Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 24;84(6):1887-1901.
doi: 10.1128/IAI.00097-16. Print 2016 Jun.

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

Guiling Li  1 , Zhuowen Liang  2 , Xiatai Wang  1 , Yonghong Yang  3 , Zhujun Shao  4 , Machao Li  4 , Yueyun Ma  2 , Fen Qu  5 , Donald A Morrison  6 , Jing-Ren Zhang  7   8
Affiliations

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

Guiling Li et al. Infect Immun. .

Abstract

Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S. pneumoniae clinical isolates representing at least 30 serotypes. While the vast majority of these isolates (94.7%) were transformable, the transformation frequency differed by up to 5 orders of magnitude between the least and most transformable isolates. The strain-to-strain differences in transformation frequency were observed among many isolates producing the same capsule types, indicating no general association between transformation frequency and serotype. However, a statistically significant association was observed between the levels of transformation and colonization fitness/virulence in the hypertransformable isolates. Although nontransformable mutants of all the selected hypertransformable isolates were significantly attenuated in colonization fitness and virulence in mouse infection models, such mutants of the strains with relatively low transformability had no or marginal fitness phenotypes under the same experimental settings. This finding strongly suggests that the pneumococci with high transformation capability are "addicted" to a "hypertransformable" state for optimal fitness in the human host. This work has thus provided an intriguing hint for further investigation into how the competence system impacts the fitness, virulence, and other transformation-associated traits of this important human pathogen.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Diversity of S. pneumoniae isolates in natural transformability. The impact of donor DNA concentration on natural transformability of the six initially nontransformable isolates (A) or common laboratory strains (B) was determined as described in Materials and Methods. (C) A similar approach was used to assess potential dose effect of the CSP on transformability of the selected laboratory strains. The values represent the numbers of transformants for each strain (per ∼108 cells) ± standard errors from three replicates in the presence of various concentrations of donor DNA (in panels A and B) or CSP (in panel C). (D) The transformation levels of the 208 pneumococcal isolates were similarly determined in the presence of donor DNA (rpsL1 amplicon, 500 ng/ml) and CSP (500 ng/ml) and are indicated by colors as follows: none (RTF = 0, dark blue), low (0.001 ≥ RTF > 0, light blue-light yellow), intermediate (0.03 ≥ RTF > 0.001, yellow-orange), and high (RTF > 0.03, dark orange-red). Each rectangle represents a strain. The relative transformation frequency (RTF) value of each isolate was calculated by dividing its transformants with that of strain R6. The number of strains in each transformability category is indicated at the top. The transformation frequency and associated information of the isolates are described in Table S1 in the supplemental material.
FIG 2
FIG 2
Transcription of the early and late com genes in the selected strains. The transcriptional reporter constructs were generated by fusion of the promoters of the early (comX) or late (ssbB) com genes to the 5′ end of the luciferase gene in pIB166 and electroporation into 11 nontransformable isolates. The promoter sequence of spxB was used as a positive control for transcription in the nontransformable isolates. The low-transformable (TH883 and TH2741), intermediate-transformable (TH906 and TH2835), and high-transformable (TH867 and TH2912) isolates, as well as strain R6, were used as positive controls for com gene transcription. The reporter strains were grown in THY to an OD620 of ∼0.1 and treated with CSP (final concentration, 500 ng/ml) for 30 min before the measurement of the luciferase activities for the strains carrying the reporter construct of spxB (A), comX (B), or ssbB (C). The values are the means ± the standard errors of the results from triplicate samples. LT, low transformability; IT, intermediate transformability; HT, high transformability; NT, no transformability.
FIG 3
FIG 3
Genetic complementation of the mutations in the essential com genes of the selected nontransformable isolates. Genetic complementation was performed in the nontransformable isolates or corresponding mutants of R6 with the pIB166 carrying the wild-type comGC (A), coiA (B), and comE (C, D, and E). The transformants are presented as the mean CFU ± the standard errors from triplicate samples. R6 mutants with a deletion in each of the corresponding com genes and their complementation counterparts were included as controls.
FIG 4
FIG 4
Differential impact of natural transformation on the colonization fitness of pneumococcal isolates with diverse transformation capacity. (A) The transformation frequency values of the 18 pneumococcal isolates selected for comparative analysis of the in vivo fitness and virulence traits in the mouse models. The strains in each of three transformability groups are indicated by blue (low), black (intermediate), and red (high) bars. The transformants are presented as the mean CFU ± the standard errors in the presence of selection antibiotic from triplicate samples. The identification and serotype information of each strain are indicated below each bar. NT, nontypeable capsular serotype. (B) The contribution of the transformation to the nasal colonization fitness of the 18 selected pneumococcal isolates with different transformability capacity during the coinfection of each isolate and its ΔcomGC mutant. The competitive index (CI) or CFU ratio between each wild-type (WT) and isogenic mutant (MT) pair was calculated by obtaining nasal washings of the BALB/c mice infected by intranasal inoculation with the 1:1 mixture of the strain pair 4 days earlier. Each symbol represents CI value of a single mouse. The transformability category of each strain is indicated by blue (low), black (intermediate), or red (high) color. The horizontal bars indicate the medians in individual groups of mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) The colonization levels of the 18 pneumococcal isolates. The total CFU value of the wild-type strain in the output pool from each mouse in the coinfection experiment (described in panel B) is presented as in panel B.
FIG 5
FIG 5
Biased impact of natural transformation on the virulence of the hypertransformable pneumococcal isolates. (A) The contribution of the transformation to the virulence (lung infectivity) of the 18 selected S. pneumoniae isolates with different transformability capacities during the coinfection of each isolate and its ΔcomGC mutant. The experimental design and result display are the same as in Fig. 4B except for the use of CD1 mice and a 48-h infection duration. (B) The lung infectivity levels of the 18 S. pneumoniae isolates. The total CFU value of the wild-type strain in the lungs from each mouse is presented as in Fig. 4C.
FIG 6
FIG 6
Significant attenuation of the ΔcomGC and ΔssbB mutants in the hypertransformable pneumococcal backgrounds in a single-strain infection mouse model of acute pneumonia. The impact of natural transformation on pneumococcal virulence was evaluated with the ΔcomGC and ΔssbB mutants of two selected isolates with low (A)-, intermediate (B)-, or high (C)-transformability phenotype in a single-strain infection mouse model of acute pneumonia. Each CD1 mouse was intranasally infected with either the wild type (WT) or an isogenic mutant lacking comGC or ssbB (essential genes for natural transformation) and then sacrificed to quantify the bacterial burden in the lung 48 h later as described in Materials and Methods. Bacterial burden is presented as a CFU value per lung (or mouse); each symbol represents the CFU value of a single mouse. The horizontal bars indicate the medians in individual groups of mice. ***, P < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bogaert D, De Groot R, Hermans PW. 2004. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis 4:144–154. doi:10.1016/S1473-3099(04)00938-7. - DOI - PubMed
    1. Johnston C, Campo N, Berge MJ, Polard P, Claverys JP. 2014. Streptococcus pneumoniae, le transformiste. Trends Microbiol 22:113–119. doi:10.1016/j.tim.2014.01.002. - DOI - PubMed
    1. Yother J. 2006. Genetics of Streptococcus pneumoniae, p 275–288. In Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI (ed), Gram-positive pathogens. ASM Press, Washington, DC.
    1. Chen I, Dubnau D. 2004. DNA uptake during bacterial transformation. Nat Rev Microbiol 2:241–249. doi:10.1038/nrmicro844. - DOI - PubMed
    1. Bikard D, Hatoum-Aslan A, Mucida D, Marraffini LA. 2012. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12:177–186. doi:10.1016/j.chom.2012.06.003. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources