The PhoP-PhoR two-component system is essential for virulence and intracellular growth of Mycobacterium tuberculosis (MTB) in human and mouse macrophages or in mice. Here, PhoP and truncated PhoR sensor proteins were shown to participate in phosphotransfer reactions using conserved residues characteristic of two-component signaling systems. bGalactosidase activity originating from phoP promoter-lacZ construct was inhibited in presence of PhoP, suggesting transcriptional auto-inhibition by the response regulator. In vitro binding of PhoP is consistent with the in vivo transcriptional repression, indicating phosphorylation-independent assembly of the transcription initiation complex at elevated concentrations of PhoP. DNaseI protection studies reveal a consensus recognition sequence within the phoP promoter that includes three 9-bp direct repeat units. Each repeat unit adjusts to the consensusAlteration in the sequence of the newly-identified direct repeat units relieved phoP transcriptional repression in presence of PhoP, suggesting that PhoP represses its own expression by sequence-specific interaction(s) with the repeat units. Together, these results identify so far unknown PhoPregulated genetic determinants in the regulatory region of the phoP promoter that are central to understanding of how PhoP may possibly function as a global regulator in MTB.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F–specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

Mycobacterium tuberculosis

Mycobacterium tuberculosis

Background: Stalled replication forks are foci for genomic instability. Results: Both RecG and RuvAB can regress stalled forks; however, RuvAB completely unwinds the nascent DNA, whereas RuvC cleaves the Holliday junctions formed by RecG. Conclusion: RecG and RuvAB activities are distinct. Significance: Replication fork regression is a major step in processing stalled forks.