No abstract

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

The Notch family of cell-surface receptors has been proposed to regulate cell-fate decisions by modulating the ability of each cell to respond to environmental cues. In vertebrates, gain-of-function and loss-of-function studies have demonstrated a requirement for Notch signaling for proper patterning of the vasculature during embryogenesis. To examine the molecular mechanisms by which Notch regulates vascular development, we analyzed changes in gene expression in response to Notch signaling. Notch signal transduction and function were assessed in primary human endothelial cells isolated from the dermal microvasculature of neonates, HMVECd. We demonstrate that HMVECd cells express a heterodimeric form of endogenous Notch4 on their cell surface. Using an in vitro coculture assay, we found that Delta4 can function as a ligand for Notch4 in HMVECd cells. Moreover, ectopic expression of an activated allele of Notch4 upregulated the expression of Delta4, suggesting that there may be a regulatory loop between Notch4 and its ligand, Delta4. Notch4 activation also induced the expression of the transcriptional repressors, HES1, HERP1, and HERP2, as well as ephrinB2, an angiogenic factor proposed to be involved in arterial/venous endothelial cell specification.

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial βlactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3′ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithionecontaining cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

No abstract