The blp Bacteriocins of Streptococcus pneumoniae Mediate IntraspeciesCompetition both In Vitro and In Vivo�
Suzanne Dawid,2 Aoife M. Roche,1 and Jeffrey N. Weiser1*Departments of Microbiology and Pediatrics, University of Pennsylvania School of Medicine,1 and Division of
Infectious Diseases, Children’s Hospital of Philadelphia,2 Philadelphia, Pennsylvania
Received 2 November 2005/Returned for modification 8 February 2006/Accepted 18 October 2006
The introduction of the conjugate seven-valent pneumococcal vaccine has led to the replacement of vaccineserotypes with nonvaccine serotypes of Streptococcus pneumoniae. This observation implies that intraspeciescompetition between pneumococci occurs during nasopharyngeal colonization, allowing one strain or set ofstrains to predominate over others. We investigated the contribution of the blp locus, encoding putativebacteriocins and cognate immunity peptides, to intraspecies competition. We sequenced the relevant regions ofthe blp locus of a type 6A strain able to inhibit the growth of the type 4 strain, TIGR4, in vitro. Using deletionalanalysis, we confirmed that inhibitory activity is regulated by the function of the response regulator, BlpR, andrequires the two putative bacteriocin genes blpM and blpN. Comparison of the TIGR4 BlpM and -N amino acidsequences demonstrated that only five amino acid differences were sufficient to target the heterologous strain.Analysis of a number of clinical isolates suggested that the BlpMN bacteriocins divide into two families. Amutant in the blpMN operon created in the clinically relevant type 19A background was deficient in bothbacteriocin activity and immunity. This strain was unable to compete with both its parent strain and a serotype4 isolate during cocolonization in the mouse nasopharynx, suggesting that the locus is functional in vivo andconfirming its role in promoting intraspecies competition.
Streptococcus pneumoniae colonizes a majority of children by1 year of age. Colonization with a single strain can last forweeks to months but is eventually cleared. Serial culturing ofsamples from the nasopharynxes of children has demonstratedthat the population of pneumococci is constantly in flux, withone strain replacing another over time (1, 2, 5, 12). In addition,some children appear to be colonized with two or more pneu-mococcal strains at any given time (7, 20). The recent intro-duction of the seven-valent conjugate pneumococcal vaccine tothe standard infant vaccination schedule has dramatically re-duced the incidence of both colonization and invasive diseasecaused by the pneumococcus (24). Because the vaccine con-tains only 7 of the �90 serotypes of pneumococci, there havebeen concerns that some of the remaining serotypes may fillthe void left by the vaccine-targeted organisms. Over the in-tervening years since its release, reports have suggested thatserotype replacement may be occurring at the levels of bothcolonization and disease (6, 9, 13). For example, the incidenceof the previously uncommon nonvaccine serotype 19A hasincreased significantly in the postvaccine era as a cause ofinvasive pneumococcal disease in children of less than 5 yearsof age (17). Whether infections with these formerly infrequentserotypes will eventually result in a similar disease spectrumwith respect to numbers and severity has yet to be determined.The fact that serotype replacement has occurred suggests thatcompetition between the predominant vaccine strains and thereplacement strains was occurring at some level prior to vac-cination of the population. Understanding the dynamics that
occur between distinct strains of pneumococci within thepolymicrobial environment of the human nasopharynx mayhelp to better predict the outcome of vaccination. These inter-actions are likely to include bacterial factors that allow onestrain to gain a foothold in the competitive environment of thenasopharynx.
Bacteriocins are small antimicrobial peptides produced bymany bacterial species that have been implicated in intra- andinterspecies competition. Bacteriocins typically target organ-isms that are either closely related to or within the same spe-cies as the producer bacteria (4). Producer bacteria are pro-tected from the effects of their own bacteriocins via productionof a specific immunity protein. This protein is typically cotran-scribed with the genes encoding the bacteriocins. The blp locusof pneumococcus encodes a number of putative bacteriocin-like peptides (3, 19). Upstream of the bacteriocin genes, thelocus contains open reading frames for a typical two-compo-nent regulatory system (blpR and blpH), a small peptide pher-omone (blpC), and a dedicated ABC transporter (blpA and-B). The ABC transporter is thought to recognize the N ter-mini of both the pheromone and the bacteriocins and to trans-port these peptides across the cytoplasmic membrane, concur-rent with cleavage at a conserved double-glycine motif.Cleaved extracellular BlpC can then bind to the sensor kinase,BlpH. This interaction results in the activation of BlpR andupregulation of the entire gene cluster via binding to consensussequences within each promoter. Transcriptional analysis ofthe locus in the two fully sequenced pneumococcal strains R6and TIGR4 demonstrated that application of chemically syn-thesized BlpC resulted in upregulation of a number of operonsonly within the locus, including those encoding the regulatoryproteins, transport apparatus, and putative bacteriocins (3).The transcript level of a downstream operon encoding BlpXY
* Corresponding author. Mailing address: Departments of Microbi-ology and Pediatrics, University of Pennsylvania, 402A Johnson Pavil-ion, Philadelphia, PA 19104-6076. Phone: (215) 573-3511. Fax: (215)573-4856. E-mail: [email protected].
and -Z was also upregulated by the addition of BlpC. Thisoperon encodes proteins proposed to be involved in immunity.Analysis of a number of pneumococcal strains demonstratedthat there are at least four different pheromones produced andthat each is specific for its cognate BlpR/H protein (3, 19).
In this study, we characterized the blp locus in a clinicalisolate of pneumococcus that demonstrates an in vitro pheno-type consistent with bacteriocin activity and further defined theimportance of the locus in competition during model murinenasopharyngeal colonization. In addition, we sequenced thebacteriocin genes of a number of clinical isolates and deter-mined which amino acids are important in dictating inhibitionin vitro.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The S. pneumoniae strains used inthis study are described in Table 1. An array of clinical strains were analyzed andconsidered unrelated based on differences in capsular type and time and locationof isolation. All strains were grown in tryptic soy broth (TS) or on tryptic soy agar(TSA) supplemented with catalase (4,741 U/plate; Worthington, Lakewood, NJ),except where indicated. TSA was supplemented with streptomycin (100 �g/ml),kanamycin (500 �g/ml), or erythromycin (1 �g/ml) where indicated. Cultureswere grown on agar plates at 37°C in 5% CO2 or anaerobically using a BBL gaspack system. Broth cultures were grown at 37°C without agitation. For transfor-mation, bacteria were grown from plates at low inocula in C�Y (pH 8.0) (11a)at 37°C until the optical density at 620 nm reached 0.150. One-hundred-micro-liter aliquots were removed and placed at 30°C with 10 ng/ml of purified com-petence-stimulating peptides 1 and 2. After 10 min, approximately 100 pg/ml ofDNA was added to the mixture and incubated at 30°C for an additional 40 min.The culture was then transferred to 37°C and incubated for 2 h before beingplated on selective medium.
Bacteriocin assay. Pneumococci grown on TSA plates overnight were resus-pended in phosphate-buffered saline (PBS) to an optical density at 595 nm of0.700. Test strains were then stabbed into TSA plates and allowed to growanaerobically at 37°C for 6 h. Plates were carefully overlaid with 105 CFU/ml ofa mid-log-phase broth-grown overlay strain in 7 ml of TS containing 0.5% agarwhich had been maintained at 37°C before application and returned to ananaerobic environment for overnight growth at 37°C. Test strains that scoredpositive for bacteriocin activity had a clear zone of complete inhibition of theoverlay strain surrounding the area of test strain growth.
blp sequence analysis. Primers 1 and 2 (Table 2) were used to PCR amplifyand sequence the region of DNA likely to contain blpM and blpN based onsequence comparison. The blp locus from the type 6A strain was amplified usingprimers 14 and 15, which amplified a 6,600-bp fragment. An extension of the 3�region of the locus, including the downstream gene SP0547, was amplified byprimers 16 and 17. PCR was performed with Pfx high-fidelity polymerase (In-vitrogen, Carlsbad, CA), using the following cycling parameters: 30 cycles of 95°Cfor 30 s, 52°C for 30 s, and 72°C for 1 min/kb. PCR products were purified andsequenced using a BigDye Terminator v3.1 cycle sequencing kit from AppliedBiosystems. Sequencing products were analyzed on a 3730 DNA analyzer fromApplied Biosystems.
Generation of defined blp mutants. Pneumococcal mutants were created asfollows. The blpMNO region was cloned into Escherichia coli plasmid pUC19 atthe SmaI site, using primers 1 and 11. The resulting plasmid was designatedpBlpAL. The janus insertion was created by amplifying the cassette from strainCP1296 (21) by PCR and engineering BstBI and NheI sites into the 5� and 3�regions, respectively (primers 12 and 13). This product was ligated into pBlpALcut with BstBI and NheI at unique sites that span blpM through blpO. Theresulting plasmid, called pBlpAL�MNOjanus, was transformed into 6ASmr andselected for kanamycin resistance and streptomycin sensitivity. This strain wasnamed 6A�MNOjanus. The remaining blpMNO mutants were constructed byreplacing the janus cassette in this strain. The blpM and -N deletions wereconstructed by performing inverse PCR on pBlpAL, using primers 3 and 4 for theblpM deletion and primers 5 and 6 for the blpN deletion. These primers wereengineered to create a unique NsiI site between the stop and start codons of therespective genes. The resulting PCR products were then cut with NsiI, ligated,
TABLE 1. Bacterial strains used in this study
Strain Serotype Characteristic or genotypea Source or reference
P376 6A Opaque variant of clinical isolate P303 10TIGR4 4 Clinical isolate 22P29 7F Clinical isolate R. AustrianP32 19A Clinical isolate R. AustrianP35 19F Clinical isolate R. AustrianP56 12F Clinical isolate R. AustrianP631 5 Clinical isolate R. AustrianP1121 23F Recovered from human colonization study 156ASmr 6A 6A � D39Smr (backcrossed twice) This study6A�blpR 6A 6A � 0100993/486rr (23) (backcrossed three times); Emr This study6A�blpMNOjanus 6A 6ASmr � pBlp�MNOjanus; selected for Knr Sms This study6A�blpMNO 6A 6A�blpMNOjanus � pBlpAL cut with BstBI and NheI, with ends
blunted and religated, removing blpMN and -O coding sequence;selected for Smr Kns
This study
6A�blpM 6A 6A�blpMNOjanus � pBlpAL derivative containing an in-frame deletionof blpM; selected for Smr Kns
This study
6A�blpN 6A 6A�blpMNOjanus � pBlpAL derivative containing an in-frame deletionof blpN; selected for Smr Kns
This study
6A�blpO 6A 6A�blpMNOjanus � pBlpAL derivative containing a complete deletionof blpO; selected for Smr Kns
This study
6AblpMNOWT 6A 6A�blpMNOjanus � PCR product from type 6A strain using primers 1and 11; selected for Smr Kns
This study
6AblpMNOTIGR4 6A 6A�blpMNOjanus � pBlpAL derivative containing the chimeric form ofblpMN (see Materials and Methods for details); selected for Smr Kns
This study
19ASmr 19A 19A � D32Smr (backcrossed twice) This study19A�blpMNOjanus 19A 19ASmr � pBlp�MNOjanus; selected for Knr Sms This study19A�blpMNO 19A 19A�blpMNOjanus � pBlpAL cut with BstBI and NheI, with ends
blunted and religated, removing blpMN and -O coding sequence;selected for Smr Kns
This study
19AblpMNOWT 19A 19A�blpMNOjanus � pBlpAL; selected for Smr Kns This study
a Abbreviations: Kn, kanamycin; Sm, streptomycin; Em, erythromycin.
and transformed into E. coli Top10 cells (Invitrogen, Carlsbad, CA). The blpOdeletion was created by performing inverse PCR on pBlpAL, using primers 7 and2. The resulting product was phosphorylated using T4 DNA kinase, blunt endligated, and transformed into E. coli Top10 cells. The chimeric protein wascreated by amplification of DNA from TIGR4, using primers 8 and 9, whichintroduced a BstBI site at the 5� end. This product was digested with BstBI andNheI and ligated to the 1,022-kb BstBI/NheI fragment of pBlpAL. All plasmidswere verified by restriction digestion. The janus cassette was replaced in strain6A�MNOjanus by transforming the strain with the PCR product produced byprimers 1 and 11 and selecting colonies on streptomycin plates. Deletion of theblpMN operon in the serotype 19A strain was performed by amplifying the januscassette insertion in strain 6A�MNOjanus, using primers 1 and 11, and trans-forming the product into a streptomycin-resistant derivative of 19A. An un-marked mutation deleting the entire blpMNO region was created in this strain asdescribed above. The janus cassette in 19A�MNOjanus was replaced with thewild-type locus by transforming cells with the plasmid pBlpAL. All constructswere verified for a double-crossover event by a loss of kanamycin resistance andby PCR. 6A�blpR was created by isolating DNA from the type 3 isolate con-taining an erythromycin cassette, replacing the blpR gene (23), and transforming
the construct into the type 6A strain. This mutation was backcrossed three timesto reduce the possibility of transformation occurring with unlinked DNA.
Mouse colonization assay. All mice were purchased from Taconic and werehoused in accordance with Institutional Animal Care and Use Committee pro-tocols. Five to 7-week-old BALB/c mice were inoculated intranasally with 10 �lcontaining 2 � 107 to 4 � 107 CFU of a recently animal-passaged pneumococcusstrain resuspended in PBS. All suspensions were plated for colony counts fol-lowing inoculation to ensure that no inhibition had occurred in suspension priorto intranasal instillation. At 4 days postinoculation, a time point shown in pilotstudies to provide a stable level of colonization, the mice were sacrificed by CO2
asphyxiation, the trachea of each was exposed, 200 �l of sterile PBS was instilledinto it, and the lavage fluid exiting the nares was collected. The lavage fluid wasthen serially diluted in PBS and plated on TSA. Plates were supplemented withneomycin (5 �g/ml) to prevent the growth of contaminants or with neomycin plusstreptomycin to select for growth of the 19A derivatives. Results of antibioticselection were verified using colony immunoblotting with a rabbit polyclonalantibody against capsular serotype 4 on neomycin-only plates. The lower limit ofdetection of this assay was 100 CFU/ml of lavage fluid.
Nucleotide sequence accession numbers. The GenBank accession number forthe type 6A blp locus is DQ323933. Accession numbers for the blpM and blpNgenes from clinical isolates are as follows: 19F, ABD03964; 18C, ABD03966;23F, ABD03970; 5, ABD03968; 7F, ABD03958; 12F, ABD03962; and 19A,ABD03960.
RESULTS
Bacteriocin-like activity in a clinical isolate of the pneumo-coccus. In order to characterize the functional significance ofthe blp locus in pneumococci, we screened an array of clinicalisolates (n � 9) for their relative abilities to inhibit growth ofan unrelated strain in an agar overlay assay. In order to removethe inhibitory effects of pneumococcal hydrogen peroxide pro-duction, assays were done either aerobically with catalaseadded to both agar layers or under anaerobic conditions, withequivalent results. This screening procedure identified isolatesof type 6A and 19A with inhibitory activity in vitro when testedagainst a number of other pneumococcal isolates, including thefully sequenced type 4 strain TIGR4 (Table 3 and Fig. 1C).The TIGR4 strain had no reciprocal activity against type 6A or19A or any other clinical isolate tested under these assay con-ditions (Table 3).
BlpR regulates expression of in vitro bacteriocin activityand immunity. In order to confirm that the blp locus wasresponsible for intraspecies inhibition, the blpR regulatorygene homologue of the type 6A strain was deleted by replace-ment of an internal fragment of the gene with the erythromycinresistance cassette. The resulting strain, 6A�blpR, was ana-lyzed for loss of its inhibitory activity and immunity, using theplate overlay method. As predicted, 6A�blpR was deficient in
TABLE 2. Primers used in this study
Primer Sequence and description
1...................TTCCTTTCATATAGTGGATAGGTC; 275 bpupstream of blpM ATG
2...................CAGTTTACGGAACAAGTTTTAATAT; 40 bpupstream of blpO ATG
3...................CCAATGCATTAACAAAAGGAGACTTGTATG;construction of blpM mutation, forward primercontaining NsiI site separating start and stopcodons of blpM
4...................CCAATGCATAACAAATACTCCTTTTTTA;construction of blpM mutation, reverse primercontaining NsiI site separating start and stopcodons of blpM
5...................CCAATGCATTAAAAATGAAAGCTAAATTTT;construction of blpN mutation, forward primercontaining NsiI site separating start and stopcodons of blpN
6...................CCAATGCATACAAGTCTCCTTTTGTTA;construction of blpN mutation, reverse primer withNsiI site separating start and stop codons of blpN
7...................TTAATTTACAGGGGAGTTTCTTT; forward primer56 bp from 3� end of blpO ORF
8...................TGTCTTCGAAGGTGGTGGTGCTGCTTTTG;forward primer engineering BstBI site intoTIGR4 blpM for construction of chimera
9...................CCCTGTAAATTAAGCTAGCAAATAC; reverseprimer for amplification of blpMNO from TIGR4for construction of chimera
10.................GAAGAGATTAGGGTTTTGTGCC; forward primer35 bp upstream of stop codon of blpA
11.................TCTCGCAAGGAAGATGTTCCG; reverse primer 22bp upstream of stop codon of SP0535
12.................GGCCGCTTTCGAAGGATCCGTTTGATTTTTAATGGATAAT; forward primer for amplification of thejanus cassette with engineered BstBI site
13.................ACCTGCTAGCGGGCCCCTTTCCTTATGCTTTTGGAC; reverse primer for amplification of januscassette with engineered NheI site
14.................GTGAGCGACTTTATAGTTTCAATCC; forwardprimer for sequencing of type 6A blp locus 240 bpupstream of the stop codon of blpA
15.................CTGAAAATGAGTTCCTCCTGG; reverse primer forsequencing of type 6A blp locus within SP0547
16.................CTGAAAATGAGTTCCTCCTGG; forward primerfor sequencing of entire SP0547 locus 427 bpdownstream of start codon of blpY
17.................GCCTCTGGATTGGCTTGGGTATCA; reverseprimer for sequencing of SPO547 within SP0548
FIG. 1. (A) Table summarizing results of agar overlay assays with deletion mutants of the blp locus. Plus signs designate definite zones ofinhibition, and empty cells designate combinations that were not tested. (B) Alignment of BlpM and BlpN amino acid sequences from type 6Aand TIGR4 strains and the chimeric proteins from 6AblpMNOTIGR. Shaded amino acids are nonconserved, and arrows designate putative cleavagesites of preproteins. (C) Photographs of results of selected overlay assays. Pictures a to g demonstrate test strains with zones of inhibition, whilepictures h to m demonstrate test strains lacking inhibition. Pictures a and b are shown with a TIGR4 overlay, and pictures c to m are shown withan overlay of 6A�blpMNO. Test strains: a and c, type 6A; b and f, type 19A; d, 6AblpMNOWT; e, 6A�blpO; g, 19AblpMNOWT; h, 6A�blpMNO;i, 6A�blpM; j, 6A�blpN; k, TIGR4; l, 6AblpMNOTIGR4; m, 19A�blpMNO.
in vitro intraspecies inhibition when tested against TIGR4. Inaddition, as expected, 6A�blpR was killed by its parent strain,suggesting a deficiency in expression of its immunity phenotype(Fig. 1A). Therefore, deletion of the BlpR response regulatorled to a defect in both killing and immunity. These observa-tions provided the first direct functional demonstration thatthe blp locus is involved in intraspecies competition in vitro.
Sequence analysis of the 6A blp locus. Given the preliminaryresults for the type 6A strain, we sequenced its blp locus fromthe N terminus of the blpA gene to the end of the previouslydefined locus, SP0547 (Fig. 2). This region was predicted tocontain the genes encoding the bacteriocins based on the ar-rangement of the locus in other previously sequenced strains(8, 22). As in the TIGR4 locus, putative bacteriocin genes inthe type 6A locus were preceded by a highly conserved con-sensus sequence for BlpR binding. The type 6A strain’s blplocus contains homologues for the predicted bacteriocin genesblpM, -N, and -O. These genes would be predicted to encodethree proteins, with each containing a conserved N-terminalsignal sequence followed by a double-glycine motif, consistentwith the sequences of previously described bacteriocins. Sur-prisingly, similar genes were also found in the TIGR4 genome,although TIGR4 did not inhibit the growth of the type 6Astrain. The type 6A strain encodes BlpM, -N, and -O proteinsthat have 6 of 84, 2 of 67, and 2 of 49 residues, respectively, thatdiffer from the TIGR4 sequence. The type 6A locus containstwo operons downstream of the putative BlpM, -N, and -Obacteriocins, preceded by two additional BlpR consensus bind-ing sequences that contain open reading frames (ORFs) en-coding proteins of unknown function. The final operon con-tains homologous ORFs for the genes blpX, -Y, and -Z and,unlike TIGR4, is predicted to include the downstream ORFSP0547 due to deletion of a transcriptional terminator se-quence. BlpX, -Y, and -Z and SP0547 are 100%, 96%, 98%,and 99% identical to the TIGR4 sequence, respectively, at theamino acid level.
Deletional analysis of blpM, -N, and -O. Using chromosomalallelic replacement, the 6A blpM, -N, and -O ORFs were de-leted both individually and in combination. In order to createin-frame, unmarked deletions in these small, closely approxi-mated genes, the janus cassette (21) was inserted betweenunique restriction sites, replacing the entire blpMNO operon.A type 6A strain derivative made streptomycin resistant wasused for insertion of the janus cassette in the blpMNO locus.The resulting isolate was resistant to kanamycin and sensitiveto streptomycin, confirming the insertion of the janus cassette.The cassette was then replaced by transforming this strain withfragments containing altered versions of the blpMNO operon.Separate deletions were created in blpM, -N, and -O by delet-
ing each gene in its entirety, leaving only its predicted stop andstart codons. The deletion of the entire blpMNO locus wascreated by introduction of a deletion spanning from the 5� endof the blpM ORF through the 3� end of the blpO ORF. In orderto determine the significance of the small number of aminoacid differences between BlpM and BlpN in the type 6A isolateand TIGR4, a chimeric gene was created by exchanging thetype 6A strain blpM and -N with TIGR4 blpM and -N. Thechimeric protein contains the N terminus of 6A blpM with allthree amino acid changes in the C terminus found in theTIGR4 locus and the entire blpN locus from TIGR4 (Fig. 1B).This chimeric construct was used to determine if the differencein killing between the two strains was the result of the differ-ences in these amino acids. To confirm this result and todemonstrate an absence of additional mutations outside thelocus explaining the phenotype, a PCR product containingthe corresponding original parental type 6A locus was usedto replace the janus cassette. All strains were tested for aloss of in vitro inhibitory activity by the plate overlay methodagainst strains TIGR4 and 6A�blpMNO and used as anoverlay against the type 6A isolate to look for a loss ofimmunity (Fig. 1A and C).
In vitro assays for bacteriocin activity demonstrated thatboth the blpM and blpN genes are required for wild-type in-traspecies inhibitory activity but not immunity. Unlike the blpMand -N deletions, the blpO deletion had wild-type levels of activityin both inhibition and immunity. Strain 6A�blpMNO, containinga deletion of the entire locus, had a deficiency in both inhibitionand immunity, suggesting that a gene in this locus contributes tothe immunity phenotype. The construct containing the cor-rected wild-type locus, 6AblpMNOWT, had parental levels ofactivity, confirming that mutations outside blpMN could notaccount for the observed phenotypes. The type 6A strain ex-pressing the chimeric form of BlpM and -N, 6AblpMNOTIGR4,was deficient in intraspecies inhibition, similar to the pheno-type of wild-type TIGR4. This strain retained the parent strainimmunity phenotype. These data suggest that both BlpM and-N are necessary for the bacteriocin activity seen in vitro.Moreover, the difference in activity between the TIGR4 andtype 6A strains could be attributed to the five amino acids thatdiffer between the two strains in the mature, processed formsof BlpM and -N.
Conservation of BlpM and -N sequences among pneumo-coccal strains. It is known that bacteriocins tend to have asignificant degree of divergence when different strains withinthe same species are compared. This divergence may allow forintraspecies competition. Small changes in the bacteriocin of-ten require reciprocal changes in the immunity protein so thatorganisms expressing similar but not identical bacteriocins are
FIG. 2. Graphical demonstration of the blp locus in a type 6A strain and comparison with the corresponding portion of the TIGR4 genome.Solid arrows represent coding sequences for double glycine-containing proteins, vertically striped arrows represent genes of unknown function,checked arrows represent transport genes, the white box represents an insertion sequence element, and gray boxes represent the conserved putativeBlpR binding sites designating the start sites of operons. The gap in 6A designates an unsequenced region.
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not protected from each other by their own immunity proteins.In order to determine the relative conservation of the BlpMand -N proteins, blpM and -N coding sequences for the nineclinical isolates were analyzed. These strains include an arrayof clinical isolates of diverse capsular types that were isolatedin different locations at different times. Seven of the nineisolates had sequences homologous to blpM and -N. The re-maining two isolates contained coding regions for other bac-teriocin-like peptides (blpI and blpK) homologous to thosefound in the TIGR4 locus. The BlpM and -N sequences werealigned and analyzed for conserved amino acids (Fig. 3). In-terestingly, the seven BlpM sequences seemed to be dividedinto two groups. Group 1 contains those with 100% identity tothe TIGR4 sequence. Group 2 comprises those with 98 to
100% identity to the type 6A strain’s sequence. In comparingthe BlpN sequences, the RL amino acid sequence at aminoacids 40 and 41 was seen in all strains containing the group 1BlpM sequence, while the KI sequence was seen in strainscontaining the group 2 BlpM sequence. In vitro inhibitionassays with the seven clinical isolates demonstrated that onlystrains in group 2 had detectible activity. One strain in thisgroup, a type 12F strain, showed no detectible inhibitory ac-tivity on overlay assays against any strain tested, and the fivestrains in group 1 also had no detectible inhibitory activity.
The blp locus is functional in vivo during colonization. Inorder for the blp locus to play a role in intra- and interspeciescompetition, not only must it be expressed in the polymicrobialenvironment of the nasopharynx, but organisms must be in
FIG. 3. Amino acid alignment of sequences of BlpM and BlpN from a selection of clinical isolates of the serotypes indicated. Shaded aminoacids are areas of nonconservation. Arrows designate putative cleavage sites of preproteins.
close enough proximity to be affected by secreted antimicrobialproteins. To determine if the blp locus is both expressed andfunctional during colonization, we performed competition ex-periments in BALB/c mice. Because the type 6A strain washighly virulent in mice in the nasal colonization model, weperformed competition experiments with the serotype 19Astrain from group 2. An unmarked mutation of the blpMNOoperon and a replacement of the wild-type operon were cre-ated in this strain and analyzed by the overlay assay for phe-notype. Like the type 6A strain, strain 19A�blpMNO was de-ficient in growth inhibition when tested against TIGR4 andhad an immunity defect when tested against the parent strain(Fig. 1A and C). The corrected mutant, 19AblpMNOWT, hadthe expected wild-type phenotype in both inhibition and im-
munity. The type 19A strain, TIGR4, 19AblpMNOWT, and19A�blpMNO were inoculated intranasally either alone or inpairs. Singly inoculated mice were colonized with TIGR4, 19A,19A�blpMNO, or 19AblpMNOWT at equivalent levels (Fig. 4Aand B). Dually colonized mice given 19A and 19A�blpMNOwere colonized predominantly with the type 19A strain (Fig.4B). Dually colonized mice given TIGR4 and 19AblpMNOWT
were colonized primarily with the 19A strain, mimicking our invitro inhibition results (Fig. 4A). The competitive advantage ofthe 19A strain was eliminated when TIGR4 was coinoculatedwith 19A�blpMNO. In fact, these animals were colonized pri-marily with TIGR4, with little detectible colonization by themutant strain. These experiments suggest that production ofbacteriocins by the wild-type strain was able to inhibit growthof immunity-deficient strains during colonization, verifying therole of these peptides in vivo.
DISCUSSION
The presence of the blp operon in pneumococci has beenknown for several years, but there exists little informationregarding the functional characteristics of the various proteinsencoded by the locus. We were unable to appreciate any bac-teriocin-like activity by using in vitro assays for either of thetwo strains for which the locus has been best characterized (R6and TIGR4). However, we identified two clinical isolates withthe ability to inhibit the growth of several other strains in vitro.Identification of such isolates allowed us to further investigatewhich genes were responsible for intraspecies inhibition andwhether the locus was important during colonization.
The requirement for two genes suggests that BlpM andBlpN comprise a type IIb, or two-component, bacteriocin (4,16). Deletion of the entire operon resulted in a loss of immu-nity, suggesting that a protein involved in immunity to theBlpMN bacteriocins is encoded in this operon. Because noneof the mutants with single deletions of blpM, blpN, or blpO wasdeficient in immunity, the gene encoding the immunity proteinis likely the ORF identified between blpN and -O. de Saizieu etal. (3) have shown using microarray analysis that application ofpurified BlpC results in the upregulation of genes within theblp locus only, with the exception of the unlinked putativebacteriocin gene blpU. The loss of intraspecies inhibition withmutations in blpMN shows that blpU or other putative bacte-riocin genes do not make a significant contribution under theseconditions in the strain backgrounds tested.
Because the type 6A strain is able to inhibit the growth ofTIGR4, either the entire blp locus in TIGR4 is inactive,including the production of immunity proteins, or the dif-ferences between the bacteriocins produced by the twostrains are sufficient to result in failure of the TIGR4 im-munity protein to protect against the type 6A BlpMN bac-teriocins. Our data suggest that the latter is true. Alignmentof the type 6A and TIGR4 BlpM and -N protein sequenceswith particular attention to the active portion following thedouble-glycine motif demonstrates that there are only threeamino acid differences in BlpM and two amino acid differ-ences in BlpN. Our chimeric protein contained the N-ter-minal processing and secretion domain of the type 6A BlpMfused to the remainder of BlpM and -N from TIGR4. Thischimeric protein was unable to restore inhibitory activity to
FIG. 4. 19A�blpMNO is outcompeted by TIGR4 (A) or its parenttype 19A strain (B) during mouse nasopharyngeal colonization. Six-week-old BALB/c mice were challenged intranasally with single ordual inoculations of the type 19A parental strain (19A; open circles),the 19AblpMNOWT corrected mutant (19A; closed circles),19A�blpMNO (closed diamonds), and TIGR4 (19Ablp-; closed trian-gles) (A) or with single or dual inoculations of the type 19A strainand19A�blpMNO (B). The colonizing strain is depicted on the x axisand was detected in lavage fluid at 4 days postinoculation at the densityindicated (y axis). Coinoculated strains are shown in parentheses. Sta-tistical analysis was done by the Mann-Whitney test, and horizontallines indicate median values. Dashed lines denote the limit of detec-tion.
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strain 6A. Expression of wild-type immunity in the chimericstrain suggests that the blpMNO operon is transcribed. Thus,our data suggest that the specificity of the in vitro activitiesof the two bacteriocins is likely dictated by the five aminoacids that differ between the two proteins in the maturepeptide. Because these chimeric proteins were unable toinhibit growth of TIGR4 when placed behind an active pro-moter, this finding suggests either that TIGR4 actively pro-duces an immunity protein that protects it from its ownbacteriocin but not that of the type 6A strain or that thebacteriocins produced by TIGR4 are nonfunctional.
Alignment of a number of clinical isolates demonstratesthat there are two subtypes of BlpMN, with one that resem-bles the TIGR4 proteins (group I) and one that is homolo-gous to the protein in the type 6A strain (group II). Exami-nation of the available fully sequenced pneumococcal genomesreveals that there are other pneumococcal strains that have nocoding sequence for BlpMN. These include the type 23F strainsequenced by the Sanger Centre (sequence data were pro-duced by the S. pneumoniae Sequencing Group at the SangerInstitute and can be obtained from http://www.sanger.ac.uk/Projects/S_pneumoniae/), which has genes homologous toblpIJ from TIGR4 (22), and the laboratory strain R6, which hasno bacteriocin gene homologues within the blp locus (8). Usingour clinical strains, we were able to show for one of the groupII isolates that the pattern of inhibition was similar to that forthe type 6A strain, while the remainder of the isolates had nodetectable activity. The lack of appreciable activity in a largenumber of strains may be due to differences in inducingconditions for these isolates in our in vitro assay. Alternatively,we may have failed to identify the correct target bacteria,whether pneumococci or other species, for their bacteriocins.It is clear that the regulation of the blp locus is complex, likelyinvolving at least two separate two-component systems and apheromone. Mascher et al. demonstrated that the blpXYZpromoter as well as the blpABC promoter contains a bindingsite for the global response regulator CiaR (14). Peterson et al.have also shown that blpABC, blpXYZ, and SP0547 are inducedearly in competence (18). In addition, there are differences intranscription as well as functional activity between the opaqueand transparent phenotypic variants of pneumococcus (11).We have found that the transparent variant of the type 6Aisolate has very little appreciable inhibitory activity comparedwith its opaque variant. Despite this difference, these variantsshow no discernible difference in immunity (data not shown).
Previous work by Throup et al. has shown that mutants inblpR and -H are attenuated in the mouse model of respiratoryinfection (23). It is unclear how a locus involved in productionof antimicrobial peptides would play a role in the typicallysterile environment of the lung. Our work addressed the effectsof mutations in the blp locus on colonization of the mousenasopharynx, a host environment where it seems most likelythat the locus may be functional. In order to remove the pos-sible influence of BlpR/H-regulated genes not involved in bac-teriocin production, we performed colonization experimentsusing a specific unmarked deletion of the blpMNO operon. The19A�blpMNO strain showed wild-type levels of colonizationwhen given alone but was outcompeted by its isogenic parentstrain when both strains were given in equivalent numbers. Inorder to address whether the production of BlpMN could
influence colonization when tested against nonisogenic strains,we performed cocolonization experiments with TIGR4 andeither 19AblpMNOWT or 19A�blpMNO. When the blpMNOmutant was compared to the parent strain for the ability tooutcompete TIGR4 during colonization, only the parent strainexpressing bacteriocins was able to suppress the levels ofTIGR4. This finding suggests that the BlpMN bacteriocins canplay a role in intraspecies competition within the polymicrobialenvironment of the nasopharynx. Elaboration of pneumococ-cal bacteriocins in the nasopharynx may contribute to definingthe organism’s microenvironment. Efficient removal of com-petitors may allow certain strains of the pneumococcus tocolonize both more efficiently and for longer periods of time,thus increasing their potential for transmission. The observa-tion that expression of bacteriocins may provide producerswith a competitive advantage in colonization of the nasophar-ynx is particularly intriguing when considering our data for theserotype 19A strain. This serotype has emerged in the post-vaccine era as an increasingly prevalent cause of invasive dis-ease. The correlation between bacteriocin production and theability of pneumococci to colonize and cause disease in a largerpanel of clinical isolates is currently being investigated.
The environment of the nasopharynx is subject to constantfluxes in the abundance of particular inhabitants as potentialcompetitors wax and wane. A better understanding of thebacterial factors driving these alterations may allow for thecreation of novel ways to block colonization, an obligatory stepto invasive disease.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants RO1-AI38446, R21-AI054647, RO1-AI44231, and T32-AI007634-05 (S.D.)and by a MedImmune Career Development Award in Pediatric Infec-tious Diseases (S.D.).
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Editor: F. C. Fang
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