A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes.

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of the Plasmodium falciparum (Pf) blood stage that causes disease as well as the gametocyte stage that is required for transmission from humans to the mosquito vector. Most currently used therapies do not kill gametocytes, a highly specialized, non-replicating sexual parasite stage. Further confounding next generation drug development against Pf is the unknown metabolic state of the gametocyte and the lack of known biochemical activity for most parasite gene products in general. Here, we take a systematic activity-based proteomics approach to survey the activity of the large and druggable ATPase family in replicating blood stage asexual parasites and transmissible, non-replicating sexual gametocytes. ATPase activity broadly changes during the transition from asexual schizonts to sexual gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. We further experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.

A highly stable blood meal alternative for rearing Aedes and Anopheles mosquitoes.

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.

Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch.

The majority of Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by drug tolerance and little or no bacterial replication. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability and gross morphological defects in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle-active disease, latency, and reactivation.

Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis.

Computational prediction of protein function is frequently error-prone and incomplete. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins, severely limiting our understanding of Mtb pathogenicity. Here, we utilize a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate ATP-binding proteins in Mtb. We experimentally validate prior in silico predictions of >240 proteins and identify 72 hypothetical proteins as ATP binders. ATP interacts with proteins with diverse and unrelated sequences, providing an expanded view of adenosine nucleotide binding in Mtb. Several hypothetical ATP binders are essential or taxonomically limited, suggesting specialized functions in mycobacterial physiology and pathogenicity.