Xuelian Zhang

Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis

The target of a first-line tuberculosis drug that acts against persister bacteria is identified. Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase (PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to RpsA (but not a clinically identified ΔAla mutant) and subsequently inhibited trans-translation rather than canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms.

Assessment of Efflux Pump Gene Expression in a Clinical Isolate Mycobacterium tuberculosis by Real-Time Reverse Transcription PCR

The importance of some putative efflux pumps in conferring drug resistance on certain strain of Mycobacterium tuberculosis was determined by using multidrug-resistant (MDR) strain 1499 bearing defined mutations in rpoB and katG. The clinical isolate 1499 was tested for drug sensitivity and expression level of five putative efflux pump genes by real-time RT-PCR. In the presence of rifampicin or isoniazid, this isolate showed increased transcription of putative efflux pump genes Rv1258c and Rv1410c. Rv1819c was overexpressed only upon isoniazid exposure (p<0.05). Overexpression of some efflux genes is associated with multidrug resistance in certain clinical isolate of M. tuberculosis.

The p.G146A and p.P125P Polymorphisms in the Steroidogenic Factor-1 (SF-1) Gene Do Not Affect the Risk for Hypospadias in Caucasians

Hypospadias is a frequent congenital malformation in boys and is characterized by incomplete fusion of the urethral folds. The steroidogenic factor-1 (SF-1, NR5A1) gene plays a key role in hypothalamic-pituitary-steroidogenic organ development, and has previously been reported to be mutated in individuals with 46,XY disorder of sex development. Here, we investigated the role of SF-1 in hypospadias, a milder form of 46,XY disorder of sex development. We performed direct sequencing analysis of the SF-1 gene in 2 male Caucasian twins exhibiting very severe hypospadias, and in 95 Caucasian boys with mild and severe hypospadias. We further extended the analysis by investigating 332 mild and severe hypospadias cases and 422 male controls using TaqMan assays. Our sequencing revealed a novel heterozygous p.R313H (c.938G>A) missense mutation in each twin, and no mutations in the 95 Caucasian cases. Instead, a missense p.G146A (c.437G>C), and a silent known p.P125P (c.375C>T) polymorphism, respectively, was found in several of the latter cases. Further investigation of the 2 polymorphisms in the larger material of cases and controls showed no significant genotypic or allelic association. In conclusion, the SF-1 gene may not play a significant role in the development of hypospadias in Caucasians.

In vitro and in vivo Activities of a New Lead Compound I2906 against Mycobacterium tuberculosis

Background: Due to the long duration of treatment and the emergence of multidrug-resistant strains, new antitubercular agents are urgently needed. I2906, as a novel lead, was screened and tested for efficacy in vitro and in vivo. Methods:To determine the efficacy of I2906,the minimum inhibitory concentrations against Mycobacterium tuberculosis and cytotoxicity were tested, and its in vivo activities were assessed by administering it to mice infected with M. tuberculosis H37Rv or multidrug-resistant strain. Results:Under in vitro conditions, I2906 showed excellent antimycobacterial activities and low cytotoxicity. In a murine model infected with M. tuberculosis H37Rv, the reductions on bacterial loads of both lungs and spleen were statistically significant (p < 0.05) between I2906-treated mice and untreated controls after 4 weeks. Further, the colony-forming unit counts in the lungs were dramatically lower (p < 0.05) than that of isoniazid-treated mice by the addition of I2906 after 8 weeks. Moreover, survival rate was increased by I2906 treatment. For multidrug-resistant strain infection, bacterial counts were reduced significantly in the lungs and spleen due to I2906 treatment in comparison with data from untreated controls (p < 0.05). Conclusions: I2906 displayed potential antimicrobial activities against M. tuberculosis H37Rv and drug-resistant strains in vitro and in vivo, and could improve efficacy of isoniazid in vivo.

Contents Vol. 6, 2012

Mechthild Krämer (address as for M. Schmid) E-mail: mechthild.kraemer@uni-wuerzburg.de Gerd Scherer Institute of Human Genetics and Anthropology University of Freiburg Breisacherstr. 33 D–79106 Freiburg (Germany) Tel. (+49) 761 270 7030; Fax (+49) 761 270 7041 E-mail: gerd.scherer@uniklinik-freiburg.de Manfred Schartl Institute of Physiological Chemistry I University of Würzburg Biozentrum, Am Hubland D–97074 Würzburg (Germany) Tel. (+49) 931 318 4148; Fax (+49) 931 318 4150 E-mail: phch1@biozentrum.uni-wuerzburg.de