- Research article
- Open Access
Identification of pneumococcal colonization determinants in the stringent response pathway facilitated by genomic diversity
© Li et al.; licensee BioMed Central. 2015
- Received: 26 November 2014
- Accepted: 24 April 2015
- Published: 9 May 2015
Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates of Streptococcus pneumoniae and their genome sequences to help identify a novel determinant of pneumococcal colonization.
A spontaneously arising laboratory variant (SpnYL101) of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonization and lower resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published genome sequences to identify pairs of closely-related, serotype-matched isolates that differ at the gene of interest, and compared their resistance to neutrophil-killing. This method allowed rapid examination of multiple candidate genes and found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil-killing and competitiveness in mouse colonization. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to alter resistance to neutrophil-killing.
These results indicate that analysis of naturally existing genomic diversity complements traditional genetics approaches to accelerate genotype-phenotype analysis.
- Genomic diversity
- Pneumococcal colonization
- RelA/ SpoT homolog
A general question in microbiology is to understand the genetic determinants underlying a microbial phenotype, such as increased virulence or transmissibility. Developments in sequencing technology now allow rapid identification of genetic differences among phenotypic variants, but it remains a time-consuming process to verify the function of each genetic variation by constructing isogenic strains (chimeras) in which only one gene of interest differs. Genomic diversity of existing microbial populations can be used to infer the consequences of polymorphisms by evaluating phenotypic differences between naturally occurring variants, if 1) the naturally occurring variants harbor the genetic variation of interest and 2) other known genetic determinants of the phenotypic difference can be accounted for. Testing existing variants can reduce the amount of time and resources spent on constructing isogenic strains, so we hypothesized that using naturally occurring genomic diversity can accelerate gene function discovery.
Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sinusitis and otitis media worldwide. Pneumococcus frequently colonizes the human nasopharynx, which precedes invasive infections, and these colonization events serve as the reservoir for bacterial transmission . There are more than 90 capsular serotypes of pneumococcus. Simultaneous colonization by multiple serotypes and competition between serotypes has been documented during nasopharyngeal carriage in humans [1,2]. Understanding the factors that determine serotype patterns of carriage is an important public health issue [3,4]. The capsule itself has a major impact on carriage: studies have shown that serotype controls resistance to surface killing mediated by human neutrophils , ability to compete against co-colonizing strains in a mouse model of multiple-strain carriage [Krzysztof Trzciński et al., unpublished data], and pneumococcal cell surface charge . All the three properties, in turn, are correlated with carriage prevalence in human populations [4,5]. For genetic variations outside the capsular polysaccharide synthesis (cps) region, much less is known about their contribution to resistance to surface killing or ability to compete during nasopharyngeal colonization.
A variant of pneumococcus with reduced fitness
It has been reported that higher carriage prevalence of S. pneumoniae in human populations is associated with higher resistance to neutrophil-killing . We therefore tested whether the SpnYL101 strain is more likely to be killed by neutrophils than the TIGR4:19F strain in a surface killing assay . As shown in Figure 2B, the survival of the SpnYL101 strain (mean = 0.39, SD = 0.084) was significantly lower than that of the TIGR4:19F strain (mean = 0.60, SD = 0.11; t-test, t(16) = −5.28, p < 0.0001).
Decreased degree of encapsulation was shown to reduce S. pneumoniae resistance to non-opsonic neutrophil-mediated killing . We therefore assessed whether the SpnYL101 strain exhibited reduced capsule production compared to TIGR4:19F. Both strains were confirmed to remain serotype 19 F by Pneumotest-latex serotyping. The cell-associated type 19 F capsular polysaccharide was quantified by an inhibition ELISA assay, and no evidence of reduction in capsule production was observed for SpnYL101 (Figure 2C). Furthermore, SpnYL101 and TIGR4:19F showed similar efficiency in colonizing C57BL/6 mice in single-strain colonization experiment (Figure 2D). Thus, the SpnYL101 strain appeared to have acquired non-capsule change(s) that can affect both competition during co-colonization and resistance to surface phagocytosis. We measured the in vitro growth rates of SpnYL101 and TIGR4:19F in THY, and no statistically significant difference was observed (Additional file 1: Figure S1).
Genetic Variations identified in the SpnYL101 strain
We hypothesized that the phenotypic differences between the SpnYL101 and the TIGR4:19F strains were caused by genetic variation. To test this hypothesis, we sequenced the whole genome of the two strains using the Illumina MiSeq system and compared the two genomes to identify SNPs distinguishing the two strains.
SNPs identified between TIGR4:19F and SpnYL101
SNP Name c
Testing the effects of SNP with naturally existing diversity
To test whether variation in a candidate gene causes a phenotypic difference, classical genetic approaches involve creating a set of isogenic strains in which only the candidate gene differs and evaluating phenotypes among the set of isogenic strains. If multiple candidate genes were to be examined, multiple sets of isogenic strains would usually need to be constructed.
The SP_1645 C1019T variation in SpnYL101 contributes to its defects
Since the SP_1645 SNP caused a change in resistance to neutrophil-killing, we hypothesized that the SP_1645:C1019T variation also contributes to the ability to establish nasal colonization. To test this hypothesis, a reference strain (TIGR4:19A) was mixed with either SpnYL104 or SpnYL105 at equal ratio and the mixtures were used to intranasally challenge C57BL/6 mice. Nasal wash samples were collected on days 1, 4, and 7 post-challenge, and the competitive index was measured as described in Materials and Methods. The SpnYL104 and the SpnYL105 showed similar competitive index on day 1 (Figure 4B, Mann–Whitney test, U = 30.00, n1 = 9, n2 = 10, p = 0.24). By day 4, the SpnYL105 strain showed a slightly higher competitive index (median = 0.86) than was observed for the SpnYL104 (median = −0.17) and the difference was significant (Mann–Whitney test, U = 6.00, n1 = 7, n2 = 6, p = 0.035). By day 7 of colonization, the competitive index for the SpnYL105 strain (median = 0.94) was significantly higher than that of the SpnYL104 strain (median = −1.63, Mann–Whitney test, U = 3, n1 = 9, n2 = 7, p = 0.003). The results indicated that the SP_1645:C1019T variation is an important determinant of pneumococcal competitiveness during nasal colonization in the mouse.
Effects of variation in other genes involved in the stringent response pathway
In this study, we observed phenotypic differences between the TIGR4:19F and SpnYL101 strains. We sequenced the whole genomes of the two strains and identified 5 SNPs as the candidate causes of the phenotypic differences. Subsequently, we prioritized hypotheses about the likely roles of genetic variation in genes affected by a SNP by testing a pair of serotype-matched, closely-related carriage isolates that differs substantially at each candidate gene. Such isolate pairs were identified by genomic analysis of a large collection of carriage isolates with diverse genetic background and evidence was found to support the phenotypic impact of variation in the gene with TIGR4 locus tag SP_1645. Although it would be ideal to compare closely-related isolate pairs that harbor the exact same SNP of interest, such pairs may not always be available for a given collection (Additional file 1: Table S2). Additionally, this method aims to make the best use of the available genomic diversity information to complement (not replace) traditional genetics approaches and accelerate genotype-phenotype analysis. It could be inappropriate if the distance between the most closely related strains happens to be quite large because more genomic changes would make it difficult to evaluate the contribution from a specific gene variation. We also acknowlodge that serotype matching may not be as informative for other studies. As each pair of isolates came from the same ‘sequence cluster’, there is unlikely to be much relevant accessory genome variation distinguishing them , making it unlikely that the differences in phenotypes are down to other genetic variation, althogh such differences cannot be formally ruled out. We therefore emphasize that the method is complementary to, rather than a direct replacement for, experimental work that can identify the causal genetic variant underlying phenotypic differences. Finally, we confirmed the effects of the SP_1645:C1019T variation by using laboratory-constructed isogenic strains. Notably, the TIGR4:19F and SpnYL101 strains became similarly susceptible to neutrophil-killing when the SP_1645 locus was knocked out from both strains (Figure 4A). This result indicated that the other 4 SNPs were unlikely to play a major role in resistance to neutrophil-killing.
Little is known about the cellular function of the SP_1645:C1019T variation we identified. When the SP_1645 locus in the TIGR4:19F strain was replaced by the Janus cassette, no significant change in resistance to neutrophil-killing was observed. This is consistent with results in a previous transposon mutagenesis study , also using variants of TIGR4 (presumably with the wild-type allele), that disruption of the SP_1645 locus by transposon insertions did not cause a detectable defect in pneumococcal colonization of mouse nasopharynx. In contrast, replacing the SP_1645 locus in the SpnYL101 strain by Janus cassette significantly increased resistance to neutrophil-killing, restoring it to a level similar to that seen in the TIGR4:19F strain. This observation suggested that the SP_1645 allele in SpnYL101 could represent a gain-of-function change in which the gained function appeared to associate with reduced resistance to surface killing. Since it has been shown that surface charge of pneumococcus influences its resistance to neutrophil-killing , we measured the zeta potential of the TIGR4:19F, SpnYL101, and the SP_1645 knock-out strains, and did not find a significant difference in surface charge among them. In addition, the protein encoded by the SP_1645 gene was not in a previously reported list of human antibody antigens  or a list of human TH-17 cell antigens . Thus, the SP_1645:C1019T variation could represent a previously unknown determinant that, directly or indirectly, influences the interaction between pneumococcus and neutrophils.
The gene with TIGR4 locus tag SP_1645 is inferred to encode a RelA/SpoT homolog protein, which is involved in the synthesis and degradation of guanosine tetraphosphate (ppGpp). In bacteria, ppGpp is a stringent-response mediator that coordinates cellular activities in response to changes in nutrient abundance [14,15]. We therefore tested the effects of variation in other genes involved in the stringent response pathway using, once again, naturally existing genomic diversity. Interestingly, evidence suggested that variation in another RelA/SpoT homolog protein encoded by the gene with TIGR4 locus tag is also associated with change in resistance to neutrophil-killing. Construction and testing isogenic strains further confirmed the causal effects of the SNP in the gene with TIGR4 locus tag SP_1097. These data suggested that RelA/SpoT homolog proteins in pneumococcus could play a critical role in regulating bacterial responses to neutrophil-killing. Interestingly, no significant difference in competitiveness against SpnYL049 observed between SpnYL106 and SpnYL107 in the mouse colonization experiment (Additional file 1: Figure S2). The results indicated that, consistent with previous studies [4,5], resistance to phagocytic killing is an important but not the only mechanism to regulate pneumococcal colonization ability. It should be noted that mouse colonization in vivo and surface phagocytosis killing in vitro are two complementary, relevant, if imperfect model systems we used to understand the pneumococcal determinants of colonization success in human populations. More studies using a variety of approaches are needed to dissect the possible links between activities of GTP pyrophosphokinase (including gene products of SP_1645 and SP_1097), levels of stringent response, and pneumococcal colonization success.
The increasing availability of large, publicly available genomic datasets, combined with relatively inexpensive sequencing of laboratory variants, provides a wealth of information that can be mined for polymorphisms with interesting phenotypic consequences. In addition to whole genome sequences, databases on transcriptomics, proteomics, protein structure, and specific functions (e.g. antibiotic resistance, immunogenicity) could also be mined to obtain information on potential role of the targets, and therefore help focusing on genetic variations of high likelihood of functional consequences. The approach used here requires access to enable us to search such data for suitably matched, closely related isolates distinguished by the polymorphism of interest, and then to the selected isolates themselves for phenotypic evaluation. In this study we used carriage isolates to which we already had access and their whole genome sequences, which are not trivial to obtain. In order to facilitate more general use at the public level, affordable publicly funded repositories (e.g. the BEI resources http://www.beiresources.org/) should be supported. The option to use an in silico search of genomic sequence database to identify comparable isolates in which the SNP has already occurred in the course natural evolution can be far more efficient than constructing an isogenic mutant in vitro. This method allows us to quickly screen multiple SNPs and narrow down a candidate list to those with high likelihood of causing a phenotypic difference. The same procedure can be applied to studying the effect of genetic variation in other microorganisms for which collections of diverse isolates and their full-genome sequences are available. It will be particularly useful when gene replacement and creation of isogenic strains are difficult to achieve, such as in studies of essential genes or microorganisms with low recombination rate. Given the fast growing database of whole genome sequencing data for many different organisms [16,17], a method that uses naturally existing diversity of microbial populations to accelerate gene function analysis could be increasingly feasible for a wide range of microorganisms. In addition, as these databases grow the chance of sampling closely related isolates that differ at the locus of interest will grow with them. To the extent isolates (or phenotypic measurements thereof) are available with genomic sequences, it will then be possible to go one step further, evaluating the extent to which genetic differences experimentally proven to cause phenotypic traits in laboratory strains are correlated with these traits in natural populations, where genetic backgrounds will be more variable. Analogous to testing animal model results in human populations by epidemiological studies, such surveys of natural genetic and phenotypic variation will provide a much fuller picture of the significance of laboratory findings.
A single nucleotide change in pneumococcal RelA/ SpoT homolog involved in the stringent response (GTP pyrophosphokinase, TIGR4 locus tag SP_1645) was identified to reduce the ability of pneumococcus to compete for colonization and decrease the resistance to killing by human neutrophils. The identification process was shown to benefit from a strategy that includes analyzing 616 previously published pneumococcal genome sequences to select closely-related strains carrying genetic variation of interest for phenotypic evaluation. The utility of this strategy was additionaly demonstrated by identifing variation in another RelA/ SpoT homolog (TIGR4 locus tag SP_1097) that causes change in resistance to neutrophil-killing. We conclude that analysis of naturally occurring genome diversity can be used to complement traditional genetics approaches to accelerate the genotype-phenotype analysis.
Human blood was obtained from healthy adult volunteers according to a protocol approved by the Office of Human Research Administration at Harvard School of Public Health (protocol number CR-10199-04). All adult subjects provided informed consent in written.
All animal work has been conducted in compliance with the Animal Welfare Act and the guidelines of the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, and specifically approved by the Institutional Animal Care and Use Committee (IACUC) of Harvard Medical School (protocol number 2991; Animal Welfare Assurance of Compliance A3431-01 and AAALAC Accreditation #000009, 6/12/13). Mice were euthanized via CO2 inhalation followed by bilateral thoracotomy.
Strains, cells, and animals
Six capsular variants of the TIGR4 strain (TIGR4:1, TIGR4:4, TIGR4:14, TIGR4:19A, TIGR4:19F, and TIGR4:23F) were constructed and reported previously . The SpnYL101 strain is a laboratory variant of the TIGR4:19F strain, which arose during subcultures of the TIGR4:19F strain. To construct the SpnYL102 and the SpnYL103 strains, the SP_1645 locus in the TIGR4:19F and SpnYL101 was replaced with a Janus-type cassette  by using the transformation protocol described previously [18,19]. The Janus cassette in the SpnYL102 strain was then replaced by the allele corresponding to SP_1645 sequence in SpnYL101 to create the SpnYL104 strain. Similarly, the Janus cassette in the SpnYL103 strain was then replaced by the SP_1645 sequence amplified from the TIGR4:19F genomic DNA by PCR to create the SpnYL105 strain.
To construct the SpnYL106 strain, the upstream sequence of the SP_1097 locus (TIGR4 reference genome position 1029105–1030387), the kanamycin-resistance marker, the allele sequence corresponding to SP_1097 in isolate 135771 (including 109-bp upstream of the start codon), and the downstream sequence of the SP_1097 locus (TIGR4 reference genome position 1031171–1032329) were amplified by PCR and the PCR products were ligated in that order by using the Gibson Assembly Kit (New England BioLabs, Ipswich, MA). The assembly product was used to transform TIGR4:6B with selection for kanamycin resistance. After transformation, the SP_1097 locus was amplified by PCR from 12 kanamycin-resistant clones and the PCR products were sequenced to identify clones with correct allele sequence corresponding to SP_1097 in isolate 135771. The same strategy was used to construct the SpnYL107 strain.
Nasopharyngeal carriage isolates were colony-purified prior to use. All strains were grown in Todd Hewitt Broth with 0.5% yeast extract (THY) (BD, Franklin Lakes, NJ) at 37°C with 5% CO2. Strains and PCR primers used in this study are listed in Additional file 1: Table S3.
Neutrophils were isolated from human blood using Histopaque 10771, 11191 gradient reagents (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions and used immediately.
Wild-type C57BL/6 mice were obtained from the Jackson ImmunoResearch Laboratories, Bar Harbor, ME. All mice were female, 9 to 10 weeks old at the start of experiments, and were kept in a BSL2 facility.
Bacterial growth rate
Strains were streaked onto blood agar plates and cultured at 37°C in 5% CO2 overnight. Twelve colonies from each strain were subcultured in Todd–Hewitt medium with 0.5% yeast extract (THY; Becton Dickinson and Company, Sparks, MD) until O.D. 620 reached ~ 0.4 and then diluted into THY medium at a starting culture O.D. of ~0.005. Growth was monitored in sterile flat-bottomed 96-well microtitre plates (Nunc, Denmark) containing 200 μl culture each well every 30 minutes using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA) over 6 hours. The growth curves were fitted to an exponential growth equation and the growth rate was estimated using Graphpad Prism software (GraphPad Software, Inc., CA).
Mouse carriage model and competitive index quantification
In the single-strain colonization experiment, C57BL/6 mice (n = 5 for each group) were inoculated intranasally with in 10 μl of PBS containing approximately 1 × 107 CFU of either TIGR4:19F or SpnYL101. After inoculation, nasal wash samples were collected on day 1 by live sampling and on day 4 by post-mortem tracheal wash as previously described . Aliquots of each sample were titered on gentamicin plate (2.5 mg/L) to determine the colony forming unit (CFU) density.
The multi-strain pneumococcal colonization experiments were performed essentially as previously described . In 6-strain colonization experiments, five TIGR4 capsular variants (TIGR4:1, TIGR4:4, TIGR4:14, TIGR4:19A, TIGR4:23F) were mixed with either TIGR4:19F or SpnYL101 at equal ratio. C57BL/6 mice were inoculated intranasally with the mixtures in 10 μl of PBS containing approximately 1 × 106 CFU of each strain. In 2-strain colonization experiments, a reference strain (TIGR4:19A) was mixed with either SpnYL104 or SpnYL105 at equal ratio. C57BL/6 mice were inoculated intranasally with the mixtures in 10 μl of PBS containing approximately 5 × 106 CFU of each strain. Nasal wash samples were collected up to 7 days after challenge as previously described . Aliquots of each sample were titered to determine the colony forming unit (CFU) density in sample. The remaining samples were cultured overnight on blood agar plates supplemented with gentamicin to a final concentration of 2.5 mg/L, and all bacterial growth was harvested for genomic DNA extraction.
Genomic DNA was purified from cultures of samples collected from animals using DNeasy Blood and Tissue kit (QIAGEN, Valencia, CA). The relative abundance of each strain in a sample was determined by a relative quantification protocol of the 7300 Real Time PCR System (Applied Biosystems). Serotype-specific primers were adopted from a previous study  and are listed in Additional file 1: Table S3. The calibrator was composed of 6 types of genomic DNA (TIGR4:1, TIGR4:4, TIGR4:14, TIGR4:19A, TIGR4:19F, and TIGR4:23F) with equal concentration (0.25 ng/μl each). The DNA sequence specifically amplified by primers 19 F-forward and 19 F-reverse was used as the endogenous control while DNA sequences amplified by other serotype-specific primers were treated as targets. Total reaction volume of 25 μl was composed of 1 × SYBR GREEN PCR Master Mix (Applied Biosystems), 2 ng of genomic DNA and 400 nM of each primer. Two replicates reactions for each sample and the calibrator were performed. The relative level of each target in a sample was calculated by the RQ Study software (Applied Biosystems) according to manufacturer’s protocol. The frequency of the TIGR4:19F or SpnYL101 specific DNA in a sample was calculated as 1/(1+ the sum of all targets level). The competitive index of a 19 F variant was calculated as A1-A0, where A1 and A0 are the log10 transformation of the ratio of the 19 F specific DNA frequency to the (1-19 F specific DNA frequency) in a nasal wash sample and in the inoculation mixture, respectively. This relative quantification method has been validated by using DNA samples of known composition (Additional file 1: Figure S3 and Table S4). According to the validation, we set 3 and −3 as the high and low detection limits for the competitive index, respectively. Calculated competitive index values beyond the limit of detection were rounded to the nearest limit value.
Competitive indexes for strains SpnYL106 and SpnYL107 were measured against a reference strain of the same serotype but distinct antibiotic resistance. The reference strain, SpnYL049 (serotype 6B, Kanamycin sensitive (Kans) and Trimethoprim resistant (Trir)), was mixed with either SpnYL106 (serotype 6B KanrTris) or SpnYL107 (serotype 6B, Kanr Tris) at equal ratio. C57BL/6 mice (n = 10 for each group) were inoculated intranasally with the mixtures in 10 μl of PBS containing approximately 5 × 106 CFU of each strain. After inoculation, nasal wash samples were collected on day 1 by live sampling and on day 4 by post-mortem tracheal wash as previously described . Aliquots of each sample were titered on two types of blood agar plates: Tri plate (containing 3.2 mg/mL Trimethoprim and 2.5 mg/L gentamicin) and Kan plate (containing 500 mg/L Kanamycin). Titer below the detection limit was denoted as one-half on the detection limit. For each sample in which at least one strain was detectable, the competitive index was calculated as: log10(Kanr CFU density/Trir CFU density) in the sample - log10(Kanr CFU density/Trir CFU density) in the inoculation mixture.
Neutrophil surface killing assay
Neutrophil surface killing assays were performed as described previously . Briefly, bacteria were grown to mid-log phase and frozen in THY/10% glycerol at −80°C. On the day of the experiment, bacteria were thawed and diluted to 5 × 103 CFU/mL in saline, and 10 μL of this suspension was spotted and allowed to dry at room temperature on trypticase soy agar with 5% defibrinated sheep blood, with 10 replicates per plate. Twenty microliters of neutrophils (2 × 106 cells/mL) were then overlaid, allowed to dry, and incubated overnight at 37°C with 5% CO2. Percent survival was calculated by comparing killing of each strain to a duplicate control plate with no neutrophils.
Genomic DNA from isolates TIGR4:19F and SpnYL101 were used to generate multiplexed Illumina libraries using Nextera DNA sample preparation kits. These were sequenced on the Illumina MiSeq platform to produce 151 nt paired-end reads, generating a total of 213,083 reads (64.4 Mb of data) for TIGR4:19F and 383,055 reads (115.7 Mb of data) for SpnYL101. This equates to over 30-fold and over 50-fold mean coverage of a typical pneumococcal genome, respectively. Illumina sequence data were mapped against the complete genome of S. pneumoniae TIGR4 (GenBank accession: AE005672) as paired end reads using SMALT v0.6.1. Bases were called using Samtools  and VCFtools  using the criteria described previously . These sequence data have been submitted to the Sequence Read Archive (SRA) with accession number SRX535517.
In silico screen of carriage isolates
The collection of carriage isolates used in this study correspond to 616 de novo assemblies of asymptomatically carried S. pneumoniae isolates, in which 5,442 clusters of orthologous genes (COGs) were identified and were previously reported . BLASTP was used to identify the COG corresponding to each TIGR4 gene that is affected by SNPs between TIGR4:19F and SpnYL101. To identify an isolate pair varied substantially at the target gene, coding sequences in the COG were aligned by CLUSTAL 2.1 . After alignment, two sequences showed the least percent identity at the target gene and belonged to isolates of the same serotype were chosen to form a pair. In case of a tie (two or more serotype-matched pairs with equally low within-pair sequence identity), the pair that represents the closest relatives, as ascertained through the shortest phylogenetic distance calculated from a whole genome alignment , was used in subsequent phenotypic evaluations. A flowchart of the porecess is shown in Additional file 1: Figure S4.
Cell-associated type 19 F capsular polysaccharide was quantified based on a previously published inhibition ELISA protocol [4,26]. Briefly, immunolon ELISA plates (Thermo Scientific, Waltham, MA) were coated by incubating with 5 μg/mL type 19 F capsular polysaccharide (ATCC, Manassas, VA; 100 μL/well) overnight. Either standard dilutions of type 19 F polysaccharide or serial dilutions of mid-log phase bacteria were mixed 1:1 with typing serum 19b (Statens, 1:5000–1:10,000). The mixtures were incubated in the coated ELISA plates for two hours. Typing sera captured by the coated plates were detected with goat anti-rabbit IgG-HRP (Pierce, 1:60,000) and the TMB developing substrate (KPL, Gaithersburg, MD). After developing was terminated by addition of 1 N HCl, the absorbance at 450 nm was measured using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA). The software accompanied the microplate reader was used to calculate the capsular polysaccharide concentration in each sample by a standard-curve method (4-parameter fit).
Selection of putitative pneumococcal SR pathway genes
To select a pneumococcal gene putatively corresponding to Lon in the SR pathway, a text search of “PDZ domain-containing protein” in KEGG ORTHOLOGY (http://www.genome.jp/kegg/) identified orthologous group K07177, from which the only gene found in S. pneumoniae TIGR4 strain, SP_1967, was chosen. Similarly, to select a pneumococcal gene putatively corresponding to rpoS in the SR pathway, a text search of “RNA polymerase primary factor” in KEGG ORTHOLOGY identified orthologous group K03086, from which the only gene found in S. pneumoniae TIGR4 strain, SP_1073, was chosen. To select a pneumococcal gene putatively corresponding to spoT in the SR pathway, a text search of “putative GTP pyrophosphokinase” in KEGG ORTHOLOGY identified orthologous group K03086, from which the only gene found in S. pneumoniae TIGR4 strain, SP_1073, was chosen.
Competitive index between two strains was compared using a Mann–Whitney test. Survival rate, growth rate, or capsular polysaccharide production between two strains was compared using a t-test. All p-values were calculated for two-tailed tests and p < 0.05 was considered as significant. Statistical analyses were conducted using the GraphPad Prism V5.0 software (GraphPad Software, San Diego, CA, USA) and the R software package (http://www.r-project.org/).
We thank Daniel Weinberger (Yale School of Public Health) and Eric Rubin (Harvard School of Public Health) for helpful discussions about this project. We are grateful to Bernice Sim and Lisa Kagedan at Harvard School of Public Health for technical assistance.
This work was supported by awards 5R01AI048935 to ML and 5R01AI106786 to WPH from the United States National Institute of Allergy and Infectious Diseases (http://www.niaid.nih.gov). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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