Mycobacterial biotin synthases require an auxiliary protein to convert dethiobiotin into biotin.

Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Clinical characteristics and outcomes after new-onset seizure among Zambian children with HIV during the antiretroviral therapy era.

OBJECTIVE: This study describes clinical profiles including human immunodeficiency virus (HIV) disease history and seizure etiology among children living with HIV presenting with new-onset seizure during the era of antiretroviral therapy (ART) in Zambia. 30-day mortality and cause of death are also reported. METHODS: Children living with HIV (CLWHIV) with new-onset seizures were prospectively evaluated at one large urban teaching hospital and two non-urban healthcare facilities. Interviews with family members, review of medical records, and where needed, verbal autopsies were undertaken. Two clinicians who were not responsible for the patients' care independently reviewed all records and assigned seizure etiology and cause of death with adjudication as needed. RESULTS: From April 2016 to June 2019, 73 children (49 urban, 24 rural) were identified. Median age was 6 years (IQR 2.2-10.0) and 39 (53%) were male children. Seizures were focal in 36 (49%) and were often severe, with 37% presenting with multiple recurrent seizures in the 24 hours before admission or in status epilepticus. Although 36 (49%) were on ART at enrollment, only 7 of 36 (19%) were virally suppressed. Seizure etiologies were infectious in over half (54%), with HIV encephalitis, bacterial meningitis, and tuberculous meningitis being the most common. Metabolic causes (19%) included renal failure and hypoglycemia. Structural lesions identified on imaging accounted for 10% of etiologies and included stroke and non-accidental trauma. No etiology could be identified in 12 (16%) children, most of whom died before the completion of clinical investigations. Twenty-two (30%) children died within 30 days of the index seizure. SIGNIFICANCE: Despite widespread ART roll out in Zambia, new-onset seizure in CLWHIV occurs in the setting of advanced, active HIV disease. Seizure severity/burden is high as is early mortality. Enhanced programs to assure early ART initiation, improve adherence, and address ART failure are needed to reduce the burden of neurological injury and premature death in CLWHIV.