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Identification of PblB mediating galactose-specific adhesion in a successful Streptococcus pneumoniae clone.

Hsieh YC, Lin TL, Lin CM, Wang JT - Sci Rep (2015)

Bottom Line: Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner.Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung.PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chang Gung Children's Hospital, Chang Gung Memorial Hospital, Chang Gung University, College of Medicine, Taoyuan, Taiwan.

ABSTRACT
The pneumococcal genome is variable and there are minimal data on the influence of the accessory genome on phenotype. Pneumococcal serotype 14 sequence type (ST) 46 had been the most prevalent clone causing pneumonia in children in Taiwan. A microarray was constructed using the genomic DNA of a clinical strain (NTUH-P15) of serotype 14 ST46. Using DNA hybridization, genomic variations in NTUH-P15 were compared to those of 3 control strains. Microarray analysis identified 7 genomic regions that had significant increases in hybridization signals in the NTUH-P15 strain compared to control strains. One of these regions encoded PblB, a phage-encoded virulence factor implicated (in Streptococcus mitis) in infective endocarditis. The isogenic pblB mutant decreased adherence to A549 human lung epithelial cell compared to wild-type NTUH-P15 strain (P = 0.01). Complementation with pblB restored the adherence. PblB is predicted to contain a galactose-binding domain-like region. Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner. Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung. PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success.

No MeSH data available.


Related in: MedlinePlus

Mouse challenge.A. Intranasal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. Seven days later, the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15, but not in the lungs. Complementation with pblB restored bacterial titers in the nasopgaryngx. Intranasal challenge of 3-weeks-old female BALB/c mice (n = 6–10 per group in the competition model) with equal inocula of bacterial strains. Each symbol represents the competitive index (CI) value for an individual animal. CI was calculated as described in Methods. Briefly, CI indicates the log10 normalized ratio. Horizontal bars indicate the median. B. Seven days later, NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx and lung. A CI below 0 indicates a competitive disadvantage of the mutant in relation to the wild-type strain. When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx and lung. C. Galactose preincubation before intranasal inoculation. Seven days later, NTUH-P15 wild-type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx, but out-competed the NTUH-P15 pblB mutant in the lung. ND: not done (can’t be calculated). Intratracheal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. D. Twenty-four hours after inoculation, the pblB mutant had a lower bacterial titers in the lung than that of mice infected with strain NTUH-P15. Complementation with pblB restored bacterial titers in the lung. E. Twenty-four hours after inoculation, strain NTUH-P15 had extensive and increased infiltration of inflammatory cells infiltration in the lung than that of mice infected with the pblB mutant. Complementation with pblB restored the severity of pneumonia. H&E-stained tissue samples.
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f4: Mouse challenge.A. Intranasal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. Seven days later, the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15, but not in the lungs. Complementation with pblB restored bacterial titers in the nasopgaryngx. Intranasal challenge of 3-weeks-old female BALB/c mice (n = 6–10 per group in the competition model) with equal inocula of bacterial strains. Each symbol represents the competitive index (CI) value for an individual animal. CI was calculated as described in Methods. Briefly, CI indicates the log10 normalized ratio. Horizontal bars indicate the median. B. Seven days later, NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx and lung. A CI below 0 indicates a competitive disadvantage of the mutant in relation to the wild-type strain. When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx and lung. C. Galactose preincubation before intranasal inoculation. Seven days later, NTUH-P15 wild-type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx, but out-competed the NTUH-P15 pblB mutant in the lung. ND: not done (can’t be calculated). Intratracheal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. D. Twenty-four hours after inoculation, the pblB mutant had a lower bacterial titers in the lung than that of mice infected with strain NTUH-P15. Complementation with pblB restored bacterial titers in the lung. E. Twenty-four hours after inoculation, strain NTUH-P15 had extensive and increased infiltration of inflammatory cells infiltration in the lung than that of mice infected with the pblB mutant. Complementation with pblB restored the severity of pneumonia. H&E-stained tissue samples.

Mentions: Intranasal challenge of mice infected with the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15 (P = 0.03), but not in the lungs (Fig. 4A). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, bacterial titers in the nasopgaryngx could be restored (Fig. 4A). To reduce the variance in the experimental setup caused by variation between individual mice and inoculation efficacy, we performed competitive experiment. Seven days after intranasal inoculation, the NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx (P = 0.003) and lung (P = 0.001) (Fig. 4B). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx (P = 0.5) and lung (P = 0.9) (Fig. 4B). Galactose was preincubated with strain NTUH-P15 and the NTUH-P15 pblB mutant strain before mixing together and intranasal inoculation. Seven days after intranasal inoculation, the NTUH-P15 wild type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx (P = 0.4) (Fig. 4C). In the lung, the NTUH-P15 wild type strain still out-competed the NTUH-P15 pblB mutant (P value can’t be calculated) (Fig. 4C). Comparing to strain NTUH-P15 competing with the NTUH-P15 pblB mutant strain without galactose pre-incubation, a significantly lower percentage of mice was colonizing with pneumococci in the lung when strain NTUH-P15 competing with the NTUH-P15 pblB mutant strain with galactose pre-incubation (37.5% (3/8, Fig. 4C) vs 90% (9/10, Fig. 4B); P = 0.04). Intratracheal challenge of mice infected with the pblB mutant had a significantly lower bacterial titers in the lung than that of mice infected with strain NTUH-P15 at 24 hour post-challenge (P = 0.03) (Fig. 4D), but not at 48 hour (data not shown). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, bacterial titers in the lung could be restored at 24 hour post-challenge (Fig. 4D). Microscopic evaluation show extensive and increased infiltration of inflammatory cells in the lung caused by strain NTUH-P15 at 24 hour post-challenge compared to that caused by the NTUH-P15 pblB mutant strain (Fig. 4E). PblB complementation strain restored the severity of pneumonia (Fig. 4E). The survival rates between wild type and mutant strains were not significantly different.


Identification of PblB mediating galactose-specific adhesion in a successful Streptococcus pneumoniae clone.

Hsieh YC, Lin TL, Lin CM, Wang JT - Sci Rep (2015)

Mouse challenge.A. Intranasal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. Seven days later, the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15, but not in the lungs. Complementation with pblB restored bacterial titers in the nasopgaryngx. Intranasal challenge of 3-weeks-old female BALB/c mice (n = 6–10 per group in the competition model) with equal inocula of bacterial strains. Each symbol represents the competitive index (CI) value for an individual animal. CI was calculated as described in Methods. Briefly, CI indicates the log10 normalized ratio. Horizontal bars indicate the median. B. Seven days later, NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx and lung. A CI below 0 indicates a competitive disadvantage of the mutant in relation to the wild-type strain. When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx and lung. C. Galactose preincubation before intranasal inoculation. Seven days later, NTUH-P15 wild-type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx, but out-competed the NTUH-P15 pblB mutant in the lung. ND: not done (can’t be calculated). Intratracheal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. D. Twenty-four hours after inoculation, the pblB mutant had a lower bacterial titers in the lung than that of mice infected with strain NTUH-P15. Complementation with pblB restored bacterial titers in the lung. E. Twenty-four hours after inoculation, strain NTUH-P15 had extensive and increased infiltration of inflammatory cells infiltration in the lung than that of mice infected with the pblB mutant. Complementation with pblB restored the severity of pneumonia. H&E-stained tissue samples.
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f4: Mouse challenge.A. Intranasal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. Seven days later, the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15, but not in the lungs. Complementation with pblB restored bacterial titers in the nasopgaryngx. Intranasal challenge of 3-weeks-old female BALB/c mice (n = 6–10 per group in the competition model) with equal inocula of bacterial strains. Each symbol represents the competitive index (CI) value for an individual animal. CI was calculated as described in Methods. Briefly, CI indicates the log10 normalized ratio. Horizontal bars indicate the median. B. Seven days later, NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx and lung. A CI below 0 indicates a competitive disadvantage of the mutant in relation to the wild-type strain. When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx and lung. C. Galactose preincubation before intranasal inoculation. Seven days later, NTUH-P15 wild-type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx, but out-competed the NTUH-P15 pblB mutant in the lung. ND: not done (can’t be calculated). Intratracheal challenge of 3-week-old female BALB/c mice (n = 4–6 per group in the separate model) with 5 × 107 colony-forming units (CFUs) of wild-type NTUH-p15, pblB mutant, and pblB complementation strains. D. Twenty-four hours after inoculation, the pblB mutant had a lower bacterial titers in the lung than that of mice infected with strain NTUH-P15. Complementation with pblB restored bacterial titers in the lung. E. Twenty-four hours after inoculation, strain NTUH-P15 had extensive and increased infiltration of inflammatory cells infiltration in the lung than that of mice infected with the pblB mutant. Complementation with pblB restored the severity of pneumonia. H&E-stained tissue samples.
Mentions: Intranasal challenge of mice infected with the pblB mutant had a significantly lower bacterial titers in the nasopharynx than that of mice infected with strain NTUH-P15 (P = 0.03), but not in the lungs (Fig. 4A). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, bacterial titers in the nasopgaryngx could be restored (Fig. 4A). To reduce the variance in the experimental setup caused by variation between individual mice and inoculation efficacy, we performed competitive experiment. Seven days after intranasal inoculation, the NTUH-P15 wild-type strain significantly out-competed the NTUH-P15 pblB mutant in the nasopharynx (P = 0.003) and lung (P = 0.001) (Fig. 4B). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, NTUH-P15 wild-type strain did not out-compete the complementation strain in the nasopharynx (P = 0.5) and lung (P = 0.9) (Fig. 4B). Galactose was preincubated with strain NTUH-P15 and the NTUH-P15 pblB mutant strain before mixing together and intranasal inoculation. Seven days after intranasal inoculation, the NTUH-P15 wild type strain did not significantly out-compete the NTUH-P15 pblB mutant in the nasopharynx (P = 0.4) (Fig. 4C). In the lung, the NTUH-P15 wild type strain still out-competed the NTUH-P15 pblB mutant (P value can’t be calculated) (Fig. 4C). Comparing to strain NTUH-P15 competing with the NTUH-P15 pblB mutant strain without galactose pre-incubation, a significantly lower percentage of mice was colonizing with pneumococci in the lung when strain NTUH-P15 competing with the NTUH-P15 pblB mutant strain with galactose pre-incubation (37.5% (3/8, Fig. 4C) vs 90% (9/10, Fig. 4B); P = 0.04). Intratracheal challenge of mice infected with the pblB mutant had a significantly lower bacterial titers in the lung than that of mice infected with strain NTUH-P15 at 24 hour post-challenge (P = 0.03) (Fig. 4D), but not at 48 hour (data not shown). When the NTUH-P15 pblB mutant strain was complemented with the pblB gene, bacterial titers in the lung could be restored at 24 hour post-challenge (Fig. 4D). Microscopic evaluation show extensive and increased infiltration of inflammatory cells in the lung caused by strain NTUH-P15 at 24 hour post-challenge compared to that caused by the NTUH-P15 pblB mutant strain (Fig. 4E). PblB complementation strain restored the severity of pneumonia (Fig. 4E). The survival rates between wild type and mutant strains were not significantly different.

Bottom Line: Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner.Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung.PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Chang Gung Children's Hospital, Chang Gung Memorial Hospital, Chang Gung University, College of Medicine, Taoyuan, Taiwan.

ABSTRACT
The pneumococcal genome is variable and there are minimal data on the influence of the accessory genome on phenotype. Pneumococcal serotype 14 sequence type (ST) 46 had been the most prevalent clone causing pneumonia in children in Taiwan. A microarray was constructed using the genomic DNA of a clinical strain (NTUH-P15) of serotype 14 ST46. Using DNA hybridization, genomic variations in NTUH-P15 were compared to those of 3 control strains. Microarray analysis identified 7 genomic regions that had significant increases in hybridization signals in the NTUH-P15 strain compared to control strains. One of these regions encoded PblB, a phage-encoded virulence factor implicated (in Streptococcus mitis) in infective endocarditis. The isogenic pblB mutant decreased adherence to A549 human lung epithelial cell compared to wild-type NTUH-P15 strain (P = 0.01). Complementation with pblB restored the adherence. PblB is predicted to contain a galactose-binding domain-like region. Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner. Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung. PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success.

No MeSH data available.


Related in: MedlinePlus