More than two components: complexities in bacterial phosphosignaling.

For over 40 years, the two-component systems (TCSs) have taken front and center in our thinking about the signaling mechanisms by which bacteria sense and respond to their environment. In contrast, phosphorylation on Ser/Thr and Tyr (-phosphorylation) was long thought to be mostly restricted to eukaryotes and a somewhat accessory signaling mechanism in bacteria. Several recent studies exploring systems aspects of bacterial -phosphorylation, however, now show that it is in fact pervasive, with some bacterial proteomes as highly phosphorylated as those of eukaryotes.

Regulatory Intersection of Two-component System and Ser/Thr Protein Kinase Signaling in Mycobacterium tuberculosis.

Phosphosignaling in bacteria is mediated by two distinct systems, the two-component systems (TCSs) and the protein Ser/Thr/Tyr, or O-phosphorylation systems. These two arms of phosphosignaling are currently thought to be largely independent from one another. We mined a deep Mycobacterium tuberculosis (Mtb) phosphoproteome and identified over 170 O-phosphorylation sites on histidine kinases and response regulators of TCSs, suggesting that the two signaling pathways extensively intersect.

Dual Inhibition of and the Host TGFBR1 by an Anilinoquinazoline.

Tuberculosis (TB) control is complicated by the emergence of drug resistance. Promising strategies to prevent drug resistance are the targeting of nonreplicating, drug-tolerant bacterial populations and targeting of the host, but inhibitors and targets for either are still rare. In a cell-based screen of ATP-competitive inhibitors, we identified compounds with in vitro activity against replicating (), and an anilinoquinazoline (AQA) that also had potent activity against nonreplicating and persistent .

The Mycobacterium tuberculosis protein O-phosphorylation landscape.

Bacterial phosphosignalling has been synonymous with two-component systems and their histidine kinases, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignalling enzymes in eukaryotes but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is untested. Here we explored the global signalling capacity of the STPKs in Mtb using a panel of STPK loss-of-function and overexpression strains combined with mass spectrometry-based phosphoproteomics.

The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane.

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca2+) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca2+ signaling systems is not well understood. To understand the basis for Ca2+ transport and signaling in Mtb, we determined Mtb's transcriptional response to Ca2+.

Activity-based annotation: the emergence of systems biochemistry.

Current tools to annotate protein function have failed to keep pace with the speed of DNA sequencing and exponentially growing number of proteins of unknown function (PUFs). A major contributing factor to this mismatch is the historical lack of high-throughput methods to experimentally determine biochemical activity. Activity-based methods, such as activity-based metabolite and protein profiling, are emerging as new approaches for unbiased, global, biochemical annotation of protein function.

A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria.

Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays.

Transcriptional regulator-induced phenotype screen reveals drug potentiators in Mycobacterium tuberculosis.

Transposon-based strategies provide a powerful and unbiased way to study the bacterial stress response, but these approaches cannot fully capture the complexities of network-based behaviour. Here, we present a network-based genetic screening approach: the transcriptional regulator-induced phenotype (TRIP) screen, which we used to identify previously uncharacterized network adaptations of Mycobacterium tuberculosis to the first-line anti-tuberculosis drug isoniazid (INH). We found regulators that alter INH susceptibility when induced, several of which could not be identified by standard gene disruption approaches.

Anti-tubercular activity of novel 4-anilinoquinolines and 4-anilinoquinazolines.

We screened a series of 4-anilinoquinolines and 4-anilinoquinazolines and identified novel inhibitors of Mycobacterium tuberculosis (Mtb). The focused 4-anilinoquinoline/quinazoline scaffold arrays yielded compounds with high potency and the identification of 6,7-dimethoxy-N-(4-((4-methylbenzyl)oxy)phenyl)quinolin-4-amine (34) with an MIC value of 0.63-1.

To fight tuberculosis, fund basic research.

Tuberculosis (TB) is now the leading cause of death from infectious disease. On September 26, 2018, the United Nations (UN) General Assembly holds its first high-level meeting on TB, a once-in-a-lifetime chance to commit governments around the world to redouble their TB control efforts. Here I share impressions from a preparatory meeting at the UN in June and make the case for basic research as a central component of any future TB control strategy.

Mycobacterium tuberculosis Rv3651 is a triple sensor-domain protein.

The genome of the human pathogen Mycobacterium tuberculosis (Mtb) encodes ∼4,400 proteins, but one third of them have unknown functions. We solved the crystal structure of Rv3651, a hypothetical protein with no discernible similarity to proteins with known function. Rv3651 has a three-domain architecture that combines one cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA (GAF) domain and two Per-ARNT-Sim (PAS) domains.

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

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of the () 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 is the unknown metabolic state of the gametocyte and the lack of known biochemical activity for most parasite gene products in general.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence.

The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics.

Crystal structures of Mycobacterial MeaB and MMAA-like GTPases.

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis.

Agents of change - concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling.

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two-component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O-phosphorylation) in prokaryotes such as Mtb.

Mycobacterium tuberculosis supports protein tyrosine phosphorylation.

Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb.

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).

Mycobacterium tuberculosis Rv2179c protein establishes a new exoribonuclease family with broad phylogenetic distribution.

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease.

Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase.

Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress.

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.

Mycobacterium thermoresistibile as a source of thermostable orthologs of Mycobacterium tuberculosis proteins.

The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims ~1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging.

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA.

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution.

Design and synthesis of nonpeptidic, small molecule inhibitors for the Mycobacterium tuberculosis protein tyrosine phosphatase PtpB.

The design and synthesis of new inhibitor analogues for the Mycobacterium tuberculosis (Mtb) phosphatase PtpB is described. Analogues were synthesized by incorporation of two common and effective phosphate mimetics, the isothiazolidinone (IZD) and the difluoromethylphosphonic acid (DFMP). The basic scaffold of the inhibitor was identified from structure-activity relationships established for a previously published isoxazole inhibitor, while the phosphate mimetics were chosen based on their proven cell permeability and activity when incorporated into previously reported inhibitors for the phosphatase PTP1B.

The Trypanosoma brucei life cycle switch TbPTP1 is structurally conserved and dephosphorylates the nucleolar protein NOPP44/46.

Trypanosoma brucei adapts to changing environments as it cycles through arrested and proliferating stages in the human and tsetse fly hosts. Changes in protein tyrosine phosphorylation of several proteins, including NOPP44/46, accompany T. brucei development.

Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA.

The development of low muM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K(i)=1.4+/-0.