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 (O-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 O-phosphorylation, however, now show that it is in fact pervasive, with some bacterial proteomes as highly phosphorylated as those of eukaryotes. Labile, non-canonical protein phosphorylation sites on Asp, Arg, and His are now also being identified in large numbers in bacteria and first cellular functions are discovered. Other phosphomodifications on Cys, Glu, and Lys remain largely unexplored. The surprising breadth and complexity of bacterial phosphosignaling reveals a vast signaling capacity, the full scope of which we may only now be beginning to understand but whose functions are likely to affect all aspects of bacterial physiology and pathogenesis.

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. Several TCSs were phosphorylated on multiple sites, and many by multiple Ser/Thr protein kinases, suggesting convergent and cooperative regulatory interactions. To test in which way these O-phosphorylation sites affect TCS activity, we reconstituted the NarSL phosphorelay in vitro. The Ser/Thr protein kinase PknL phosphorylated the histidine kinase NarS and activated its autophosphorylating activity. A phosphoablative mutation at the PknL phosphorylation site Thr380 resulted in low autophosphorylating activity, whereas a phosphomimetic mutation strongly activated autophosphorylation. The phosphomimetic mutation also resulted in more efficient phosphotransfer from NarS to the response regulator NarL and suppression of gene expression. These data show control of NarSL signaling by STPKs through a phosphoswitch and point to extensive, functional crosstalk between TCSs and O-phosphorylation.

Dual Inhibition of Mycobacterium tuberculosis 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 Mycobacterium tuberculosis (Mtb), and an anilinoquinazoline (AQA) that also had potent activity against nonreplicating and persistent Mtb. AQA was originally developed to inhibit human transforming growth factor receptor 1 (TGFBR1), a host kinase that is predicted to have host-adverse effects during Mtb infection. The structure-activity relationship of this dually active compound identified the pyridyl-6-methyl group as being required for potent Mtb inhibition but a liability for P450 metabolism. Pyrrolopyrimidine (43) emerged as the optimal compound that balanced micromolar inhibition of nonreplicating Mtb and TGFBR1 while also demonstrating improved metabolic stability and pharmacokinetic profiles.

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. A deep phosphoproteome with >14,000 unique phosphosites shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >80% of the proteome and extensively interfaces with the transcriptional machinery. Mtb O-phosphorylation gives rise to an expansive, distributed and cooperative network of a complexity that has not previously been seen in bacteria and that is on par with eukaryotic phosphosignalling networks. A resource of >3,700 high-confidence direct substrate-STPK interactions and their transcriptional effects provides signalling context for >80% of Mtb proteins and allows the prediction and assembly of signalling pathways for mycobacterial physiology.

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+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca2+ homeostasis. To test whether the PE15/PPE20 complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+, show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.

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 response1-8, 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. We then focused on a specific regulator, mce3R, which potentiated INH activity when induced. We compared mce3R-regulated genes with baseline INH transcriptional responses and implicated the gene ctpD (Rv1469) as a putative INH effector. Evaluating a ctpD disruption mutant demonstrated a previously unknown role for this gene in INH susceptibility. Integrating TRIP screening with network information can uncover sophisticated molecular response programs.

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.

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. GAF and PAS domains are sensor domains that are typically linked to signaling effector molecules. Unlike these sensor-effector proteins, Rv3651 is an unusual sensor domain-only protein with highly divergent sequence. The structure suggests that Rv3651 integrates multiple different signals and serves as a scaffold to facilitate signal transfer.

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. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection.

Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment­the inclusion­and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.

A Hyperhemolytic/Hyperpigmented Group B Streptococcus Strain with a CovR Mutation Isolated from an Adolescent Patient with Sore Throat.

Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated with infections in human newborns or immunocompromised adults. However, mutation in the two-component regulator CovR/S relieves repression of hemolysin, potentially increasing virulence of GBS. We report the isolation of hyperhemolytic/hyperpigmented GBS strain from an adolescent patient who presented to the University of Washington clinic with symptoms of sore throat. While the patient also tested positive for mononucleosis, a GBS strain with increased hemolysis was isolated from the throat swab obtained from the patient. As hyperhemolytic/hyperpigmented GBS strains are typically associated with mutations in the regulator CovR/CovS, we sequenced the covR/S loci in the clinical isolate. An adenine to cytosine mutation resulting in a change in amino acid coding sequence from glutamine at position 120 to proline in CovR (Q120P) was identified. Introduction of the Q120P amino acid substitution in a CovR complementation plasmid abolished complementation of a ΔcovR mutant derived from the wild type GBS serotype Ia strain A909; these results confirm that the hyperhemolysis observed in the clinical isolate is due to the Q120P substitution in CovR. Antibiotic was prescribed and the patient's symptoms resolved without reported complications. This study represents the first report of the isolation of a hyperhemolytic/hyperpigmented GBS strain due to a covR/S mutation from an adolescent patient with persistent sore throat who was also diagnosed with mononucleosis. The isolation of GBS CovR/S mutants indicates their presence in settings of co-infections and includes adolescents.