Characterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole

Objectives The characterization of Giardia lamblia WB C6 strains resistant to metronidazole and to the nitro-thiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] as the parent compound of thiazolides, a novel class of anti-infective drugs with a broad spectrum of activities against a wide variety of helminths, protozoa and enteric bacteria. Methods Issuing from G. lamblia WB C6, we have generated two strains exhibiting resistance to nitazoxanide (strain C4) and to metronidazole (strain C5) and determined their susceptibilities to both drugs. Using quantitative RT-PCR, we have analysed the expression of genes that are potentially involved in resistance formation, namely genes encoding pyruvate oxidoreductases (POR1 and POR2), nitroreductase (NR), protein disulphide isomerases (PDI2 and PDI4) and variant surface proteins (VSPs; TSA417). We have cloned and expressed PDI2 and PDI4 in Escherichia coli. Using an enzyme assay based on the polymerization of insulin, we have determined the activities of both enzymes in the presence and absence of nitazoxanide. Results Whereas C4 was cross-resistant to nitazoxanide and to metronidazole, C5 was resistant only to metronidazole. Transcript levels of the potential targets for nitro-drugs POR1, POR2 and NR were only slightly modified, PDI2 transcript levels were increased in both resistant strains and PDI4 levels in C4. This correlated with the findings that the functional activities of recombinant PDI2 and PDI4 were inhibited by nitazoxanide. Moreover, drastic changes were observed in VSP gene expression. Conclusions These results suggest that resistance formation in Giardia against nitazoxanide and metronidazole is linked, and possibly mediated by, altered gene expression in drug-resistant strains compared with non-resistant strains of Giardi

Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli

Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism. In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model. To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings

Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli

Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism. In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model. To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings

Ribosome standby sites and other structural aspects of translation initiation regions in Escherichia coli

Translation initiation, which is rate-limiting in protein synthesis, is often the step at which regulation occurs. Here, we investigated several mechanisms of translation initiation in Escherichia coli, including their control. First, we showed that translation of the transcriptional regulator CsgD is inhibited by two sRNAs through a direct antisense mechanism. In some bacterial mRNAs, the ribosome binding site (RBS) is sequestered in a stable structure, which generally generates very low protein output. Yet, these mRNAs are often efficiently translated, which suggested the requirement for “ribosome standby sites”. Here, we investigated the structure and sequence features of an effective standby site using plasmid-borne GFP reporter constructs, and showed that relatively short, single-stranded regions near a structurally sequestered RBS can profoundly increase translation rates. Both the length and the sequence of these single-stranded regions are important for standby site efficiency, and the standby site needs to be single-stranded. This work serves as a proof-of-principle study of the ribosome standby model. To investigate the sequence-dependency of standby sites further, we used an unbiased approach, creating plasmid libraries containing millions of different standby sites in the same reporter plasmid as before. Cells were sorted by fluorescence according to translational levels, and standby sites analyzed by deep sequencing. This analysis showed that efficient standby sites have a low GC-content and rarely contain Shine-Dalgarno sequences. Additionally, nucleotides near the 3’-border of the standby region affect translation efficiency more than those closer to the 5’-end. Mutational and structure-probing experiments are planned to verify these findings

Unstructured 5'-tails act through ribosome standby to override inhibitory structure at ribosome binding sites.

Initiation is the rate-limiting step in translation. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. The ribosome standby model of de Smit and van Duin, based on studies of the MS2 phage coat cistron, proposed how high translation rates can be reconciled with stable, inhibitory structures at an RBS. Here, we revisited the coat protein system and assessed the translation efficiency from its sequestered RBS by introducing standby mutations. Further experiments with gfp reporter constructs assessed the effects of 5-tails-as standby sites-with respect to length and sequence contributions. In particular, combining in vivo and in vitro assays, we can show that tails of CA-dinucleotide repeats-and to a lesser extent, AU-repeats-dramatically increase translation rates. Tails of increasing length reach maximal rate-enhancing effects at 16-18 nucleotides. These standby tails are single-stranded and do not exert their effect by structure changes in the neighboring RBS stem-loop. In vitro translation and toeprinting assays furthermore demonstrate that standby effects are exerted at the level of translation initiation. Finally, as expected, destabilizing mutations within the coat RBS indicate an interplay with the effects of standby tails

Characterization of a Giardia lamblia WB C6 clone resistant to the isoflavone formononetin

Giardia lamblia is a common intestinal-dwelling protozoan and causes diarrhoea in humans and animals worldwide. For several years, a small number of drugs such as the 5-nitroimidazole metronidazole (MET) or the thiazolide nitazoxanide (NTZ) have been used for chemotherapy against giardiasis. However, various pre-clinical and clinical investigations revealed that antigiardial chemotherapy may be complicated by emergence of giardial resistance to these drugs. The present study addressed the question if isoflavones with antigiardial activity, such as daidzein (DAI) or formononetin (FOR), may serve as alternative compounds for treatment of giardiasis. For this purpose, the potential of G. lamblia clone WB C6 to form resistance to FOR and related isoflavones was tested in vitro. In the line of these experiments, a clone (C3) resistant to isoflavones, but sensitive to MET and NTZ, was generated. Affinity chromatography on DAI-agarose using cell-free extracts of G. lamblia trophozoites resulted in the isolation of a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). In a nucleoside hydrolase assay, recombinant NH hydrolysed all nucleosides with a preference for purine nucleosides and was inhibited by isoflavones. Using quantitative RT-PCR, the expression of genes that are potentially involved in resistance formation was analysed, namely NH and genes encoding variant surface proteins (VSPs, TSA417). The transcript level of the potential target NH was found to be significantly reduced in C3. Moreover, drastic changes were observed in VSP gene expression. This may indicate that resistance formation in Giardia against isoflavones is linked to, and possibly mediated by, altered gene expression. Taken together, our results suggest FOR or related isoflavones as an alternative antigiardial agent to overcome potential problems of resistance to drugs like MET or NTZ. However, the capacity of Giardia to develop resistance to isoflavones can potentially interfere with this alternative treatment of the disease

Characterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole

OBJECTIVES: The characterization of Giardia lamblia WB C6 strains resistant to metronidazole and to the nitro-thiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] as the parent compound of thiazolides, a novel class of anti-infective drugs with a broad spectrum of activities against a wide variety of helminths, protozoa and enteric bacteria. METHODS: Issuing from G. lamblia WB C6, we have generated two strains exhibiting resistance to nitazoxanide (strain C4) and to metronidazole (strain C5) and determined their susceptibilities to both drugs. Using quantitative RT-PCR, we have analysed the expression of genes that are potentially involved in resistance formation, namely genes encoding pyruvate oxidoreductases (POR1 and POR2), nitroreductase (NR), protein disulphide isomerases (PDI2 and PDI4) and variant surface proteins (VSPs; TSA417). We have cloned and expressed PDI2 and PDI4 in Escherichia coli. Using an enzyme assay based on the polymerization of insulin, we have determined the activities of both enzymes in the presence and absence of nitazoxanide. RESULTS: Whereas C4 was cross-resistant to nitazoxanide and to metronidazole, C5 was resistant only to metronidazole. Transcript levels of the potential targets for nitro-drugs POR1, POR2 and NR were only slightly modified, PDI2 transcript levels were increased in both resistant strains and PDI4 levels in C4. This correlated with the findings that the functional activities of recombinant PDI2 and PDI4 were inhibited by nitazoxanide. Moreover, drastic changes were observed in VSP gene expression. CONCLUSIONS: These results suggest that resistance formation in Giardia against nitazoxanide and metronidazole is linked, and possibly mediated by, altered gene expression in drug-resistant strains compared with non-resistant strains of Giardia

Blood pressure influences end-stage renal disease of Cd151 knockout mice

Podocytes of the kidney adhere tightly to the underlying glomerular basement membrane (GBM) in order to maintain a functional filtration barrier. The clinical importance of podocyte binding to the GBM via an integrin-laminin-actin axis has been illustrated in models with altered function of α3β1 integrin, integrin-linked kinase, laminin-521, and α-actinin 4. Here we expanded on the podocyte-GBM binding model by showing that the main podocyte adhesion receptor, integrin α3β1, interacts with the tetraspanin CD151 in situ in humans. Deletion of Cd151 in mouse glomerular epithelial cells led to reduced adhesive strength to laminin by redistributing α3β1 at the cell-matrix interface. Moreover, in vivo podocyte-specific deletion of Cd151 led to glomerular nephropathy. Although global Cd151-null B6 mice were not susceptible to renal disease, as has been shown previously, increasing blood and transcapillary filtration pressure induced nephropathy in these mice. Importantly, blocking the angiotensin-converting enzyme in renal disease–susceptible global Cd151-null FVB mice prolonged their median life span. Together, these results establish CD151 as a crucial modifier of integrin-mediated adhesion of podocytes to the GBM and show that blood pressure is an important factor in the initiation and progression of Cd151 knockout–induced nephropathy