Environmental and genetic regulation of Streptococcus pneumoniae galactose catabolic pathways.

Efficient utilization of nutrients is crucial for microbial survival and virulence. The same nutrient may be utilized by multiple catabolic pathways, indicating that the physical and chemical environments for induction as well as their functional roles may differ. Here, we study the tagatose and Leloir pathways for galactose catabolism of the human pathogen Streptococcus pneumoniae. We show that galactose utilization potentiates pneumococcal virulence, the induction of galactose catabolic pathways is influenced differentially by the concentration of galactose and temperature, and sialic acid downregulates galactose catabolism. Furthermore, the genetic regulation and in vivo induction of each pathway differ, and both galactose catabolic pathways can be turned off with a galactose analogue in a substrate-specific manner, indicating that galactose catabolic pathways can be potential drug targets.

The single D380 amino acid substitution increases pneumolysin cytotoxicity toward neuronal cells.

Bacterial meningitis, frequently caused by Streptococcus pneumoniae (pneumococcus), represents a substantial global health threat leading to long-term neurological disorders. This study focused on the cholesterol-binding toxin pneumolysin (PLY) released by pneumococci, specifically examining clinical isolates from patients with meningitis and comparing them to the PLY-reference S. pneumoniae strain D39. Clinical isolates exhibit enhanced PLY release, likely due to a significantly higher expression of the autolysin LytA. Notably, the same single amino acid (aa) D380 substitution in the PLY D4 domain present in all clinical isolates significantly enhances cholesterol binding, pore-forming activity, and cytotoxicity toward SH-SY5Y-derived neuronal cells. Scanning electron microscopy of human neuronal cells and patch clamp electrophysiological recordings on mouse brain slices confirm the enhanced neurotoxicity of the PLY variant carrying the single aa substitution. This study highlights how a single aa modification enormously alters PLY cytotoxic potential, emphasizing the importance of PLY as a major cause of the neurological sequelae associated with pneumococcal meningitis.

Pneumococcal capsule expression is controlled through a conserved, distal cis-regulatory element during infection.

Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial pneumonia in the US and worldwide. Studies have shown that the differing chemical make-up between serotypes of its most important virulence factor, the capsule, can dictate disease severity. Here we demonstrate that control of capsule synthesis is also critical for infection and facilitated by two broadly conserved transcription factors, SpxR and CpsR, through a distal cis-regulatory element we name the 37-CE. Strikingly, changing only three nucleotides within this sequence is sufficient to render pneumococcus avirulent. Using in vivo and in vitro approaches, we present a model where SpxR interacts as a unique trimeric quaternary structure with the 37-CE to enable capsule repression in the airways. Considering its dramatic effect on infection, variation of the 37-CE between serotypes suggests this molecular switch could be a critical contributing factor to this pathogen's serotype-specific disease outcomes.

Structure-function analysis for the development of peptide inhibitors for a Gram-positive quorum sensing system.

The Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilization, oxidative stress response, and virulence. Here, we characterized this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with a 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilization of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystallization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologs suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species.

Siglec-5 is an inhibitory immune checkpoint molecule for human T cells.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5 suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of NFAT and AP-1. Furthermore, we show that GBS ß-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5 ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

Glutamate Dehydrogenase (GdhA) of Streptococcus pneumoniae Is Required for High Temperature Adaptation.

During its progression from the nasopharynx to other sterile and nonsterile niches of its human host, Streptococcus pneumoniae must cope with changes in temperature. We hypothesized that the temperature adaptation is an important facet of pneumococcal survival in the host. Here, we evaluated the effect of temperature on pneumococcus and studied the role of glutamate dehydrogenase (GdhA) in thermal adaptation associated with virulence and survival. Microarray analysis revealed a significant transcriptional response to changes in temperature, affecting the expression of 252 genes in total at 34°C and 40°C relative to at 37°C. One of the differentially regulated genes was gdhA, which is upregulated at 40°C and downregulated at 34°C relative to 37°C. Deletion of gdhA attenuated the growth, cell size, biofilm formation, pH survival, and biosynthesis of proteins associated with virulence in a temperature-dependent manner. Moreover, deletion of gdhA stimulated formate production irrespective of temperature fluctuation. Finally, ΔgdhA grown at 40°C was less virulent than other temperatures or the wild type at the same temperature in a Galleria mellonella infection model, suggesting that GdhA is required for pneumococcal virulence at elevated temperature.

A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa.

Heme is an essential metabolite for most life on earth. Bacterial pathogens almost universally require iron to infect a host, often acquiring this nutrient in the form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is no exception, where heme acquisition and metabolism are known to be crucial for both chronic and acute infections. To unveil unknown genes and pathways that could play a role with heme metabolic flux in this pathogen, we devised an omic-based approach we dubbed "Met-Seq," for metabolite-coupled transposon sequencing. Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) and massively parallel sequencing, allowing for direct identification of genes associated with metabolic changes. In this work, we first construct and validate a heme biosensor for use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentially influence intracellular heme levels. Identified genes largely consisted of metabolic pathways not previously associated with heme, including many secreted virulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitches whose functions are not currently understood. We verify that five Met-Seq hits affect intracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipid acquisition system, heme biosynthesis regulator Dnr, and two predicted antibiotic monooxygenase (ABM) domains of unknown function (PA0709 and PA3390). Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins. Our data suggest that Met-Seq could be extrapolated to other biological systems and metabolites for which there is an available biosensor, and provides a new template for further exploration of iron/heme regulation and metabolism in P. aeruginosa and other pathogens.IMPORTANCE The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub "Met-Seq" (metabolite-coupled Tn sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS), and next-generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept, we create and test a heme biosensor and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme-binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and shed additional light on the balance between infection, heme uptake, and heme biosynthesis.

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization.

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus.

The aspartate-less receiver (ALR) domains: distribution, structure and function.

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.

Dynamic structural changes underpin photoconversion of a blue/green cyanobacteriochrome between its dark and photoactivated states.

The phytochrome superfamily of photoreceptors exploits reversible light-driven changes in the bilin chromophore to initiate a variety of signaling cascades. The nature of these alterations and how they impact the protein moiety remain poorly resolved and might include several species-specific routes. Here, we provide a detailed picture of photoconversion for the photosensing cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain from Thermosynechococcus elongatus (Te) PixJ, a member of the cyanobacteriochrome clade. Solution NMR structures of the blue light-absorbing dark state Pb and green light-absorbing photoactivated state Pg, combined with paired crystallographic models, revealed that the bilin and GAF domain dynamically transition via breakage of the C10/Cys-494 thioether bond, opposite rotations of the A and D pyrrole rings, sliding of the bilin in the GAF pocket, and the appearance of an extended region of disorder that includes Cys-494. Changes in GAF domain backbone dynamics were also observed that are likely important for inter-domain signal propagation. Taken together, photoconversion of T. elongatus PixJ from Pb to Pg involves complex structural changes within the GAF domain pocket that transduce light into a mechanical signal, many aspects of which should be relevant to others within the extended phytochrome superfamily.

Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens.

Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential.

Phytochrome structure and photochemistry: recent advances toward a complete molecular picture.

Phytochromes are nature's primary photoreceptors dedicated to detecting the red and far-red regions of the visible light spectrum, a region also essential for photosynthesis and thus crucial to the survival of plants and other photosynthetic organisms. Given their roles in measuring competition and diurnal/seasonal light fluctuations, understanding how phytochromes work at the molecular level would greatly aid in engineering crop plants better suited to specific agricultural settings. Recently, scientists have determined the three-dimensional structures of prokaryotic phytochromes, which now provide clues as to how these modular photoreceptors might work at the atomic level. The models point toward a largely unifying mechanism whereby novel knot, hairpin, and dimeric interfaces transduce photoreversible bilin isomerization into protein conformational changes that alter signal output.

Structural basis for the photoconversion of a phytochrome to the activated Pfr form.

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB'. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Cyanochromes are blue/green light photoreversible photoreceptors defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore.

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3(1) carbon of the ethylidene side chain and the C4 or C5 carbons in the A-B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb --> Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.

Phosphorylation of the RitR DNA-binding domain by a Ser-Thr phosphokinase: implications for global gene regulation in the streptococci.

We report selective phosphorylation of the DNA-binding domain of the Streptococcus pneumoniae transcriptional regulator RitR. RitR is annotated as a two-component response regulator, but lacks a cognate His kinase as a neighbouring locus in the genome. In addition, Asn replaces Asp at the expected acceptor site. By the use of combinatorial phage display, we identified PhpP, a S. pneumoniae Ser-Thr eukaryotic-like PP2C phosphatase as an interacting partner of RitR. RitR interacts with the phage-displayed peptide VADGMGGR which forms a part of the active-site sequence of PhpP. RitR is phosphorylated in vitro by StkP, the presumed cognate kinase of PhpP, and the site on RitR that is phosphorylated has been localized to the RitR DNA-binding domain. PhpP together with its cognate kinase StkP appear to be necessary for Piu haem transporter expression. In vitro studies suggest that PhpP and StkP interact competitively with RitR in that RitR-PhpP-piu promoter ternary complexes are disrupted by StkP. Our findings indicate a regulatory link between RitR and Ser-Thr kinase-phosphatase-based bacterial signal transduction.

Solution structure of a cyanobacterial phytochrome GAF domain in the red-light-absorbing ground state.

The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B' cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80 degrees relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B' GAF domain can, by itself, complete the Pr --> Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.

Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion.

Photointerconversion between the red light-absorbing (Pr) form and the far-red light-absorbing (Pfr) form is the central feature that allows members of the phytochrome (Phy) superfamily to act as reversible switches in light perception. Whereas the chromophore structure and surrounding binding pocket of Pr have been described, those for Pfr have remained enigmatic for various technical reasons. Here we describe a novel pair of Phys from two thermophilic cyanobacteria, Synechococcus sp. OS-A and OS-B', that overcome several of these limitations. Like other cyanobacterial Phys, SyA-Cph1 and SyB-Cph1 covalently bind the bilin phycocyanobilin via their cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains and then assume the photointerconvertible Pr and Pfr states with absorption maxima at 630 and 704 nm, respectively. However, they are naturally missing the N-terminal Per/Arndt/Sim domain common to others in the Phy superfamily. Importantly, truncations containing only the GAF domain are monomeric, photochromic, and remarkably thermostable. Resonance Raman and NMR spectroscopy show that all four pyrrole ring nitrogens of phycocyanobilin are protonated both as Pr and following red light irradiation, indicating that the GAF domain by itself can complete the Pr to Pfr photocycle. (1)H-(15)N two-dimensional NMR spectra of isotopically labeled preparations of the SyB-Cph1 GAF domain revealed that a number of amino acids change their environment during photoconversion of Pr to Pfr, which can be reversed by subsequent photoconversion back to Pr. Through three-dimensional NMR spectroscopy before and after light photoexcitation, it should now be possible to define the movements of the chromophore and binding pocket during photoconversion. We also generated a series of strongly red fluorescent derivatives of SyB-Cph1, which based on their small size and thermostability may be useful as cell biological reporters.

Screening for compounds that affect the interaction between bacterial two-component signal transduction response regulator protein and cognate promoter DNA.

Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups--sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination were found to act elsewhere instead; a possible reason for this is that full-length sensor kinases are integral membrane proteins whose activity might reflect interaction with the cell membrane or with membrane components. We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems.

Differential assay for high-throughput screening of antibacterial compounds.

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.

Bacterial 2-component signal transduction systems: a fluorescence polarization screen for response regulator-protein binding.

Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.