacteria in untreated hospital waste. METHOD: Waste water samples were collected from sewerage disposal points of two renowned hospital of Dhaka city and a total of 59 Escherichia coli and 29 Klebsiella pneumonia isolates were isolated. Antibiotic susceptibility were measured by disc diffusion method. RESULTS: Resistance among E. coli and K. pneumoniae to 10 frequently used antibiotic tested were respectively as follows: cefotaxime (CTX) 48 and 45%; ceftazidime (CAZ) 40 and 38%; ampicillin (AMP) 71 and 100%; streptomycin (S) 50 and 34%; sulfamethoxazole-trimethoprim (SXT) 58 and 28%; ciprofloxacin (CIP) 71 and 48%; kanamycin (K) 38 and 10%; chloramphenicol (C) 28 and 10%; gentamycin (CN) 19 and 16% and imipenem (IPM) 12 and 7%. Results also demonstrate that the sites that were at the disposal point of hospital waste have higher degree of resistance. High degree of resistance was observed when 23 high-resistant E. coli isolates were further tested with 15 additional antibiotics. CONCLUSION: This study revealed a significant rise of MDR bacteria in the hospital waste and underscore necessity of hospital waste treatment.

5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus.

Interferon (IFN) exerts its antiviral effect by inducing a large family of cellular genes, named interferon (IFN)-stimulated genes (ISGs). An intriguing member of this family is indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of the main branch of tryptophan (Trp) degradation, the kynurenine pathway. We recently showed that IDO strongly inhibits human parainfluenza virus type 3 (PIV3), a significant respiratory pathogen. Here, we show that 5-hydoxytryptophan (5-HTP), the first product of an alternative branch of Trp degradation and a serotonin precursor, is essential to protect virus growth against IDO in cell culture. We also show that the apparent antiviral effect of IDO on PIV3 is not due to the generation of the kynurenine pathway metabolites, but rather due to the depletion of intracellular Trp by IDO, as a result of which this rare amino acid becomes unavailable for the alternative, proviral 5-HTP pathway.

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-ß. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. IMPORTANCE: The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.