The benzothiazinone lead compound, BTZ043, kills Mycobacterium tuberculosis by inhibiting the essential flavo-enzyme DprE1, decaprenylphosphoryl-beta-D-ribose 2-epimerase. Here, we synthesized a new series of piperazine-containing benzothiazinones (PBTZ) and show that, like BTZ043, the preclinical candidate PBTZ169 binds covalently to DprE1. The crystal structure of the DprE1-PBTZ169 complex reveals formation of a semimercaptal adduct with Cys387 in the active site and explains the irreversible inactivation of the enzyme. Compared to BTZ043, PBTZ169 has improved potency, safety and efficacy in zebrafish and mouse models of tuberculosis (TB). When combined with other TB drugs, PBTZ169 showed additive activity against M. tuberculosis in vitro except with bedaquiline (BDQ) where synergy was observed. A new regimen comprising PBTZ169, BDQ and pyrazinamide was found to be more efficacious than the standard three drug treatment in a murine model of chronic disease. PBTZ169 is thus an attractive drug candidate to treat TB in humans.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Pharmacology & Drug Discovery

The central nervous system (CNS) has specific barriers that protect the brain from potential threats and tightly regulate molecular transport. Despite the critical functions of the CNS barriers, the mechanisms underlying their development and function are not well understood, and there are very limited experimental models for their study. Claudin 5 is a tight junction protein required for blood brain barrier (BBB) and, probably, choroid plexus (CP) structure and function in vertebrates. Here, we show that the gene claudin 5a is the zebrafish orthologue with high fidelity expression, in the BBB and CP barriers, that demonstrates the conservation of the BBB and CP between humans and zebrafish. Expression of claudin 5a correlates with developmental tightening of the BBB and is restricted to a subset of the brain vasculature clearly delineating the BBB. We show that claudin 5a-expressing cells of the CP are ciliated ependymal cells that drive fluid flow in the brain ventricles. Finally, we find that CP development precedes BBB development and that claudin 5a expression occurs simultaneously with angiogenesis. Thus, our novel transgenic zebrafish represents an ideal model to study CNS barrier development and function, critical in understanding the mechanisms underlying CNS barrier function in health and disease.

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.

Correspondence: a.vandersar@vumc.nl Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of mediumthroughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish-Mycobacterium marinum infection model and its added value for tuberculosis research.

Chicken cathelicidin-2 (CATH-2) is a host defense peptide that exhibits immunomodulatory and antibacterial properties. Here we examined effects of CATH-2 in zebrafish embryos in the absence and presence of infection. Yolk-injection of 0.2-1.5 h post-fertilized (hpf) zebrafish embryos with 2.6 ng/kg CATH-2 increased proliferation of phagocytic cells at 48 hpf by 30%. A lethal infection model was developed to test the prophylactic protective effect of CATH-2 peptide. Embryos (0.2-1.5 hpf) were injected with 2.6 ng/kg CATH-2, challenged with a lethal dose of fluorescently labeled Salmonella enteritidis pGMDs3 at 28 hpf and monitored for survival. Prophylactic treatment with CATH-2 was found to delay infection starting at 22 h post-infection (hpi). At 18-20 hpi, significantly lower (2-fold) fluorescence intensity and decreased bacterial loads were detected in peptide-treated embryos. Thus prophylactic administration of low CATH-2 concentrations confer partial protection in zebrafish embryos by boosting the innate immune system.