ABSTRACT
The blp locus of Streptococcus pneumoniae secretes and regulates bacteriocins, which mediate both intra- and interspecific competition in the human nasopharynx. There are four major alleles of the gene blpH, which encodes the receptor responsible for activating the blp locus when bound to one of four distinct peptide pheromones (BlpC). The allelic variation of blpH is presumably explained by a need to restrict cross talk between competing strains. The BlpH protein sequences have polymorphisms distributed throughout the sequence, making identification of the peptide binding site difficult to predict. To identify the pheromone binding sites that dictate pheromone specificity, we have characterized the four major variants and two naturally occurring chimeric versions of blpH in which recombination events appear to have joined two distinct blpH alleles together. Using these allelic variants, a series of laboratory-generated chimeric blpH alleles, and site-directed mutants of both the receptor and peptide, we have demonstrated that BlpC binding to some BlpH types involves an electrostatic interaction between the oppositely charged residues of BlpC and the first transmembrane domain of BlpH. An additional recognition site was identified in the second extracellular loop. We identified naturally occurring BlpH types that have the capacity to respond to more than one BlpC type; however, this change in specificity results in a commensurate drop in overall sensitivity. These natural recombination events were presumably selected for to balance the need to sense bacteriocin-secreting neighbors with the need to turn on bacteriocin production at a low density.
IMPORTANCE Bacteria use quorum sensing to optimize gene expression to accommodate for local bacterial density and diffusion rates. To prevent interception of quorum-sensing signals by neighboring strains, the genomes of single species often encode strain-specific signal/receptor pairs. The blp locus in Streptococcus pneumoniae that drives bacteriocin secretion is controlled by quorum sensing that involves the interaction of the signal/receptor pair BlpC/BlpH. We show that the pneumococcal population can be divided into several distinct BlpC/BlpH pairs; however, there are examples of naturally occurring chimeric receptors that can bind to more than one BlpC type. The trade-off for this broadened specificity is a loss of overall receptor sensitivity. This suggests that under certain conditions, the advantage of signal interception can trump the requirements for self-induction.
INTRODUCTION
Streptococcus pneumoniae (pneumococcus) is a common pediatric pathogen that colonizes the nasopharynx of a majority of children by 1 year of age (1, 2). During this first year of life, children may be colonized at any point with as many as four different pneumococcal serotypes (3) and even multiple strains of the same pneumococcal serotype (4). The introduction of the multivalent conjugate pneumococcal vaccine has resulted in decreased colonization with the seven targeted serotypes. These strains were rapidly replaced with previously rare serotypes, suggesting that intraspecies competition was keeping the replacement strains at low levels in the population (5, 6). Competition between pneumococcal strains in the nasopharynx has important implications for the epidemiology of pneumococcal infection in the postvaccine era (7, 8).
One mechanism for competition is through the elaboration of bacteriocins, small antimicrobial peptides that are involved in both inter- and intraspecific bacterial competition. Bacteriocin production is commonly controlled by quorum sensing, which involves the secretion and extracellular detection of a signaling molecule or pheromone (reviewed in references 9 and 10). The local concentration of pheromone is used by bacteria as an indication of bacterial density and local diffusion rates. Quorum sensing presumably prevents bacteria from initiating costly bacteriocin production during times when the achievable concentration would be too dilute to be effective in inhibiting competitors (11). The secretion of pheromones into the extracellular environment can be disadvantageous if the signal is intercepted by neighboring strains. To decrease the likelihood of signal interception by unrelated strains, bacterial systems controlled by quorum sensing are often separated into pherotypes that are defined by the expression of restricted pheromone receptors/pheromone pairings.
Bacteriocin production in pneumococcus is controlled by a quorum-sensing system encoded by the blp locus. This locus also encodes bacteriocin peptides and specific immunity proteins that protect producer strains from the antagonistic effects of their own bacteriocins (12–14). Activation of the locus occurs as a result of the accumulation of a peptide pheromone (BlpC) that is processed and secreted out of the cell by the BlpAB complex (Fig. 1A). BlpC binds to and activates the receptor, BlpH, which is the histidine kinase of a two-component regulatory system. Receptor activation results in the phosphorylation of the response regulator, BlpR (12–15). Phospho-BlpR then binds to a consensus sequence found in promoters in the blp locus and induces the upregulation of all the genes in the locus, resulting in the expression of the bacteriocins and cognate immunity proteins encoded in the bacteriocin immunity region (BIR). The BIR of each strain contains the sequences for an assortment of as many as 11 distinct bacteriocin-like peptides, with the peptides encoded by blpMN and blpIJK being experimentally implicated in interstrain inhibitory activity (12, 16, 17).
Blp signaling and chimera construction strategy. (A to C) Diagrammatic representation of the BlpC-initiated signaling cascade in strains with an intact blpA gene (A), strains with a disrupted blpA gene during an encounter with a matched BlpC-secreting strain (B), or strains with a disrupted blpA gene during an encounter with a mismatched BlpC-secreting strain (C). BlpC (blue ovals) is processed and secreted out of the cell only in strains that encode an intact BlpAB complex (black box). Accumulated BlpC binds to and activates cognate but not noncognate BlpH (blue and yellow transmembrane proteins, respectively). Activated BlpH becomes phosphorylated, resulting in the activation of BlpR (black oval). BlpR binds to and upregulates promoters in the blp locus (D), resulting in the production of bacteriocins (triangles) and associated immunity proteins (brown ovals). A strain with a nonfunctional blpA can still produce immunity, but only if it is signaled by a cognate BlpC. (D) Diagrammatic representation of the blp locus and the strategy used for the construction of chimeric blpH genes. A recipient strain containing a lacZ gene driven by the BIR promoter and the exchangeable Janus cassette in place of blpC is transformed with a PCR product containing the region from blpR to blpC of a noncognate strain. Any crossovers that replaced this cassette would occur upstream and downstream of the Janus cassette, inserting the blpC gene and some amount of the blpH gene, depending on the region of crossover. Black arrows, genes that are relatively highly conserved between the two strains; colored arrows, divergent genes. The BIR contains a variable array of bacteriocins (dark brown squares) and immunity proteins (light brown squares). Promoter regions are marked by right-angle arrows, and pBIR denotes the promoter driving expression of the first set of bacteriocins. The integrated plasmid that contains the lacZ gene and a chloramphenicol resistance cassette are denoted by white arrows or boxes and dashed lines. The BlpC-regulated genes, blpT (T) and blpS (S), are shown as white arrows.
To control the potential for cross stimulation, at least four different alleles of the blpC gene encode the peptide pheromone, and these correspond to the four major alleles of the blpH gene encoding the pheromone receptor (13, 14, 16, 18). The BlpC peptides are characterized by a highly conserved N-terminal signal sequence region followed by a double glycine motif. The signal sequence is cleaved from the active peptide during transport by the peptidase domain of the BlpA transporter. Genomes that encode a particular blpC allele typically encode the same histidine kinase allele, suggesting that a particular histidine kinase type will respond only to its cognate pheromone type (13, 14, 18). The BlpH receptor is a member of the class 10 receptor-histidine protein kinase family that includes ComD, involved in the induction of the pneumococcal competence state, and AgrC, involved in the regulation of virulence factors in Staphylococcus aureus. AgrC and ComD are also involved in the recognition of secreted peptides and are characterized by a similar degree of diversity in allelic variants that exists to restrict cross talk between neighboring strains (19–21). The N-terminal 70 amino acids (aa) of ComD have been implicated in peptide recognition, because this is where all the amino acid differences between the two major pherotypes lie (20). For AgrC, the recognition domain seems to depend on an interaction of the secreted peptides with amino acids in the first and second extracellular loops of the sensor domain (22, 23). No variant of the autoinducing peptide (AIP), which interacts with AgrC, is larger than 9 amino acids, and both competence-stimulating peptide (CSP) alleles that interact with ComD are 17 aa. These peptides are significantly smaller than three of the four BlpC types (27 aa), suggesting that the interaction of BlpC with BlpH may involve more than one site.
The blp locus appears to be absolutely conserved in the pneumococcal genome; however, we have shown previously that 50 to 70% of pneumococcal strains are unable to secrete their own pheromones or bacteriocins due to a disruption of the gene encoding the pheromone/bacteriocin transporter, blpA (17). These strains are still able to sense exogenous pheromone via BlpH binding, allowing production of bacteriocin immunity proteins that can be used to protect the organism from bacteriocin-mediated killing (Fig. 1B). These so-called cheater strains are at a competitive disadvantage if they are unable to recognize a pheromone produced by a neighboring inhibitory strain (Fig. 1C). Given the abundance of cheater genotypes in the population, pheromone specificity may act to limit cross talk between strains from different genetic backgrounds, preventing the induction of immunity. In this study, we demonstrate that in addition to the four major blpH alleles, there are three subtypes of one of the major alleles, blpH6A, which differ in their response to exogenous BlpC. Although restriction of cross talk between mismatched BlpC/BlpH types is the rule, some naturally occurring alleles can respond to more than one BlpC type. Using sequence information from naturally occurring chimeras and the pheromone responsiveness of laboratory-created chimeric BlpH sequences, we demonstrate that pheromone specificity is influenced by interactions with distinct regions in the N-terminal 250 aa of the BlpH protein. In particular, we have identified a key charge-charge interaction between aa 17 of BlpH and aa 14 of mature BlpC that plays a major role in peptide recognition and signaling in some BlpH/BlpC pairs. We have identified chimeric peptides that have the ability to respond to more than one BlpC type; however, these chimeras have reduced levels of responsiveness to their cognate pheromone, suggesting that there is a trade-off between the ability to detect multiple signals and the threshold for signaling.
MATERIALS AND METHODS
Bacterial strains and growth conditions.The S. pneumoniae strains used in this study are detailed in Table 1. All S. pneumoniae strains were grown at 37°C in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) or at 37°C in 5% CO2 on tryptic soy agar (TSA) plates supplemented with catalase (4,741 U/plate; Worthington, Lakewood, NJ) or 5% sheep blood (SBA). Escherichia coli strains were grown in Luria-Bertani (LB) broth or LB agar supplemented with the appropriate antibiotics at 37°C. The antibiotic concentrations used were as follows: for S. pneumoniae, 500 μg/ml kanamycin, 100 μg/ml streptomycin, and 200 μg/ml spectinomycin; for E. coli, 50 μg/ml kanamycin and 100 μg/ml spectinomycin.
Pneumococcal strains and plasmids used in this studya
Prediction of a proposed structure of BlpH.The amino acid sequence of BlpH was submitted to Stockholm University's TOPCONS program (http://topcons.cbr.su.se/), and the consensus prediction was used to determine proposed transmembrane portions. The information from TOPCONS was used to create a diagram of the transmembrane domains using the program Protter (http://wlab.ethz.ch/protter) (24).
Whole-genome sequence analysis.The region from blpT to blpA from the prototypic strain R6 was used to identify by a BLAST search contigs containing the blp locus in 229 publically available whole-genome sequence strains (see Table S1 in the supplemental material). This region was annotated, and the BlpC, BlpH, and BlpA protein sequences were aligned using the program CLC Sequence Viewer (CLCbio).
Determination of transcriptional activity of strains.The transcriptional activity of the reporter strains was determined by assaying β-galactosidase activity using the substrate o-nitrophenyl-β-d-galactopyranoside (ONPG). Strains were streaked onto SBA, inoculated into THY medium, and grown to an optical density at 620 nm (OD620) of 0.5. Cultures were frozen in 20% glycerol. For the assays, 100 μl of thawed culture was added to 15 ml of THY and allowed to grow to an OD620 of 0.20. Aliquots (90 μl) of cells were added to a 96-well plate containing 2-fold dilutions of synthetic active BlpC (sBlpC) in 10 μl of sterile water. Blank wells with medium alone and cells exposed to water only were used as controls. Plates were incubated for 1 h at 37°C before lysis with 10 μl of 1% Triton X-100. A 25-μl volume of ONPG in 5× Z buffer (5 mM MgCl, 50 mM KCl, 0.3 M Na2HPO4, 0.2 M NaH2PO4, 250 mM β-mercaptoethanol, 4 mg/ml ONPG) was added, and the reaction was allowed to continue until a discriminatory color change was observed. A 50-μl volume of 1 M NaCO3 was added to stop the reaction, and the plates were read at OD415 and OD550. Miller units were determined as previously described (25). Sample procedures were performed in triplicate, and each assay was performed at least three times, with representative results being shown. Statistically significant differences between the log values at which 50% of maximal activation is reached (log 50% effective concentrations [EC50s]) were determined by the F test using GraphPad Prism (version 6.0) software.
Construction of blpH reporter strains.Reporter constructs representing blpH types P164 and R6 were previously described (17). The 6A.1, 6A.2, 6A.3, and T4/R6 reporter strains were constructed as follows: PSD106, a type R6 lacZ reporter strain with a Janus cassette in place of blpC, was transformed with a PCR fragment of blpRHCB amplified from strains P1, P795, P802, and P801, respectively, using primers 5 and 12 (primer sequences are listed in Table 2). The Janus cassette contains the gene for kanamycin resistance and the rpsL+ gene for streptomycin sensitivity (26). Removal of the Janus cassette by allelic exchange results in kanamycin-sensitive/streptomycin-resistant transformants. The T4 reporter strain was constructed as follows: a lysate from strain P139 was used to transform PSD106. Allelic replacements were identified on streptomycin-containing plates and confirmed to be kanamycin sensitive. Colonies were screened on medium containing X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), and blue colonies were selected and verified by sequencing. To construct reporters that could not self-signal, a deletion in blpC created with allelic replacement with a spectinomycin cassette was amplified from strain PSD110 and transformed into each reporter strain to replace blpC by homologous recombination. All constructs were verified by sequencing.
Primers used in this study
Construction of chimeric strains.Pneumococcal transformations were performed using type R6 and type 6A blpH reporter strains with a Janus cassette in place of blpC. The process for the creation of chimeric blpH strains is depicted in Fig. 1D. blpRHC of strain P800 was PCR amplified using conserved-sequence primers specific for sequences within blpR and blpB (primers 1 and 4). This PCR product was purified and transformed into a BlpHR6- or BlpH6A.1-expressing strain with the Janus cassette in place of blpC, PSD106, or PSD108 with a lacZ reporter gene fused to the first BIR promoter. Pneumococcal transformations were performed as previously described (12). Any transformants in which the Janus cassette was removed by allelic exchange with P800 DNA (streptomycin resistant, kanamycin sensitive) were sequenced to identify exactly where fusion between the two sequence types occurred. A similar approach was used to construct the blpH6A.3-blpHP164 fusions, except that sequentially smaller PCR products derived from amplification off the blpHP164-containing strain, PSD121, using primers 13, 14, and 15 with common downstream primer 5 were used to transform blpH6A.3 reporter strain PMP103 to determine the minimal sequence required to recover a strain with a BlpHP164-like phenotype. These transformants were selected directly on spectinomycin to screen for integration of the spectinomycin resistance gene in blpC. To determine responsiveness, chimeric strains were streaked on medium containing X-Gal and 500 ng/ml of sBlpC types P164 and R6 or type 6A (GenScript, Piscataway, NJ). The BlpC and sBlpC sequences are listed in Table 3. Each chimera's phenotypic response to sBlpC was correlated with the chimeric blpH nucleotide sequences to identify patterns. For any strains that self-induced on medium containing X-Gal, the blpC gene of that chimera was deleted via transformation with a PCR product from the corresponding reporter strains containing the blpC::aap9 insertion to determine its true phenotypic response to sBlpC.
sBlpc sequences used in this study
Construction of site-directed mutant strains.The site-directed mutant strains PMP110 and PMP111 were created using strains PMP106 and PMP107, respectively. A portion of the blp locus containing the C-terminal region of blpR, the entire blpH gene, the spectinomycin cassette in place of blpC, and the N-terminal region of blpB was cloned from each strain using primers 1 and 4. To construct site-directed mutants, these fragments were TA cloned (TOPO TA Cloning kit [version 2.1]; Invitrogen, Grand Island, NY) and transformed into E. coli DH5α cells, selected on spectinomycin, and then sequenced to ensure that no mutations had been introduced, creating plasmids pE161 and pE162. These plasmids were then used as a template for site-directed mutagenesis (QuikChange II site-directed mutagenesis kit; Agilent Technologies, Santa Clara, CA) using the manufacturer's instructions. Two synthetic oligonucleotide primers that contained the mutation of interest were designed. The resulting mutagenized plasmids, pE163 and pE164, containing mutations that changed aa 17 of each BlpH sequence, were sequenced to confirm the presence of the mutation and transformed into PMP109. Colonies were selected for streptomycin and spectinomycin resistance and screened for the loss of kanamycin resistance to identify strains that had replaced the Janus cassette and integrated the blpC deletion. The resulting strains were sequenced to ensure integration of the mutation of interest. Site-directed mutant strains were tested for pheromone responsiveness on medium containing X-Gal or by Miller assay, as described above.
RESULTS
The major allelic variants of blpH have sequences that differ throughout the length of the protein.BlpH is predicted to have seven transmembrane regions and an intracellular enzymatic kinase domain (see Fig. S1 in the supplemental material). Only short segments of the protein are predicted to be located outside the cell. This predicted topology is remarkably similar to the recently refined topology map of AgrC that was verified using substituted cysteine accessibility and green fluorescent protein fusions (27). A survey of the region from blpH through blpA in 229 strains for which the whole-genome sequence (WGS) was available (WGS strains) confirmed that there are four major allelic variants of blpH (blpHP164, blpHR6, blpH6A, and blpHT4). The gene product encoded by blpHR6 has the most divergent sequence, sharing only 86% identity at the amino acid level with the other alleles (Table 4; see also Fig. S2 in the supplemental material). BlpHP164 and BlpH6A have much more similar sequences, sharing approximately 92% amino acid sequence identity. Alignment of the blpH6A alleles associated with the blpC6A gene revealed three suballeles of the blpH6A gene; we refer to these as blpH6A.1, blpH6A.2, and blpH6A.3. The gene products of these alleles share approximately 98% sequence identity, with most of the differences in the 6A subset occurring in the first 60 amino acids (see Fig. S2 in the supplemental material). BlpH6A.3 is a naturally occurring P164/6A-type chimera; the first 49 amino acids are 100% identical to the BlpHP164 sequence, and the remainder of the protein sequence is BlpH6A-like. Unlike the 6A minor variants, the sequence differences between the four major allelic variants of BlpH are distributed throughout the first 214 amino acids of the protein (see Fig. S2 in the supplemental material). The four most common allelic variants of the pheromone BlpC also have highly divergent sequences following the double glycine motif at which the prepeptide is cleaved. BlpCT4 is truncated at its C terminus, missing the final 9 amino acids present in the other three variants (Table 3).
Percent identity among the amino acid sequences of the most common types of BlpH
Analysis of the upstream region of the blp locus from the WGS strains demonstrated that the four major blpH alleles are relatively evenly distributed in this collection (Table 5; see also Table S1 in the supplemental material). The presence or absence of a disrupting mutation in the transporter gene was determined for each of the 229 WGS strains. Over 90% of the strains expressing the BlpHT4 type are predicted to have cheater phenotypes because the blp locus encodes a disrupted version of the BlpA transporter (Table 5; see also Table S1 in the supplemental material). This finding was unexpected because it suggests that very few pneumococci would be expected to secrete the BlpCT4 peptide, which has implications for the survival of BlpHT4 cheater strains. The remaining BlpH types are represented by more equal distributions of disrupted and nondisrupted transporter genotypes.
Distribution of BlpH types in 229 WGS strains and association with nonfunctional blpA sequencesa
To determine the pheromone specificity for each of the blpH alleles, we performed stimulation assays using increasing concentrations of each of the four BlpC peptides on reporter strains constructed to encode each of the six primary blpH alleles and a naturally occurring BlpH chimera, BlpHP801. Reporter strains were constructed to have the 5′-most BIR promoter driving the lacZ gene and a spectinomycin cassette in place of the blpC gene to prevent stimulation through the production of endogenous pheromone. Maximal stimulation levels were assessed for all strains, and the values at which 50% of maximal activation was reached (EC50) were calculated for specific 6A subtype/BlpC pairings (Fig. 2A to H).
Specificity and degree of response of BlpH types to cognate and noncognate BlpC types. (A to H) Reporter strains with a deletion in blpC were tested for responsiveness to increasing concentrations of the four major BlpC types. Strains were stimulated with increasing concentrations of BlpC ranging from 0 ng/ml to 500 ng/ml. (A to G) Results are displayed on a log10 scale x axis, with the concentration of BlpC increasing from left to right for each peptide/reporter combination. The BlpC types used are noted in the key. The blpH type of the reporter strain is noted above each set of graphs. The expected BlpC responsiveness of each blpH allele on the basis of the cooccurring blpC allele is shown as a colored line below the blpH designation. (H) EC50s for 6A.1, 6A.2, and 6A.3 BlpH reporter strains when induced by the BlpC types noted. The mean and 95% confidence intervals are shown. *, P < 0.0001. All EC50s were determined by normalizing values to range from 0 to 100% followed by nonlinear fit analysis. (I) Natural induction profile of two of the BlpH6A reporters with an intact blpC gene during growth in broth over time after initiating growth at an OD600 of 0.05.
BlpHP164- and BlpHR6-expressing reporters responded only to their cognate pheromone, with no evidence of cross talk (Fig. 2A and B). BlpH6A.1- and BlpH6A.2-expressing reporters responded to BlpCT4 in addition to BlpC6A; however, the maximal responsiveness to the noncognate peptide was low (Fig. 2C and D). The reporter expressing the naturally occurring BlpHP164/6A chimera, BlpH6A.3, responded to both BlpC6A and BlpCP164 (Fig. 2E). The maximal activation level was lower for BlpC6A in this reporter than for the other 6A suballeles, and the EC50 was significantly higher (179 ng/ml compared with 12 and 15 ng/ml for BlpH6A.1 and BlpH6A.2, respectively) (Fig. 2H), consistent with an overall trend of broadened peptide specificity correlating with decreased sensitivity to peptide stimulation. In fact, the BlpH6A.3 chimera actually responded more robustly to the noncognate peptide BlpCP164 than to its cognate peptide (the EC50 for BlpCP164 was 45 ng/ml, whereas that for BlpC6A was 179 ng/ml) (Fig. 2H). Like the 6A.1 and 6A.2 alleles of BlpH, the reporter strain expressing BlpHT4 responded to both BlpC6A and BlpCT4 (Fig. 2F), although in this case, the response to both cognate and noncognate peptides was characterized by low maximal activation levels (a 3-fold increase from uninduced samples for both BlpC types compared with 5- to 20-fold increases for other receptor/pheromone pairs).
The survey of the WGS strains identified an additional naturally occurring BlpH chimera, BlpHP801, in which the N-terminal 33 aa were derived from BlpHT4 and the remainder of the sequence was derived from BlpHR6. The genome of the strain with this allele, P801, encodes a blpCR6 allele. Unlike the BlpH6A.3-expressing chimera, which demonstrated dual responsiveness, the BlpHP801-expressing reporter strain responded only to BlpCR6 without any appreciable response to BlpCT4 (Fig. 2G); the pattern of response was very similar to that noted with the full-length BlpHR6-expressing strain (Fig. 2B), suggesting that in this case, the first 33 amino acids are not involved in peptide discrimination.
To demonstrate the in vivo consequences of the diminished response noted with dually responsive receptors, reporter strains with either the blpH6A.1 (singly responsive receptor) or the blpH6A.3 (dually responsive receptor) allele with an intact blpC6A gene were grown in broth culture, and samples were taken at 30-min intervals (Fig. 2I). The blpH6A.1-containing strain induces earlier and to a greater extent than the blpH6A.3-containing strain, demonstrating that the increased EC50 for this strain translates into a diminished ability to self-induce. These data confirm that the decreased specificity for pheromone signaling comes at a cost of decreased overall sensitivity and that this change translates into a decreased and delayed response to self-signaling.
The N-terminal domain of BlpH is responsible for pheromone responsiveness.Because the naturally occurring chimeric strains expressing the BlpH6A.3 and BlpHP801 types provided conflicting results about the importance of the N terminus to pheromone responsiveness, we created a series of laboratory-generated reporter strains expressing chimeric BlpH types to clarify the sequences that are involved in peptide recognition. Because chimeras were obtained in an unbiased fashion by allowing recombination between the two alleles at any point of homology along the gene, we recovered a large number of blpH alleles with distinct junctions between the two alleles.
We first created fusions between the highly divergent blpHR6 and blpHP164 alleles. The resulting BlpHR6/BlpHP164 chimeras had variable amounts of BlpHR6 sequence in the N terminus joined to the C terminus of BlpHP164 and expressed BlpCP164. There were three primary phenotypic outcomes when these chimeras were plated on medium with X-Gal and peptide pheromone (Fig. 3A to D). Any chimera with a fusion of the two BlpH types within the first 167 amino acids of the protein did not respond to either sBlpC peptide pheromone, suggesting that a fusion in this region disrupts all signaling (Fig. 3B). Any chimera with a fusion of the two BlpH types between amino acids 182 and 228 was active, even without the addition of sBlpC of either pherotype. When the blpC gene of two of these chimeras was deleted, the locus was still highly active even without the addition of sBlpC (Fig. 3C), suggesting that the fusion resulted in a constitutively activated form of the protein. Any chimera with a fusion of the two BlpH types downstream of amino acid 250 responded only to exogenous sBlpCR6 and not to BlpCP164 (Fig. 3D).
Phenotypes of BlpHR6 and BlpHP164 chimeric strains. (A) Diagrammatic representation of the topology of BlpH predicted by TOPCONS shown with the phenotypic result of fusions between the N terminus of BlpHR6 and the C terminus of BlpHP164. Predicted transmembrane regions are shown as rounded rectangles. The region from aa 16 to 170 in orange denotes the span of the fusion points that resulted in a nonfunctional BlpH. The region from aa 182 to 229 in gray denotes the location of the fusions that resulted in a constitutively active BlpH phenotype. The region from aa 251 to 446 in red denotes the region of fusions that resulted in a BlpHR6-responsive phenotype. Regions in black were not specifically tested because no chimeric fusions between the strains with two distinct phenotypes were identified. (B to D) The graphs show the Miller units of a representative chimera in response to 2-fold-increasing concentrations of BlpCP164 or BlpCR6 ranging from 0 to 500 ng/ml. The data on the x axis are on a logarithmic scale, and both axes are identical in each graph. Graphs outlined in orange (B), gray (C), and red (D) depict the response of a representative chimera from the color-matched region. Points of chimeric fusion that were tested are noted by amino acid number.
These data suggest that the sensor domain of BlpH lies in the region of the transmembrane domains within the first 250 aa of the protein (Fig. 3A). The isolation of chimeric strains that had a constitutively active phenotype consistent with altered signaling suggests that a signal transduction domain lies in the region including aa 180 to 228, which falls in the 7th transmembrane segment.
Because all fusions in the N-terminal 167 aa of BlpHR6 resulted in nonfunctional histidine kinases, we chose to create fusions between two more closely related blpH alleles in an attempt to better delineate the amino acids involved in pheromone specificity. The BlpH6A.1/BlpHP164 chimeras had variable amounts of the BlpH6A.1 sequence in the N-terminal region of BlpH fused to a C-terminal portion of the BlpHP164 sequence and an intact BlpCP164 (Fig. 4). The only chimera that responded to BlpCP164 contained 7 amino acids of BlpH6A.1 in the N terminus, followed by the BlpHP164 sequence for the remainder of the protein (Fig. 4A and B). Any chimera with at least 17 amino acids of the 6A.1 sequence in the N terminus responded only to BlpC6A, although maximal activity and the EC50 were significantly reduced compared with those for the wild type (Fig. 4A and C). A fusion of BlpH6A.1 to BlpHP164 at aa 159 had an EC50 that was equivalent to that of the wild type (Fig. 4D to F). There were no nonfunctional chimeras isolated. These chimeras demonstrated that 17 amino acids of the BlpH6A sequence joined to the remainder of the BlpHP164 sequence were sufficient to remove responsiveness to BlpCP164 and promote responsiveness to BlpC6A. This fusion, however, did not restore wild-type activity, suggesting that additional sites important for binding or signal transduction lie between aa 17 and 159.
The first 17 amino acids of BlpH6A are sufficient to change pheromone responsiveness in BlpH6A.1/BlpHP164 chimeras. (A to E) Dose-response curves for full-length BlpHP164, BlpH6A.1, and chimeras consisting of portions of BlpH6A.1 joined to BlpHP164. The number of N-terminal amino acids derived from BlpH6A.1 is shown in white. Reporters were incubated with 2-fold dilutions of sBlpC from 0 to 500 ng/ml. The resulting fold change from the result obtained with no peptide is shown on a log10 x axis. All y-axis scales are identical. (F) The EC50s of the BlpC6A-responsive chimeras and the wild-type strain are shown with 95% confidence intervals.
Changing a single amino acid of BlpH is sufficient to change pheromone responsiveness.Data from the chimeric BlpH6A/BlpHP164-expressing reporters suggested that the amino acids between aa 7 and 17 of BlpH play an important role in pheromone binding. Examination of the two sequences up to aa 17 demonstrated two positively charged amino acids at positions 14 and 17 in BlpHP164 that were replaced with a neutral amino acid and a negatively charged amino acid in the BlpH6A.1 sequence (Fig. 5A). Both BlpHP164 and BlpH6A.3, which has a type P164 sequence in its N terminus, have a positively charged histidine at position 14 and a lysine at position 17. All the other BlpH types (6A.1, 6A.2, R6, and T4) have an asparagine at position 14 and a negatively charged glutamate at position 17. These differences appear to correlate with an opposite amino acid difference in the cognate BlpC sequences at position 14. At position 14 of sBlpC, type P164 has a negatively charged glutamate, while types R6 and 6A have a positively charged lysine residue (Fig. 5A). Since these two amino acids are oppositely charged in cognate pairs, we hypothesized that aa 17 of BlpH might interact with aa 14 of sBlpC, resulting in an electrostatic interaction that influences binding.
The interaction between aa 17 in BlpH and aa 14 in BlpC plays an important role in peptide specificity. (A) The amino acid sequences of the active forms of BlpCP164 and BlpC6A and the first 50 aa of the noted BlpH types are shown as an alignment. Differences in amino acid sequences between the types are shaded in gray, and the region implicated to be responsible for the pheromone specificity identified in Fig. 4 is shown as a box from aa 8 to 17 of BlpH. The locations of the oppositely charged amino acids in BlpC and BlpH are marked with arrows. (B to D) Dose-response curves for reporter strains with and without charge switch mutations at aa 17. Twofold dilutions of each of the two BlpC peptides BlpCP164 and BlpC6A and their derivatives, BlpCP164E14K and BlpC6AK14E, respectively, were used to stimulate reporter strains. The blpH allele in each reporter is shown above the graphs. All x and y axes are identical in scale. y axes show the fold change from the result for no peptide added for each strain, and x axes are shown in log10 scale. (E) EC50s and 95% confidence intervals for all dose-response curves with R2 values over 0.8. Colors designating BlpC types are identical to those described for panels B to D. P values compare log EC50 values. *, P < 0.001; **, P < 0.0001.
To address the role of aa 17 in pheromone specificity, we created two new blpH alleles in the blpH6A.2 and blpH6A.3 backgrounds in which aa 17 was switched to the oppositely charged residue. The two site-directed mutants were created in a reporter background with a deletion in blpC so that the influence of endogenously produced peptide did not interfere with the dose-response curves. This approach allowed us to examine the impact of amino acid 17 of BlpH in determining the change in responsiveness between types 6A.2 and 6A.3 specifically. We found that an E17K change in a BlpH6A.2 background was sufficient for this mutant to gain responsiveness to BlpCP164, although, like the naturally occurring BlpH6A.3 allele, its response to BlpC6A suffered in comparison to the BlpH6A.2 wild-type response profile (Fig. 5B). The K17E change in the dually responsive BlpH6A.3 background (BlpH6A.3K17E) was not sufficient to completely remove BlpCP164 responsiveness (Fig. 5C). This is most likely due to the presence of the neighboring positively charged histidine residue at position 14 in BlpH6A.3 that may partially compensate for the loss of the lysine residue. The BlpH6A.3K17E mutant did strongly respond to very low concentrations of BlpC6A compared to the response in the wild-type background (Fig. 5C). These findings are supported by calculation of the EC50s for the two mutants (Fig. 5E), which demonstrated that the EC50 of BlpCP164 stimulation of BlpH6A.2E17K was similar to that seen in wild-type BlpHP164-expressing reporters (Fig. 5D and E) and that the K17E mutation in BlpH6A.3 resulted in a significant drop in the EC50 in response to BlpC6A compared with the EC50 in response to BlpC6A of the parental strain. The fact that responsiveness to BlpC6A was not completely lost in the BlpH6A.2E17K-expressing strains demonstrates that additional regions of the N-terminal 250 aa are involved in dictating specificity.
An electrostatic interaction between BlpH and BlpC determines pheromone specificity in some BlpC/BlpH interactions.To determine if the importance of aa 17 in BlpH was due to an electrostatic interaction between aa 17 of BlpH and aa 14 of BlpC, we created synthetic signaling peptides in which aa 14 in BlpCP164 and BlpC6A was exchanged for the oppositely charged amino acid. These peptides were used to stimulate the BlpH6A.2, BlpHP164, and BlpH6A.3 reporters. The BlpCP164 peptide with the E14K mutation (the BlpCP164E14K peptide) lost the ability to stimulate the BlpHP164 or BlpH6A.3 allele, and the change was not sufficient to allow stimulation of the remaining BlpC6A alleles (Fig. 5B to D). The BlpC6AK14E peptide was able to stimulate transcription from all BlpC reporters that were tested. The response of the BlpHP164 reporter strain to this peptide, however, was significantly less than the response to cognate BlpCP164, reflected both in a higher EC50 and in lower maximal stimulation values (Fig. 5D and E), suggesting again that further interactions with the peptide are involved in receptor binding and stimulation. These data support the role of the electrostatic interaction between BlpH aa 17 and sBlpC aa 14 in type-specific binding, in particular, influencing the ability of the BlpCP164 peptide to bind to its cognate receptor. The electrostatic interaction at aa 17 appears to be less critical for BlpC6A receptor binding, as these receptors could still be stimulated even when the C/H interactions would involve electrostatic repulsion due to changes in either the receptor or the peptide.
Additional regions of peptide recognition exist within the sensor domain.To identify additional areas of peptide recognition, we took advantage of the observation that the BlpH6A.3 receptor was activated by both BlpCP164 and BlpC6A but the BlpHP164 receptor was activated only by BlpCP164. Because these two proteins are identical in the first 49 aa, the region involved in the restriction of BlpC6A activation of BlpHP164 must lie within the sensor domain but after aa 49. Comparison of the sequence differences in this region between all BlpC6A-responsive alleles (all BlpH6A subtypes and BlpHT4) and BlpCP164 demonstrated that there were charge or polarity differences at aa 79, 119, 152, 157, 225, and 229 that might explain the BlpC6A restriction (see Fig. S2 and S3 in the supplemental material).
To better delineate the region involved, the BlpH6A.3 reporter strain was transformed with a PCR product amplified from the BlpHP164 reporter. Transformants would contain a chimeric BlpH with an N terminus derived from BlpH6A.3 and a C terminus derived from BlpHP164. Transformants were screened on medium containing X-Gal and either BlpCP164 or BlpC6A. Five transformants with the BlpHP164 phenotype and nine transformants with the BlpH6A.3 phenotype were sequenced to delineate the region of BlpHP164 that resulted in BlpC6A exclusion. BlpH6A.3/BlpHP164 fusions that occurred after aa 124 had the BlpH6A.3 phenotype (blue on both peptides), effectively eliminating the region downstream of aa 124 in BlpC6A restriction (Fig. 6A). All colonies that had the BlpHP164 phenotype (white on BlpC6A and blue on BlpCP164) had an identical fusion point just before aa 119 (Fig. 6A). This suggests that the region of BlpHP164 that restricts the response to BlpC6A spans the region between and including aa 119 and aa 124. This region is adjacent to the second extracellular loop within the fifth transmembrane domain and contains three differences between the BlpCP164- and BlpC6A-responsive BlpH alleles: aa 119, G → D; aa 123, I → T; and aa 124, W → G (Fig. 6B; see also Fig. S2 in the supplemental material). This region is in a location nearly identical to the region identified to be involved in AIG specificity in the S. aureus histidine kinase AgrC (22).
The fifth transmembrane domain of BlpH contains a region important in pheromone specificity. (A) Diagrammatic representation of the first 250 aa of BlpH containing the region-defining fusions between BlpH6A.3 and BlpHP164 and their respective responsiveness to BlpC. Blue, BlpHP164 sequence; green, BlpH6A sequence. The numbers over fusion points signify the first amino acid of the C-terminal type. As noted in the text, BlpH6A.3 is a naturally occurring chimera that contains the first 49 aa of BlpHP164 fused to a BlpH6A-derived sequence. Chimeras made from the two alleles have a BlpHP164 phenotype if the fusion occurred N terminal to aa 119 and a BlpH6A.3 phenotype if the fusion occurred C terminal to aa 124. Plus and minus signs denote the phenotype (blue and white, respectively) of strains expressing the noted BlpH types on plates supplemented with X-Gal and sBlpC. (B) The predicted topography of BlpH and the locations implicated in peptide recognition from chimeric studies up to the intracellular kinase are shown. Orange, the area of fusion found in either inactive chimeras (when fusing divergent alleles) or chimeras with decreased function (when fusing more similar alleles); red, the predicted location of aa 17 within the first transmembrane domain; yellow, aa 119 to 124 implicated in BlpC6A exclusion in BlpHP164.
DISCUSSION
We have shown that there are six primary allelic variants of the histidine kinase BlpH of the blp locus in Streptococcus pneumoniae. Some of these receptors exhibit typical pheromone specificity, activating the blp locus only when bound by their cognate pheromone. Analysis of naturally occurring chimeras and laboratory-derived fusion proteins has demonstrated that for some peptide/receptor interactions, recognition involves an electrostatic interaction between aa 14 of the pheromone and aa 17 of the receptor. Transmembrane prediction programs place aa 17 of BlpH within the first transmembrane domain, suggesting that the peptide must associate intimately with the membrane for this interaction to occur (Fig. 6B). A similar mechanism of interaction of CSP with its receptor, ComD, in the membrane has been hypothesized to involve a hydrophobic patch in an alpha-helical stretch within the signaling peptide (28). The interaction between aa 17 of BlpH and aa 14 of BlpC is necessary for BlpCP164-mediated signaling, but our data for BlpC6A recognition and the phenotype of the naturally occurring BlpHT4/BlpHR6 chimera make it clear that additional interactions occur between the peptide and receptor in the first 250 aa. One interaction seems to involve amino acids adjacent to the second extracellular loop, similar to the region identified in AgrC specificity (22) (Fig. 6B). At this point, it is not clear whether there is direct peptide interaction with this region and, if so, what sequence in the peptide is responsible for this binding. Interestingly, unlike the interaction of noncognate AIG with AgrC, there is no evidence of antagonism between noncognate BlpC peptide/receptor pairs (data not shown), suggesting that the inability of certain pheromones to signal noncognate receptors is a result of low affinity for the receptor rather than a difference in signal transmission.
In this study, we found that almost all BlpHT4 variants in the WGS strains were predicted to encode nonfunctional BlpA proteins, meaning that most BlpHT4 strains are cheater strains that are unable to secrete their own bacteriocins but would retain the ability to respond to pheromone secreted by other strains, allowing the production of immunity. If receptor specificity were restricted to only cognate pheromones, the lack of BlpCT4-secreting strains in the population would make this group of strains vulnerable to killing by any bacteriocin-secreting strain. Perhaps as a means for survival, BlpHT4 strains are capable of responding to both cognate BlpCT4 and noncognate BlpC6A, a common pherotype that is found in both producer and cheater backgrounds. The ability of BlpHT4 strains to respond to two pheromone types provides additional evidence of the relevance of pheromone specificity to pneumococcal competition.
Fusions in this N-terminal region of interest between the two most genetically different allelic variants, BlpHP164 and BlpHR6, were nonfunctional, while fusions between types more similar in this region (BlpHP164 and BlpH6A) were both functional and informative. These findings suggest that fusions between more similar BlpH types are more compatible with a functional receptor. In the case of the completely nonfunctional fusions, we were unable to determine if the lack of activity was due to poor peptide binding or to an unstable protein that was being rapidly degraded. We attempted to address this issue by generating polyclonal antibodies against a conserved region of BlpH; however, the resultant antibodies were unable to recognize BlpH in Western blots of whole-cell lysates or concentrated membrane preparations (not shown), either because of poor antibody sensitivity or because histidine kinases are generally made in very small quantities.
Our study of the WGS strains identified several unique naturally occurring blpH chimeras that resulted from recombination events between blpH alleles. One of these fusions consisted of a receptor that contained the first 33 aa of BlpHT4 joined to the remainder of the BlpHR6 sequence (BlpHP801). Unlike the naturally occurring P164/6A fusion, BlpH6A.3, in which an important region of specificity seems to lie in the extreme N terminus, the BlpHP801 receptor responded only to BlpCR6. This is likely due to the lack of a charge difference at aa 17 between BlpHT4 and BlpHR6 (and a lack of a charge difference in the corresponding aa 14 of the BlpC peptides) (Table 3; see also Fig. S2 in the supplemental material), suggesting that, in this case, peptide interactions after aa 33 are responsible for peptide specificity.
Fusions between the two dissimilar BlpH types resulted in a series of chimeras that had a constitutively active phenotype. All the fusion points occurred just before and in the linker domain that joins the receptor domain of the histidine kinase to the kinase domain. Studies in the structurally homologous AgrC receptor have demonstrated that this region is important in the presentation of the kinase domain of one member of a histidine kinase dimer to the phosphate-accepting histidine residue of the other member (29). The change in the relative position of these two domains between members of the dimer upon peptide binding has been shown to be responsible for histidine phosphorylation. Given their location in this critical domain, the fusion point of our constitutively active BlpH alleles is likely to have altered the relative position of the kinase domain, resulting in histidine phosphorylation in the absence of peptide binding.
In the competitive polymicrobial environment of the human nasopharynx, the ability to limit cross talk between strains might improve the survival of one strain over the other. For a bacteriocin producer, it would be advantageous to secrete a peptide that can bind only to other members of the clonal population to restrict the ability of neighboring strains to sense the signal and produce protective immunity. However, while peptide specificity provides an advantage for producer strains, the ability to intercept noncognate peptides would be beneficial for cheater strains that cannot secrete their own peptide pheromone. On the basis of our data, there appears to be a trade-off; BlpH types that benefit from the ability to respond to two different pheromone types have a lower sensitivity and a lower magnitude of response to either pheromone. These complex interactions involve a balance between the energetic cost of bacteriocin/immunity production and the benefit derived from their activity. We identified four unique chimeric BlpH types in addition to those described in this work in the blp loci of the 229 WGS strains (see Table S1 in the supplemental material), suggesting that the creation of chimeric receptors is a relatively common occurrence. With a continuous supply of DNA to support the development of novel chimeric proteins, strains can change the type and degree of response to secreted peptides, as well as change the ability to secrete pheromone signals by modification of blpA and the repertoire of bacteriocin and immunity proteins produced, to better compete within the nasopharynx.
ACKNOWLEDGMENTS
This work was supported by grant number R01AI101285 from the NIAID.
We thank Michael Watson for his careful reading of the manuscript.
FOOTNOTES
- Received 22 October 2014.
- Accepted 21 January 2015.
- Accepted manuscript posted online 26 January 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.02432-14.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
