Identification of Widespread Adenosine Nucleotide Binding in Mycobacterium tuberculosis

SummaryComputational 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

Data from: Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture

RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant’s proficiency at cell entry with the other’s proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies

Figure_3-source_data_1

Figure 3 – source data 1. This 7-zip archive contains the data and source code used for Figure 3 (the analysis of mutation frequencies at site 151 after serial passage in the lab)

Figure_1-source_data_1

Figure 1 – source data 1. This 7-zip archive contains the data and source code used for Figure 1 (the analysis of mutation frequencies at site 151 in naturally occurring sequences)

<i>Mycobacterium tuberculosis</i> Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

<div><p>The majority of <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) 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 <i>Mtb</i> response to altered oxygen status, using an <i>in vitro</i> model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of <i>Mtb</i> in reaeration. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. <i>Mtb</i> 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 <i>Mtb</i> life cycle—active disease, latency, and reactivation.</p></div

PknB transcript and protein levels change in response to oxygen.

<p>(A) Microarray analysis of STPK transcript levels during a hypoxia time course. (B) Quantitative MS analysis of STPKs in wild-type <i>Mtb</i> in log phase, day 7 of hypoxia, and day 2 of reaeration. (C) Quantitative MS analysis of PknB abundance in wild-type <i>Mtb</i>.</p

PknB overexpression leads to elongated bacilli in hypoxia.

<p>Scanning electron microscopy of (A) wild-type and (B) <i>tet-pknB</i> on day 0 (log phase), and (C) wild-type and (D) <i>tet-pknB</i> after 7 d of hypoxia. (E) The percent of elongated bacilli for each strain in log phase and hypoxia.</p

Chemical inhibition of PknB compromises viability in reaeration.

<p>(A) Wild-type <i>Mtb</i> in log phase was treated with sub-MIC concentrations of K252a, and growth in aerated culture was measured for 7 d. (B) Viability during a hypoxia time course was determined on day 0, hypoxia day 7, and reaeration day 1 and 2 by CFU analysis. Error bars represent standard deviation.</p

Broad STPK inhibition compromises <i>Mtb</i> growth in reaeration.

<p>(A) Wild-type <i>Mtb</i> in aerated log phase culture was treated with sub-MIC concentrations (0.1, 1, and 10 µM) of staurosporine, and growth was measured over 7 d. (B) Viability was determined on day 0, day 7 of hypoxia, and day 1 of reaeration by plating for CFU analysis. Error bars represent standard deviation.</p

Effects of PknB dysregulation in hypoxia and reaeration.

<p>(A) <i>Mtb</i> growth (dashed line) is regulated by PknB (solid line) in response to oxygen tension. Under low oxygen conditions in hypoxia, PknB activity is decreased to facilitate growth arrest, but returns upon reaeration to support regrowth. PknB dysregulation by (B) overexpression or (C) inhibition interferes with survival at different stages. (D) <i>Mtb</i> growth is most vulnerable to PknB targeting in hypoxia and reaeration. Elevated PknB levels in hypoxia and reduced levels in reaeration are most detrimental.</p