Structure-Activity Relationships of the Competence Stimulating Peptides (CSPs) in Streptococcus pneumoniae Reveal Motifs Critical for Intra-group and Cross-group ComD Receptor Activation.
ABSTRACT: Streptococcus pneumoniae is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate S. pneumoniae infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between ?-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in S. pneumoniae and as scaffolds for the design of QS-based anti-infective therapeutics against S. pneumoniae infections.
Project description:Streptococcus pneumoniae is an important pathogen that utilizes quorum sensing (QS) to regulate genetic transformation, virulence, and biofilm formation. The competence-stimulating peptide (CSP) is a 17-amino acid signal peptide that is used by S. pneumoniae to trigger QS. S. pneumoniae strains can be divided into two main specificity groups based on the CSP signal they produce (CSP1 or CSP2) and their compatible receptors (ComD1 or ComD2, respectively). Modulation of QS in S. pneumoniae can be achieved by targeting the CSP:ComD interaction using synthetic CSP analogues. However, to rationally design CSP-based QS modulators with enhanced activities, an in-depth understanding of the structural features that are required for receptor binding is needed. Herein, we report a comprehensive in-solution three-dimensional structural characterization of eight CSP1 and CSP2 analogues with varied biological activities using nuclear magnetic resonance spectroscopy. Analysis of these structures revealed two distinct hydrophobic patches required for effective ComD1 and ComD2 binding.
Project description:The competence stimulating peptide (CSP) plays a key role in the regulation of pneumococcal quorum sensing (QS), a communication system that is critical to the infectivity of pneumococci. CSP functions through binding and activating a transmembrane receptor, ComD. Molecules that can modulate pneumococcal QS through intercepting CSP:ComD interaction may serve as new generation of antibacterial agents to treat pneumococcal infections. In this work, we systematically modified the N-terminus of CSP1, a region that is essential to ComD activation, to identify detailed structural features of the N-terminus that are responsible for its function. Our results revealed structural features that are optimal to achieve receptor activation and structure-activity trends that improve our understanding of CSP:ComD interaction, all of which will contribute to the design of novel pneumococcal QS modulators with higher potency and improved pharmacological properties.
Project description:Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.
Project description:Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic alpha-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the alpha-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified alpha-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.
Project description:Streptococcus mutans ( S. mutans) is a Gram-positive human pathogen that is one of the major contributors to dental caries, a condition with an economic cost of over $100 billion per year in the United States. S. mutans secretes a 21-amino-acid peptide termed the competence stimulating peptide (21-CSP) to assess its population density in a process termed quorum sensing (QS) and to initiate a variety of phenotypes such as biofilm formation and bacteriocin production. 21-CSP is processed by a membrane bound protease SepM into active 18-CSP, which then binds to the ComD receptor. This study seeks to determine the molecular mechanism that ties 21-CSP:SepM recognition and 18-CSP:ComD receptor binding and to identify QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in both 21-CSP and 18-CSP and assessed the ability of the mutated analogs to modulate QS. We identified residues that are important to SepM recognition and ComD receptor binding. Our results shed light on the S. mutans competence QS pathway at the molecular level. Moreover, our structural insights of the CSP signal can be used to design QS-based anti-infective therapeutics against S. mutans.
Project description:Quorum sensing (QS) is a cell-cell communication mechanism that enables bacteria to assess their population density and alter their behavior upon reaching high cell number. Many bacterial pathogens utilize QS to initiate an attack on their host, thus QS has attracted significant attention as a potential antivirulence alternative to traditional antibiotics. Streptococcus pneumoniae, a notorious human pathogen responsible for a variety of acute and chronic infections, utilizes the competence regulon and its associated signaling peptide, the competence stimulating peptide (CSP), to acquire antibiotic resistance and establish an infection. In this work, we sought to define the binding pockets within the ComD1 receptor used for binding the hydrophobic side-chain residues in CSP1 through the introduction of highly-conservative point mutations within the peptide. Optimization of these binding interactions could lead to the development of highly potent CSP-based QS modulators while the inclusion of non-natural amino acids within the CSP sequence would confer resistance to protease degradation, a requirement for drug candidates.
Project description:Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.
Project description:Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.
Project description:DnaJ proteins are characterized by a 'J' domain which is homologous to a region of the Escherichia coli protein DnaJ. DnaJ has been shown to interact with the chaperone protein DnaK, and a number of eukaryotic DnaJ-like proteins have been found to interact with the 70 kDa heat-shock protein/70 kDa heat-shock cognate protein (Hsp70/Hsc70), the eukaryotic homologues of DnaK. Cysteine-string proteins (Csps) are believed to function in calcium-stimulated exocytosis and in this paper we describe a specific ATP-dependent interaction between a Csp (Csp1) and Hsc70/Hsp70. We also show that Csp1 can stimulate the ATPase activity of both Hsc70 and Hsp70 several-fold. Furthermore, we demonstrate that Csp2, a Csp variant found in adrenal chromaffin cells, can enhance the ATPase activity of Hsc70 to a similar extent as Csp1, whereas Csp(137-198), a truncated protein lacking the 'J' domain of Csp1 is unable to stimulate the ATPase activity of Hsc70. This suggests that the functions of Csp1 and Csp2 must differ in some aspect other than interaction with Hsc70. This study is also important from a general view of DnaJ/Hsc70 interactions, as Csps lack a G/F-rich region which has been suggested to be essential for activation of the ATPase activity of DnaK by DnaJ. Thus, this work would imply that a G/F-rich region is not an essential feature of DnaJ proteins for stimulation of the ATPase activity of Hsp70 proteins.
Project description:Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. ?-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity.