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Peritoneal Challenge Modulates Expression of Pneumococcal Surface Protein C during Bacteremia in Mice

Lisa R. Quin,¹ Quincy C. Moore III,¹ Justin A. Thornton,^1, and Larry S. McDaniel^1,2,3*

Departments of Microbiology,¹ Surgery,² Medicine, The University of Mississippi Medical Center, Jackson, Mississippi 39216³

Received 2 August 2007/ Returned for modification 29 August 2007/ Accepted 16 December 2007

	ABSTRACT

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Differential expression of pneumococcal virulence proteins has been demonstrated. We previously demonstrated challenge route-dependent differences in pneumococcal surface protein C (PspC) expression during bacteremia. In this study, we investigated differences in PspC expression during the transition of pneumococci from the peritoneum to the blood. Time course analysis of PspC expression using flow cytometry demonstrated that Streptococcus pneumoniae D39 collected from blood expressed significantly more PspC than did D39 collected from the peritoneum of intraperitoneally (i.p.)-infected mice. Various challenge models were then used to determine whether host responses originating from the peritoneum can influence PspC expressed by pneumococci in the blood. Using heat-inactivated D39 (HI-D39) and sterile peritoneal dialysis fluid (PDF), we investigated whether stimulation of peritoneal responses can influence PspC expression. Injection of mice i.p. with HI-D39 or PDF immediately prior to intravenous (i.v.) infection with D39 caused a significant increase in PspC expressed by D39 in the blood. Finally, we used cytokine array analysis to investigate specific inflammatory mediators that may result in differential PspC expression. Of the 96 inflammatory cytokines assayed, D39 i.p. challenge led to increased expression of 33 cytokines in serum; whereas D39 i.v. challenge led to increased expression of 15 and decreased expression of 11 cytokines relative to serum of the uninfected control. These results indicate that PspC is differentially regulated during growth in vivo and that the level of expression varies depending on the host environment.

	INTRODUCTION

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The versatile nature of the pneumococcus is central in acclimating to hostile host environments (20, 22, 28). Recent studies using various models of pneumococcal disease have demonstrated differential expression of several major virulence-associated proteins, including pneumococcal surface protein C (PspC) (15, 19, 22, 24). Recently, Northern blotting, microarray technology, and real-time PCR analysis have been performed to measure differences in pneumococcal gene induction during infections. Results of these assays have demonstrated that certain genes are not simply upregulated; rather there is tissue-specific gene activation (15, 19, 22, 24). In fact, virulence factors required for maintenance of infection are distinct from those required during migration of pneumococci throughout various host environments (20). While the mechanisms involved in differential regulation of virulence genes are not yet fully understood, the coordinated patterns of regulation suggest that within a discrete focus of infection pneumococci sense and respond to specific environmental conditions (17, 18, 24).

In a previous study, we demonstrated that PspC message and protein levels are increased during bacteremia. Specifically, greater expression of PspC was detected during bacteremia following intraperitoneal (i.p.) challenge than during that following intravenous (i.v.) challenge (24). These results indicated that, depending on the challenge route, there is differential regulation of pspC transcription and protein expression during systemic infection. Other studies by Orihuela et al. have demonstrated that peritoneal culture can alter the levels of virulence genes expressed by pneumococci (21). While the peritoneum is not a typical site of a pneumococcal infection, we have demonstrated that this environment leads to significant increases in PspC expression. Therefore, time course analyses of PspC expression were performed to investigate PspC expression during the transition of pneumococci from the peritoneum to the blood. Also, we used various pneumococcal challenge models to determine whether host factors lead to increased PspC expression. By injecting mice with heat-inactivated D39 (HI-D39) i.p. and then immediately infecting them with viable D39 i.v., we have examined the effects of host responses in the peritoneum on the expression of PspC on the surface of viable pneumococci in the blood. Furthermore, we used peritoneal dialysis fluid (PDF) to artificially stimulate i.p. innate host responses. The nonphysiological composition of PDF provides a sterile means of inducing complement activation, inflammatory cytokines, and chemoattractants within the peritoneum (3, 6). Our results suggest that soluble mediators, originating from within the peritoneum, migrate or diffuse to the blood and affect PspC expression. Cytokine antibody array analysis was used to investigate the host immune factors, specifically inflammatory cytokines, which were predominantly upregulated in the blood following i.p. or i.v. infection. Our results demonstrated important contrasts in the host responses generated as a consequence of pneumococcal infection and suggest that the regulation of PspC expression may be governed by host factors.

	MATERIALS AND METHODS

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Bacterial strains, growth conditions, and heat inactivation. The Streptococcus pneumoniae strain used in this study was D39, a mouse virulent capsular serotype 2 laboratory strain that expresses PspC of group 3.1 (10). Bacteria were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) at 37°C to mid-log phase, harvested by centrifugation, and stored in aliquots containing 10% glycerol at –80°C. The bacterial concentration was determined prior to experiments by thawing an aliquot and plating serial dilutions on blood agar. D39 was heat inactivated by incubating 1 x 10⁸ CFU/ml at 65°C for 30 min. HI-D39 was collected by centrifugation, suspended in lactated Ringer's (LR) solution, and stored at –80°C. Samples were plated on blood agar to verify inactivation.

Mouse model and harvesting of pneumococci. Naïve CBA/CAHN-XID/J (CBA/N) mice (Jackson Laboratories) expressing an X-linked immunodeficiency were used, as this mouse strain is susceptible to pneumococcal infection (31) and has been extensively used in our studies. Mice were injected i.p. or i.v. with D39 in a volume of 0.2 ml of LR solution. All challenge doses were verified by plate count. Mouse peritoneal fluid (PF) was collected by peritoneal lavage with 7 ml of cold LR solution. Blood samples were collected from each anesthetized mouse by retro-orbital bleeding using microhematocrit capillary tubes (Fisher Scientific). Bacteria were harvested from the blood as described previously (24).

Flow cytometry analysis of PspC expression in vitro. PspC expression by D39 was examined by flow cytometry as previously described using an anti-PspC monoclonal antibody, recombinant D39 PspC 87 (RDC87) (24). In vitro expression of PspC by pneumococci following exposure to mouse PF was compared to expression following incubation with THY alone. Briefly, D39 was incubated for 30 min at 25°C with 100 µl of either PF or THY. Pneumococci were washed and incubated with 10 µg/ml of RDC87 for 30 min at 25°C. Following additional washes, bacteria were incubated with 30 µg/ml biotinylated goat anti-mouse immunoglobulin G secondary antibody (Southern Biotech) for 30 min at 25°C, stained with 1.0 µg/ml of streptavidin-conjugated Alexa Fluor 488 (Molecular Probes), and suspended in 2 ml of phosphate-buffered saline for analysis. Additional in vitro experiments were performed using PF collected from mice 24 h following i.p. injection with 1 x 10⁸ CFU/ml HI-D39. In these experiments, viable D39 grown in culture was incubated with 100 µl treated PF for 30 min at 25°C and the remaining incubations were performed as described above to detect PspC expression.

Flow cytometry analysis of PspC expression by pneumococci harvested from mice. To examine in vivo expression of PspC, D39 harvested from mice at the indicated time points was immediately incubated with 10 µg/ml of RDC87 for 30 min at 25°C. Pneumococci were washed, and the remaining incubations were performed as described in the in vitro flow cytometry protocol above.

PspC expression over time was investigated using five groups of two mice that were challenged i.p. with 1 x 10⁶ CFU of D39. Pneumococci were recovered from blood and from PF at 1, 4, 8, 12, and 18 h, and PspC expression was analyzed by flow cytometry. Samples were plated to ensure that similar CFU in blood and PF were used in our flow cytometry analysis, and the relative number of D39 bacteria harvested at 1, 4, 8, 12, and 18 h was approximately 10³, 10⁴, 10⁵, 10⁷, and 10⁸ CFU/ml, respectively. This experiment was independently repeated, and each time point represents data from a total of four mice.

In other experiments five groups of four mice first received either (i) 0.2 ml LR solution injected i.p., (ii) 7 ml LR solution injected i.p., (iii) 7 ml Delflex peritoneal dialysis solution with 4.25% dextrose (Fresenius Medical; kindly provided by M. Flessner) injected i.p., (iv) HI-D39 diluted to 1 x 10⁸ CFU/ml in LR solution injected i.p., or (v) 1 x 10⁵ CFU of viable D39 injected i.p. Groups that received LR solution, Delflex, or HI-D39 i.p. (groups 1 to 4) were immediately challenged i.v. with 1 x 10⁵ CFU of viable D39. Pneumococci were recovered from the blood at 24 h, and PspC expression was analyzed by flow cytometry. Again, plate counts of all samples were performed to ensure that similar bacterial CFU in blood were used in our analyses. In all cases, the number of D39 bacteria harvested from blood at 24 h was approximately 10⁸ CFU/ml. Three independent experiments using groups of four mice were performed.

Experimental controls and data analysis. D39 grown in vitro was used as the positive control for the flow cytometry assays. The negative isotype-matched controls included D39 grown in vitro and collected from blood and from PF incubated with 10 µg/ml of an isotype-matched unrelated monoclonal antibody as a replacement for RDC87. In all cases the mean fluorescence intensity (MFI) for the negative control was less than 3.6 ± 0.3. Forward and side scatter were used to exclude aggregates, and the geometric MFI above background fluorescence (MFI for the negative controls) was calculated for each sample using a FACScan cytometer (Becton Dickinson). Results are expressed either as the calculated positive percentage or as the MFI ± standard error of three independent experiments. Student's t test was used to compare flow cytometry data.

Cytokine array analysis. Blood was collected from mice 24 h following i.v. or i.p. infection with 1 x 10⁵ CFU of D39 or from mice injected with 0.2 ml LR solution injected i.p. as an uninfected control. Serum was analyzed with mouse cytokine antibody array C Series 1000 (RayBiotech) according to the manufacturer's specifications. Briefly, antibody array membranes III and IV were placed into eight-well trays and blocked with 2 ml of blocking buffer for 30 min. Blocking buffer was decanted, and membranes were incubated at 25°C for 2 h with l ml of 10-fold-diluted serum collected from mice injected with LR solution i.p. or from mice challenged with D39 i.v. or i.p. Samples were decanted, and membranes were washed with 1x wash buffer I followed by 1x wash buffer II at 25°C with shaking. Diluted biotin-conjugated antibodies (1 ml) were added to each membrane and allowed to incubate at 4°C overnight. Membranes were again washed with 1x wash buffers I and II prior to being incubated at 25°C for 2 h with diluted horseradish peroxidase-conjugated streptavidin. Following a final wash, the membranes were incubated with detection buffers C and D. The chemiluminescent signal was then detected by exposure to film, and intensity was determined by densitometry. Signal intensities were quantitated using ImageQuant 5.0 (Molecular Dynamics), and cytokines with intensity values twofold higher or lower than those detected in serum collected 24 h following injection with LR solution i.p. were considered significant.

	RESULTS

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Time course analyses of PspC expression in vivo. We first determined that naïve mouse PF alone did not stimulate PspC expression in vitro. The MFI for D39 incubated in THY or in PF was 17.3 ± 4 or 15.6 ± 6, respectively. To further investigate whether peritoneal factors influenced PspC expression, we incubated pneumococci with PF collected from mice treated with HI-D39. Again, we did not detect a significant increase in PspC expression in vitro following incubation with the treated PF (MFI = 18 ± 3) compared to D39 incubated in THY alone (MFI = 17.3 ± 4).

Previous studies demonstrated significant differences in PspC expression by pneumococci in blood of mice challenged i.p. compared to those challenged i.v. (24). Therefore, we investigated differences in PspC expression during the transition of pneumococci from the peritoneum to the blood by infecting groups of mice i.p. with D39. D39 was collected from blood and from PF at 1, 4, 8, 12, and 18 h post-i.p. infection. Despite lower numbers of pneumococci recovered from the blood and PF at the very early time points, using flow cytometry we detected differences in the surface expression of PspC. Although there were increased levels of PspC on the surface of D39 collected from PF at 1 h postinfection, by 4 h a greater percentage of pneumococci collected from blood than of those collected from PF had increased expression of PspC (Fig. 1). This suggested that PspC expression may be increased during the transition from the peritoneum to the bloodstream. A similar trend in PspC expression was detected on pneumococci collected from blood at 8, 12, and 18 h postinfection (Fig. 1). Examining the pattern of expression, we detected a sharp rise in PspC expressed by D39 in blood by 8 h. This pattern of upregulation was not as evident in D39 collected from PF. Analysis of later time points of both D39 from blood and D39 from PF revealed a reduction in PspC expression at 12 h followed by an increase at 18 h. Taken together, these results demonstrated that PspC facilitates pneumococcal transcytosis, leading to enhanced bacteremia, and suggest that PspC is differentially regulated during growth in vivo depending on the host environment.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Time course analysis of PspC expression in vivo. Flow cytometry was used to investigate PspC expression on the surface of pneumococci collected from blood and from PF following i.p. infection. The percentages of pneumococci that were positive for PspC were calculated based on the increase in MFI above background. Two mice were used for each time point, and data are presented as an average of the data from a total of four mice from two independent experiments.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Inflammatory stimuli in peritoneum increase PspC expression in blood. (A) Graph representing the MFI ± standard error of PspC expression by D39: cultured in vitro ("a" in panel B), collected from blood 24 h following i.v. infection ("b" in panel B), collected from blood following i.p. injection with HI-D39 and then i.v. infection with D39 ("c" in panel B), collected from blood following i.p. injection with LR solution and then i.v. infection with D39 ("d" in panel C), collected from blood following i.p. injection with Delflex and then i.v. infection with D39 ("e" in panel C), and collected from blood following i.p. infection ("f" in panel C). *, P = 0.02 versus PspC expression on D39 cultured in vitro; **, P = 0.002 versus PspC expression on D39 following i.v. challenge. (B) Representative histograms of PspC expression for experiments a, b, and c. (C) Representative histograms of PspC expression for experiments d, e, and f. The positive percentage is indicated above each peak.

Inflammatory mediators may signal an increase in PspC expression. To investigate host immune factors that possibly lead to the differential regulation of PspC in vivo, we used cytokine antibody array analysis. Serum collected from mice 24 h following injection of LR solution i.p., or following i.v. or i.p. infection with D39 was analyzed using mouse cytokine antibody arrays. Radiography was used to detect signal intensities, which were then quantified by densitometry (Fig. 3A and B). The difference relative to the LR solution-injected control was determined, and cytokines with intensity values twofold higher or lower than those detected in serum collected 24 h following i.p. injection with LR solution, the uninfected control, were considered significant. The differences in intensity values relative to the control are summarized in Table 1. Of the 96 inflammatory cytokines assayed, D39 i.p. challenge led to increased expression of 33 cytokines, 18 of which were uniquely upregulated in serum following i.p. infection. In contrast, D39 i.v. challenge led to increased expression of 15 cytokines and decreased expression of 11 cytokines relative to the LR solution i.p. control. These results demonstrated important contrasts in the cytokine expression profiles in serum generated as a consequence of different routes of pneumococcal infection and again suggest that the regulation of pneumococcal virulence factors in vivo may be governed by the host environment.

View larger version (96K): [in this window] [in a new window]   FIG. 3. Film images of cytokine arrays. A total of 96 cytokines were analyzed, 62 on array III (A) and 34 on array IV (B). Serum collected either from mice challenged with D39 i.p., mice challenged with D39 i.v., or mice injected with LR solution i.p. was each incubated with the arrays. Differences in the intensities of spots in serum from D39 i.p.- or i.v.-infected mouse serum, relative to the LR solution i.p. control, indicated differential expression of the corresponding cytokines.

	DISCUSSION

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PspC is a multifunctional virulence protein that is also known as CbpA, SpsA, and Hic due its ability to bind choline, the secretory component of immunoglobulin A, and complement factor H, respectively (5, 8, 11, 24, 25). PspC is differentially regulated during the natural progression of pneumococcal disease (10, 15, 24). In vivo experiments examining the regulation of pneumococcal virulence gene expression have demonstrated that different genes are induced during pneumococcal bacteremia—that is, a planktonic state, compared to tissue infection such as pneumonia or meningitis (18). Other studies have demonstrated that pspC message levels are increased during bacteremia (15, 24) and during invasion of the lung and cerebrospinal fluid (22). Strains that have mutations in pspC have decreased proliferation on mucosal surfaces, in the lungs, and in circulation (2, 9, 25). Also, pneumococci that express PspC are resistant to phagocytosis by microglial cells and can diminish immune responses mounted by brain macrophages in vitro (23).

We have previously demonstrated that pneumococci collected from blood of i.p.-infected mice had a more substantial increase in PspC expression than did those collected from i.v.-infected mice (24). In this study, we investigated whether the peritoneum contained factors that stimulated the expression of PspC on the pneumococcal surface. In vitro experiments were performed in which pneumococci were first incubated either in naïve mouse PF or with treated PF. Our results demonstrated that PF and the treated PF did not lead to increased PspC expression in vitro. These results indicated that neither the host factors resident in the peritoneum nor those stimulated by the presence of HI-pneumococci influenced PspC expression in vitro.

Streptococci can be recovered from blood as early as 30 min following i.p. challenge (7, 27). Since previous in vivo studies were conducted on pneumococci that had been collected from blood 24 h following infections (24), we examined the expression of PspC on the surface of pneumococci collected from blood and from PF over time. Our time course analysis data suggest that after 1 h the level of PspC expressed by D39 in blood following i.p. infection is greater than that of D39 present in the peritoneum. This observation that PspC is upregulated during the transition of pneumococci from the peritoneum to the blood is supported by previous studies that indicated that pspC is induced during transcytosis (20, 22). Our results provide further evidence that PspC is differentially regulated during progression of pneumococcal disease and that upregulation of PspC may promote dissemination through specific host environments (24). Interestingly, although our experimental procedures to detect surface expression of PspC were very different from those used by Oggioni et al. (18) to examine gene expression, our results support their observation that pneumococcal gene regulation may be dependent on the growth phase and/or host environment.

To investigate the effects of in vivo host responses in the peritoneum on the expression of PspC on the surface of viable pneumococci in the blood, we used HI-pneumococci. The introduction of HI-D39 to the mouse peritoneum led to increased PspC expression by viable D39 collected from blood. Since the i.p. environment was changed by the presence of killed pneumococci, we therefore attempted to artificially and nonspecifically initiate innate responses within the peritoneum using dialysis solution. The nonphysiological composition of PDF provides a sterile means of inducing complement activation, inflammatory cytokines, and chemoattractants within the peritoneum (3, 6). Using this solution we were also able to induce PspC on viable D39 in the blood. Together, these results indicate that soluble mediators or cellular components, while initiated in the peritoneum, are able to migrate to the bloodstream where they influence PspC expression.

To further evaluate the specific influence of the innate immune response that could influence the regulation of PspC, we utilized cytokine antibody arrays. In these experiments, we have identified several host immune factors, specifically inflammatory cytokines that are predominantly upregulated in the blood following i.p. infection with D39. These included gamma interferon (IFN-) and the IFN--inducing cytokine interleukin-12 p40/p70 (IL-12 p40/p70). During an infection with extracellular bacteria, such as pneumococci, production of these cytokines stimulates neutrophil recruitment to the site of inflammation (13, 30). While local expression of IFN- and IL-12 is important in early innate host defense during pulmonary infections and pneumococcal meningitis, excess systemic expression can be harmful to the host (13, 29). Following i.p. infection, we also detected elevated expression of IL-6, monocyte chemoattractant protein 1/CCL2, intracellular adhesion molecule 1, and insulin-like growth factor binding protein 6 (IGFBP-6) in serum. Upregulation of many of these cytokines and chemokines has also been detected in brain tissue and in serum during pneumococcal meningitis (13, 29). Another interesting observation was that expression of only 15 of the 96 tested cytokines/chemokines was increased following i.v. challenge with D39. These included eotaxin, a chemoattractant that was also upregulated following i.p. infection, which is produced by epithelial and phagocytic cells and predominately functions in recruitment of eosinophils from mucosal tissues during allergic reactions (1, 4). Furthermore, in contrast to i.p.-infected mouse serum, we detected decreased expression of 11 cytokines in i.v.-infected mouse serum including IGFBP-6, L-selectin, cutaneous T-cell-attracting chemokine, and matrix metalloproteinase 3 (MMP-3). Of these, L-selectin and MMP-3 also have roles in the development of neuronal injury during pneumococcal meningitis (14, 16). Together, these results demonstrated significant contrasts in the host inflammatory responses generated as a consequence of the origin of pneumococcal infection. While pneumococcal meningitis studies have demonstrated differences between cytokine levels in the brain and those in blood (26), this study is the first to our knowledge to demonstrate dissimilar cytokine expression profiles in the serum following different modes of challenge. The contrasting pattern of cytokine expression following i.p. infection supports our hypothesis with respect to the role of soluble mediators and their ability to migrate to the blood where they influence expression of PspC. Although pneumococcal gene regulation is unlikely to be cytokine specific with regards to the host's innate immune response, our results suggest that the host factors originating from within the peritoneum lead to differential regulation of PspC. The detection of these specific cytokines in serum will allow for a more focused investigation of the host immune response that stimulates upregulation of pneumococcal surface proteins, including PspC.

	ACKNOWLEDGMENTS

 
We thank Michael Flessner for providing us with the Delflex solution used in our studies. We are also grateful to Cecile Snell and Stephanie Warren for their support with flow cytometry and densitometry analysis, respectively.

This study was partially supported by the Alliance for Graduate Education in Mississippi (AGEM) award by the National Science Foundation.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Microbiology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216. Phone: (601) 984-6880. Fax: (601) 984-1708. E-mail: LMcDaniel{at}microbio.umsmed.edu

Published ahead of print on 26 December 2007.

Editor: A. Camilli

Present address: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105.

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