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, 61 (5), 1196-210

Switch From Planktonic to Sessile Life: A Major Event in Pneumococcal Pathogenesis

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Switch From Planktonic to Sessile Life: A Major Event in Pneumococcal Pathogenesis

Marco R Oggioni et al. Mol Microbiol.

Abstract

Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT-PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence-specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum-sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction - from the pneumococcal point of view - between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm-like state.

Figures

Fig. 1
Fig. 1
In vivo and in vitro gene expression patterns of selected S. pneumoniae genes. mRNA levels were measured by quantitative real time RT-PCR and evaluated according to the 2–ΔΔCT method (Livak and Schmittgen, 2001). Gene expression of strain TIGR4 was analysed in six distinct conditions which included (A) pneumococci recovered from brain homogenates of five mice infected IC, (B) pneumococci recovered from lung washes of five mice infected IN, (C) four pools of bacteria forming biofilm on plastic, (D) pneumococci recovered from blood samples of five IV infected mice, and (E) pneumococci on agar plates. All values of fold change in gene expression are reported as change towards expression of the relative gene in liquid culture (black colour indicates similar expression as in liquid). Each line in the figure represents a different biological replica, while each column represents a single gene. The colour code used is displayed on the right.
Fig. 2
Fig. 2
Light microscopy of pneumococcal biofilm. Appearance of pneumococci attached to plastic microtiter plates in the presence of CSP. Panels A, B and C refer to strain D39, while panels D, E and F to TIGR4. Panels A and D show the glossy amorphous blebs visible by phase contrast microscopy on the bottom of microtiter wells. The images of the structures sticking to the bottom of the microtiter wells were taken from washed, but non-fixed, 6 well plates with a 40x objective. Panels B and E show methanol fixed samples stained with alcian blue for polysaccharides and counterstained with crystal violet and observed in bright filed (40× objective). Panels C and F are as B and E, but observed with a 100× objective. Noteworthy is the uneven distribution of pneumococci on the bottom of the wells (B, C, E and F), which is compatible with the structures seen in panels A and D. The blue staining surrounding pneumococci in bright field (panels B, C, E and F) is compatible with a polysaccharide extracellular matrix described generally as main constituent of bacterial biofilms.
Fig. 3
Fig. 3
Viable counts of pneumococci attached to plastic. The pneumococcal strains TIGR4 and D39 and a series of isogenic mutants were evaluated for attachment to polystyrene microtiter wells. After 18 h of incubation in TSB the plates were washed and bacterial cells were detached by sonication. Counts of bacteria from wells with medium alone are shown by empty bars while counts of cells with CSP-supplemented medium are shown in grey (100 ng ml−1 of CSP2 for TIGR4 and 30 ng ml−1 of CSP1 for D39). Strains used in the experiment were the wild-type strains TIGR4 and D39, and their rough mutants FP23 and RX1, comC mutants FP64 and FP5, and comD mutants FP184 and FP48 respectively (Table 2).
Fig. 4
Fig. 4
Concentration-dependent effect of CSP on attachment to plastic. The amount of biofilm formation of different S. pneumoniae strains incubated at increasing CSP concentrations (0, 3, 10, 30, 100 and 300 ng ml−1) was evaluated by determination of the viable counts of detached cells. A. Strains carrying the comC1 allele were incubated with CSP1; D39 (square), G54 (circle), ATCC6302 (triangle) and ATCC6305 (diamond). B. Strains carrying the comC2 allele were incubated with CSP2; TIGR4 (open square), A66 (open circle), and ATCC6307 (open triangle).
Fig. 5
Fig. 5
Impact of modulation of the competence system on pneumococcal virulence. The effect of inhibition and induction of the competence system on pneumococcal virulence was assayed in the murine infection models of sepsis and pneumonia. In both experiments three groups of mice were challenged with TIGR4 (control group, filled square), the comD mutant FP184 (competence negative strain, filled circle) and with TIGR4 treated with CSP2 (treatment group, open square). Results of the sepsis model (panel A) have been reported previously (Oggioni et al., 2004) and are reproduced with the permission of the publisher. In panel A mice were challenged IV and the treatment group (open square) received two IV doses of 1.3 μg CSP2 per mouse at time 0 and 24 h after challenge. Panel B reports IN challenge with a challenging dose of 2 × 105 bacteria. In this pneumonia model the treatment group received 1.3 μg of CSP2 once together with the IN challenge. All experiments were performed in CD1 outbred mice (n = 20 IV; n = 2 × 16 IN) using the same frozen bacterial stocks for challenge. Differences in survival are statistically significant for the treatment groups both in the IV challenge in panel A (TIGR4 versus TIGR4 treated, *P = 0.012; FP184 versus TIGR4 treated, ***P < 0.0001; FP184 versus TIGR4, ns) and the IN challenge in panel B (TIGR4 treated versus FP184, **P = 0.0017; TIGR4 treated versus TIGR4, *P = 0.042; FP184 versus TIGR4, ns).
Fig. 6
Fig. 6
The effect of physical state of pneumococci on infectivity. A challenge experiment using in parallel sessile bacteria from a biofilm (open square) and planktonic cells from liquid culture (filled square) was done in the IV sepsis model, the IN pneumonia model and the IC meningitis model. In all experiments two groups of mice were challenged either with pneumococci from liquid culture (mid log phase) or from biofilm (24 h biofilm cells detached from microtiter plates). In panel A mice were challenged IV with 2 × 103 cfu per mouse, panel B reports IC challenge with a dose of 2 × 102 cfu and panel C reports IN challenge with a dose of 105 cfu. All experiments were performed with strain TIGR4 in MF1 outbred mice (n = 6 sepsis; n = 5 meningitis; n = 10 pneumonia) using the same frozen pre-counted bacterial stocks for challenge. Differences in survival are statistically significant for the IV challenge in panel A (*P = 0.021) and for the IC challenge in panel B (*P = 0.045). The difference in virulence in panel C is not statistically significant (ns, P = 0.067).

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