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.

Mycobacterium tuberculosis H37Rv enolase (Rv1023)- expression, characterization and effect of host dependent modifications on protein functionality.

Mycobacterium tuberculosis enolase is an essential glycolytic enzyme that catalyzes the conversion of 2, phosphoglycerate (PGA) to phosphoenol pyruvate (PEP). It is also a crucial link between glycolysis and the tricarboxylic acid (TCA) pathway. The depletion of PEP has recently been associated with the emergence of non-replicating drug resistant bacteria. Enolase is also known to exhibit multiple alternate functions, such as promoting tissue invasion via its role as a plasminogen (Plg) receptor. In addition, proteomic studies have identified the presence of enolase in the Mtb degradosome and in biofilms. However, the precise role in these processes has not been elaborated. The enzyme was recently identified as a target for 2-amino thiazoles - a novel class of anti-mycobacterials. In vitro assays and characterization of this enzyme were unsuccessful due to the inability to obtain functional recombinant protein. In the present study, we report the expression and characterization of enolase using Mtb H37Ra as a host strain. Our study demonstrates that the enzyme activity and alternate functions of this protein are significantly impacted by the choice of expression host (Mtb H37Ra or E. coli). Detailed analysis of the protein from each source revealed subtle differences in the post-translational modifications. Lastly, our study confirms the role of enolase in Mtb biofilm formation and describes the potential for inhibiting this process.

Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell.

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis, has evolved mechanisms to evade host defenses and persist within host cells. Host-directed therapies against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of purinergic receptor 7, the receptor previously reported for this peptide, remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft-dependent process. We also discovered a role for GAPDH in LL-37-mediated autophagy induction and clearance of intracellular pathogens. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37-mediated autophagy which was rescued by GAPDH overexpression. This process was dependent on intracellular calcium and p38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggest a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden.

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.

Mycobacterium tuberculosis glyceraldehyde-3-phosphate dehydrogenase plays a dual role-As an adhesin and as a receptor for plasmin(ogen).

The spread of infection is directly determined by the ability of a pathogen to invade and infect host tissues. The process involves adherence due to host-pathogen interactions and traversal into deeper tissues. Mycobacterium tuberculosis (Mtb) primarily infects the lung but is unique in its ability to infect almost any other organ of the human host including immune privileged sites such as the central nervous system (CNS). The extreme invasiveness of this bacterium is not fully understood. In the current study, we report that cell surface Mtb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a virulence factor by multiple mechanisms. Firstly, it serves as a dual receptor for both plasminogen (Plg) and plasmin (Plm). CRISPRi-mediated silencing of this essential enzyme confirmed its role in the recruitment of Plg/Plm. Our studies further demonstrate that soluble GAPDH can re-associate on Mtb bacilli to promote plasmin(ogen) recruitment. The direct association of plasmin(ogen) via cell surface GAPDH or by the re-association of soluble GAPDH enhanced bacterial adherence to and traversal across lung epithelial cells. Furthermore, the association of GAPDH with host extracellular matrix (ECM) proteins coupled with its ability to recruit plasmin(ogen) may endow cells with the ability of directed proteolytic activity vital for tissue invasion.

A Luminescence-Based Coupled Enzyme Assay Enables High-Throughput Quantification of the Bacterial Second Messenger 3'3'-Cyclic-Di-AMP.

Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.

Repurposing ethyl bromopyruvate as a broad-spectrum antibacterial.

BACKGROUND: The emergence of drug-resistant bacteria is a major hurdle for effective treatment of infections caused by Mycobacterium tuberculosis and ESKAPE pathogens. In comparison with conventional drug discovery, drug repurposing offers an effective yet rapid approach to identifying novel antibiotics. METHODS: Ethyl bromopyruvate was evaluated for its ability to inhibit M. tuberculosis and ESKAPE pathogens using growth inhibition assays. The selectivity index of ethyl bromopyruvate was determined, followed by time-kill kinetics against M. tuberculosis and Staphylococcus aureus. We first tested its ability to synergize with approved drugs and then tested its ability to decimate bacterial biofilm. Intracellular killing of M. tuberculosis was determined and in vivo potential was determined in a neutropenic murine model of S. aureus infection. RESULTS: We identified ethyl bromopyruvate as an equipotent broad-spectrum antibacterial agent targeting drug-susceptible and -resistant M. tuberculosis and ESKAPE pathogens. Ethyl bromopyruvate exhibited concentration-dependent bactericidal activity. In M. tuberculosis, ethyl bromopyruvate inhibited GAPDH with a concomitant reduction in ATP levels and transferrin-mediated iron uptake. Apart from GAPDH, this compound inhibited pyruvate kinase, isocitrate lyase and malate synthase to varying extents. Ethyl bromopyruvate did not negatively interact with any drug and significantly reduced biofilm at a 64-fold lower concentration than vancomycin. When tested in an S. aureus neutropenic thigh infection model, ethyl bromopyruvate exhibited efficacy equal to that of vancomycin in reducing bacterial counts in thigh, and at 1/25th of the dosage. CONCLUSIONS: Ethyl bromopyruvate exhibits all the characteristics required to be positioned as a potential broad-spectrum antibacterial agent.

p47 licenses activation of the immune deficiency pathway in the tick Ixodes scapularis.

The E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) acts as a molecular rheostat for the immune deficiency (IMD) pathway of the tick Ixodes scapularis How XIAP activates the IMD pathway in response to microbial infection remains ill defined. Here, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine 63-dependent manner and interacts with the p47 ubiquitin-like (UBX) module. p47 also binds to Kenny (IKKγ/NEMO), the regulatory subunit of the inhibitor of nuclear factor (NF)- κB kinase complex. Replacement of the amino acid lysine to arginine within the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, mitigation of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKß (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization by the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agent Anaplasma phagocytophilum Collectively, we highlight the importance of ticks for the elucidation of paradigms in arthropod immunology. Manipulating immune signaling cascades within I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.

Mycobacterium tuberculosis Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Functions as a Receptor for Human Lactoferrin.

Iron is crucial for the survival of living cells, particularly the human pathogen Mycobacterium tuberculosis (M.tb) which uses multiple strategies to acquire and store iron. M.tb synthesizes high affinity iron chelators (siderophores), these extract iron from host iron carrier proteins such as transferrin (Tf) and lactoferrin (Lf). Recent studies have revealed that M.tb may also relocate several housekeeping proteins to the cell surface for capture and internalization of host iron carrier protein transferrin. One of the identified receptors is the glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This conserved multifunctional protein has been identified as a virulence factor in several other bacterial species. Considering the close structural and functional homology between the two major human iron carrier proteins (Tf and Lf) and the fact that Lf is abundantly present in lung fluid (unlike Tf which is present in plasma), we evaluated whether GAPDH also functions as a dual receptor for Lf. The current study demonstrates that human Lf is sequestered at the bacterial surface by GAPDH. The affinity of Lf-GAPDH (31.7 ± 1.68 nM) is higher as compared to Tf-GAPDH (160 ± 24 nM). Two GAPDH mutants were analyzed for their enzymatic activity and interaction with Lf. Lastly, the present computational studies offer the first significant insights for the 3D structure of monomers and assembled tetramer with the associated co-factor NAD+. Sequence analysis and structural modeling identified the surface exposed, evolutionarily conserved and functional residues and predicted the effect of mutagenesis on GAPDH.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv.

BACKGROUND: Obtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful. Several enzymes of the glycolytic cycle are known to be multifunctional, however relatively few enzymes from M.tb H37Rv have been characterized in the context of their enzymatic and pleiotropic roles. One of the primary reasons is the difficulty in obtaining sufficient amounts of functionally active protein. RESULTS: In the current study, using M.tb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) we demonstrate that expression in E. coli or M. smegmatis results in insolubility and improper subcellular localization. In addition, expression of such conserved multisubunit proteins poses the problem of heteromerization with host homologues. Importantly the expression host dramatically affected the yield and functionality of GAPDH in terms of both enzymatic activity and moonlighting function (transferrin binding). The applicability of this system was further confirmed using two additional enzymes i.e. M.tb Pyruvate kinase and Enolase. CONCLUSIONS: Our studies establish that the attenuated strain M.tb H37Ra is a suitable host for the expression of highly hydrophobic, conserved, multimeric proteins of M.tb H37Rv. Significantly, this expression host overcomes the limitations of E. coli and M. smegmatis expression and yields recombinant protein that is qualitatively superior to that obtained by traditional methods. The current study highlights the fact that protein functionality (which is an an essential requirement for all in vitro assays and drug development) may be altered by the choice of expression host.

Purification and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) from pea seeds.

Glyceraldehyde-3-phosphate dehydrogenase [GAPDH, NAD + oxidoreductase (phosphorylating) 1.2.1.12] catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate coupled with the reduction of NAD(+) to NADH. In addition to its role in glycolysis, this enzyme has numerous alternate functions, in both prokaryotes and eukaryotes. In plants, additional functions have been reported from multiple species including Pisum sativum. A recent study has identified that GAPDH may play an important role in seed ageing and programmed cell death. Despite this the existing purification protocols are almost 40 years old, and only partial characterization of the enzyme has been reported. In the current study, we report a modified method for purification of enzymatically active pea seed GAPDH along with the characterization of the enzyme. Using 2D gel electrophoresis our study also demonstrates that pea seeds contain four isoforms of NAD(+) dependent GAPDH.

Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment.

Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle.

Reverse overshot water-wheel retroendocytosis of apotransferrin extrudes cellular iron.

Iron (Fe), a vital micronutrient for all organisms, must be managed judiciously because both deficiency or excess can trigger severe pathology. Although cellular Fe import is well understood, its export is thought to be limited to transmembrane extrusion through ferroportin (also known as Slc40a1), the only known mammalian Fe exporter. Utilizing primary cells and cell lines (including those with no discernible expression of ferroportin on their surface), we demonstrate that upon Fe loading, the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is recruited to the cell surface, 'treadmills' apotransferrin in and out of the cell. Kinetic analysis utilizing labeled ligand, GAPDH-knockdown cells, (55)Fe-labeled cells and pharmacological inhibitors of endocytosis confirmed GAPDH-dependent apotransferrin internalization as a prerequisite for cellular Fe export. These studies define an unusual rapid recycling process of retroendocytosis for cellular Fe extrusion, a process mirroring receptor mediated internalization that has never before been considered for maintenance of cellular cationic homeostasis. Modulation of this unusual pathway could provide insights for management of Fe overload disorders.

Protein moonlighting in iron metabolism: glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Iron is essential for the survival of both prokaryotic and eukaryotic organisms. It functions as a cofactor for several vital enzymes and iron deprivation is fatal to cells. However, at the same time, excess amounts of iron are also toxic to cells due to the formation of free radicals via the Fenton reaction. As a consequence of its double-edged behaviour, the uptake and regulation of iron involves an intricate balance of acquisition, trafficking, recycling and shuffling between various tissues and organs. This is accomplished by differential regulation of genes involving numerous proteins and enzymes. Several of the proteins identified in these processes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aconitase and lactoferrin (Lf), possess multiple functions within the cell. Such proteins are referred to as moonlighting or multifunctional proteins, whereby proteins initially thought to possess a single well-established function have subsequently been discovered to exhibit alternative functions. In many cases, these multiple functions are conserved across species.

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin.

Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.