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We compared growth of Streptococcus pneumoniae mutants with disruption in the pspA (PspA-), nanA (NanA-) or hyl (Hyl-) gene to the parental D39 strain using a competitive growth model in mice with and without prior influenza infection. Total bacteria numbers recovered from influenza-infected mice were significantly greater compared to mice without influenza infection. Whereas Hyl- and NanA- mutants did not display attenuation in mice with or without prior influenza infection, the PspA- mutant exhibited attenuation in mice both with and without influenza infection. This defect was severe influenza-infected mice where PspA- growth was 1800-fold less than D39. Furthermore, PspA immunization significantly reduced secondary bacterial lung burdens and specific markers of lung damage in mice receiving serotypes 2, 3 and 4 pneumococci. Our findings indicate that PspA contributes to secondary S. pneumoniae infection following influenza and that PspA immunization mitigates early secondary pneumococcal lung infections.
Influenza A infection dramatically increases susceptibility to secondary Streptococcus pneumoniae infection resulting in significantly greater morbidity and mortality . Viral effects implicated include disruption of innate-effector responses [2, 3] and modification of respiratory mucosa [4, 5]. Whereas viral contributions to secondary pneumococcal infection have received considerable attention, contributions of S. pneumoniae virulence factors remain unclear. Much effort has focused on pneumococcal virulence factors as targets for vaccine development in primary infection [6–8]. Among those explored are hyaluronidase (Hyl), neuraminidase A (NanA) and pneumococcal surface protein A (PspA). Hyl targets hyaluronic acid in host connective tissues and extracellular matrix enhancing access for tissue invasion and colonization [8, 9]. Like influenza neuraminidase, pneumococcal NanA cleaves terminal sialic acid residues on respiratory-surface glycoconjugates promoting adhesion and colonization of bacteria [10, 11]. PspA is a choline-binding surface protein, which inhibits complement-mediated phagocytosis and binds to and prevents killing by lactoferrin [12, 13]. Numerous studies have documented attenuation in mutants lacking PspA [12, 14–16], and PspA protein is protective in a variety of delivery and challenge models [17–22].
Current pneumococcal vaccines have been effective in decreasing incidence of invasive pneumococcal disease [23, 24]; however, protecting the elderly and selection driven shifts in colonizing and disease causing serotypes remain prominent concerns [23, 25, 26]. As S. pneumoniae virulence factors are attractive candidates for vaccine development, further examination of their roles in the influenza-infected lung will help identify targets critical for secondary pneumococcal infection. This study examined the relative contributions of Hyl, NanA and PspA to S. pneumoniae virulence in healthy and influenza-infected mice. Additionally, the ability of immunization with PspA to reduce serotype 2, 3 and 4 primary and secondary pneumococcal growth and lung damage was assessed.
Healthy 6–8 week old female C57BL/6 mice were obtained from the NCI-Frederick Animal Production Area, Frederick, MD. All procedures were approved by the Montana State University Institutional Animal Care and Use Committee.
S. pneumoniae strains D39 (serotype 2), WU2 (serotype 3) and TIGR4 (serotype 4) were cultured at 37°C/5% CO2 in Todd Hewitt broth with 0.5% yeast extract (THY). Insertion-duplication mutants of D39 lacking PspA (PspA-), Hyl (Hyl-) and NanA (NanA-) were generously provided by Dr. J. C. Paton. These mutants have been previously described  and while these and other insertion-duplication mutants have been routinely employed [14, 27, 28], there exists the possibility of polar effects. Bacteria were cultured in THY supplemented with 0.2 µg/ml erythromycin when appropriate and harvested in mid- to late-log phase, and aliquots containing 20% glycerol were snap frozen and stored at −80°C. To determine stock CFU concentrations, 10-fold serial dilutions of aliquots were plated on 5% sheep blood agar plates containing 25 µg/ml neomycin, supplemented when appropriate with 0.2 µg/ml erythromycin, and incubated at 37°C/5% CO2 for 24 h. Aliquots were thawed, washed with sterile PBS and diluted to desired concentrations for inoculation.
Groups of 5–7 mice were infected via oropharyngeal aspiration  with 400 PFU of the H1N1 mouse-adapted influenza A/Puerto Rico/8/34 (PR8) influenza virus in 50 µl PBS or received a sham PBS infection. On day 6 post-influenza or control inoculation, mice were infected via oropharyngeal aspiration with a mixture of 5 × 105 CFU of D39 and 5 × 105 CFU of PspA-, Hyl- or NanA- mutant in 50 µl PBS. Numbers of mutant and total bacteria in each inoculum were determined by selective and non-selective plating, respectively, as described above. At 24 h after the pneumococcal infection, mice were euthanized and lungs were lavaged with 5 ml PBS. Lungs were removed, placed in recovered bronchioalveolar lavage fluid (BALF), homogenized, snap frozen and stored at −80°C. Numbers of mutant and total bacteria present in lung homogenates were again determined by selective and non-selective plating as described above. Competitive index (CI) values were calculated by dividing recovered mutant/total bacteria ratios in lung homogenates by mutant/total bacteria ratios present in inocula as described previously . To quantify PFU, lung homogenates were serially diluted, and plaque assay performed as previously described .
As insertion-duplication mutants are subject to reversion, in vivo stability was assessed by infecting mice with the individual Hyl-, NanA- and PspA- mutants. Lung homogenates recovered after 24 h were serially diluted and plated on both selective and non-selective blood agar plates. Bacteria numbers obtained from selective plating were divided by those recovered from non-selective plating to assess increases in non-selected populations due to revertants. Mean values calculated for the Hyl- (.9904), NanA-(1.018) and PspA- (1.025) mutants were not significantly different than the expected value of 1 when analyzed by one-sample t-test (α = .05) indicating reversion rates were not confounding during in vivo growth through 24 h.
To assess inhibition of primary and secondary pneumococcal growth by PspA immunization, mice under light isoflurane anesthesia received intranasal immunizations twice weekly for three weeks. Groups of 10–14 mice received 1 µg recombinant N-terminal His-tagged PspA protein (rPspA/Rx1), generously provided by Dr. J.C. Paton, along with 4 µg cholera toxin B subunit (CTB, List Biological Laboratories) in 20 µl sterile saline. Control groups received PBS or adjuvant alone. Mice receiving PspA were immunized with adjuvant for two weeks receiving only protein in the third week with PBS and adjuvant controls receiving only saline in the third week. Twenty days following immunization, mice in the three groups were divided into subgroups (5–7 mice) receiving either 400 PFU PR8 influenza or sham infection. Six days after influenza or sham infection, all mice received 1 × 106 CFU D39, WU2 or TIGR4 pneumococci via intratracheal instillation. Twenty-four hours following bacteria inoculation, BALF and lungs were harvested. Viral and bacterial loads in lung were determined as described above. PspA-specific IgG and IgA titers in sera were determined by ELISA.
For ELISA, high binding 96-well plates were coated with 1 µg/ml PspA in 0.05 M carbonate buffer, pH 9.6, for 3 h at 37°C and then at 4°C overnight. Plates were washed 3 times with wash buffer (0.005% Tween 20 in PBS) and non-specific binding sites were blocked with 5% nonfat dried milk in PBS at 37°C for 1 h. After 3 washes, 2-fold serial dilutions of serum samples in wash buffer were added and incubated at 37°C for 1 h. Following 3 washes, alkaline phosphatase-conjugated, anti-mouse, isotype-specific secondary Ab (IgG, Sigma-Aldrich; IgA, Serotec) was added and incubated at 37°C for 2 h. After 5 washes, p-nitrophenyl phosphate in diethanolamine buffer was added and incubated at 37°C for 30 min. Endpoint titers were defined as the highest reciprocal dilution exhibiting absorbance (405 nm) above 0.100 OD units above negative controls .
To evaluate PspA immunization effects on lung pathology, levels of albumin and lactate dehydrogenase (LDH) in recovered BALF were determined using commercially available kits (631-2; Sigma Diagnostics, St. Louis, MO, and CytoTox 96; Promega, Madison, WI).
Analyses of one-sample t-test, two-tailed Mann-Whitney, one-way ANOVA with Bonferroni post-test and two-way ANOVA with Bonferroni post-test were performed using GraphPad Prism version 4.00 (GraphPad Software, San Diego, California).
Prior to administration of mutant/D39 mixtures on day 6 after flu and control inoculations, mice having previously received influenza exhibited symptoms and behavior consistent with influenza infection including clustering, ruffled fur and a wasted appearance, whereas controls appeared symptom-free. Influenza-infected mice contained 1 × 106 – 1 × 107 PFU in their lungs day 7 after viral inoculation, whereas control mice revealed no detectable virus (data not shown). Median total bacteria numbers recovered from mice with prior influenza infection were significantly greater (Fig. 1A, 1B and 1C) compared to mice without prior influenza infection for all three mixed mutant and wildtype inocula with increases ranging from 15,000-fold (Hyl- & D39) to 29,000-fold (PspA- & D39).
Competitive growth is a common method for identifying fitness defects between co-administered bacterial strains [30, 33, 34]. To better resolve interactions of pneumococcal virulence factors with prior influenza infection, we chose this method to normalize lung deposition of mutant and wild type strains in influenza-infected mice given the synergistic nature of this superinfection can amplify small initial variation in bacteria numbers. We calculated CI values as a measure of mutant fitness and so where a CI value of 1 indicates both strains grew equally well, a CI value of 0.1 indicates a 10-fold reduction in mutant growth and/or survival relative to wild type.
Median CI values of Hyl- in mice with and without prior flu infection at 24 h were 1.39 and 1.6, respectively, indicating mutant fitness was not attenuated relative to the wild type (Fig. 1D). The difference between these CI values was not statistically significant (P = .0566), suggesting that prior influenza infection did not impact the role of this virulence factor. Similarly, NanA- exhibited median CI values of 1.37 and 1.43 in mice with and without influenza infection respectively, and thereby failed to exhibit attenuation (Fig. 1E). Again, difference in the CI values of NanA- in mice with and without flu infection was not significant (P = .9817). Contrary to Hyl- and NanA-, the PspA- mutant displayed attenuation in both mice with and without antecedent influenza infection (Fig. 1F). The median CI value of PspA- in mice without prior flu infection was 0.021, representing a 47-fold reduction in fitness relative to the D39 strain. The median CI value for PspA- in influenza-infected mice was 0.00053, representing a greater than 1800-fold reduction in relative growth of the PspA- mutant which was highly significant compared to PspA- growth in mice without prior flu infection.
Mice receiving influenza again displayed visible symptoms of infection on day 6 post-influenza infection and had 1 × 105 – 1 × 106 PFU their lung, whereas controls appeared healthy and did not have detectable virus (data not shown). PspA-specific IgG and IgA were detected in the sera of PspA-immunized mice but not in the sera of PBS or adjuvant-only treated controls (data not shown).
At 24 h following primary D39 challenge, PspA-immunized mice exhibited significant 15-fold and 14-fold reductions in mean bacterial titers when compared to PBS-treated and CTB-treated controls, respectively (Fig. 2A). In influenza-infected groups receiving secondary D39 challenges, PspA-immunized mice exhibited significant 35-fold and 53-fold reductions compared to PBS-treated and CTB-treated controls, respectively (Fig. 2A). No significant differences were detected between PBS-treated and CTB-treated groups in either primary or secondary D39 challenges. Despite significant reduction in secondary D39 infection in the PspA-immunized group, mean bacteria titers for this group remained significantly higher than PBS-treated, CTB-treated and PspA-immunized mice receiving primary D39 infections when analyzed by one-way ANOVA with Bonferroni post-test (Fig. 2A; all P < .001).
As albumin and LDH levels in BALF have been shown to be specific markers of lung pathology , they were measured to assess mitigation of lung damage by PspA immunization after 24 h primary and secondary pneumococcal infections. Albumin concentrations and LDH activity in BALF recovered from mice receiving primary D39 infection were very low, suggesting that D39 infection alone does not cause extensive lung damage after 24 h. Thus, no significant differences were detected in albumin or LDH levels between groups receiving primary D39 infections (Fig. 2B and 2C.). Mice receiving D39 infection following influenza all exhibited significantly increased levels of albumin compared to their respective immunization treatment group lacking prior influenza infection (Fig. 2B; all P < .001). Similar results were observed for LDH levels (Fig. 2C; PBS and CTB, P < .001, PspA, P < .01). Whereas both lung damage markers were pronounced in secondary D39 infections, PspA immunization significantly reduced levels of albumin (Fig. 2B) and LDH (Fig. 2C).
To determine whether PspA immunization reduces infections caused by strains of other serotypes, the immunization and challenge experiments were repeated for strains WU2 (serotype 3) and TIGR4 (serotype 4). PspA immunization significantly reduced recovered bacteria titers 3100-fold and 2800-fold from primary WU2 infection when compared to PBS-treated and CTB-treated controls, respectively (Fig. 3A). PspA-immunized mice also exhibited significant 62-fold and 91-fold reductions in secondary WU2 titers relative to PBS-treated and CTB-treated controls, respectively (Fig. 3A). No significant differences were detected between PBS-treated and CTB-treated control groups in either primary or secondary WU2 challenges. PspA-immunized mice receiving secondary WU2 challenge following influenza again exhibited significantly greater WU2 titers than PBS-treated, CTB-treated and PspA-immunized groups receiving WU2 infection alone when analyzed by one-way ANOVA with Bonferroni post-test (Fig. 3A; all P < .001). Primary WU2 infections did not yield substantial albumin or LDH levels in recovered BALF while secondary WU2 infections yielded significantly greater levels of both lung damage markers (Fig. 3B; all P < .001, and 3C; all P < .001). PspA immunization resulted in a significant reduction in albumin concentrations in mice receiving secondary WU2 infection relative to both PBS-treated and CTB-treated controls (Fig. 3B) with parallel results observed for LDH concentrations (Fig. 3C).
Primary infection with the TIGR4 pneumococcal strain was also found to be significantly reduced in PspA immunized mice 135-fold and 424-fold compared to PBS-treated and CTB-treated controls, respectively (Fig. 4A). PspA immunization significantly reduced lung burdens 12- and 6-fold in mice receiving secondary TIGR4 infections following influenza when compared to PBS-treated and CTB-treated controls (Fig. 4A). No significant differences were detected between PBS-treated and CTB- treated control groups in either primary or secondary TIGR4 challenges. Again, one-way ANOVA with Bonferroni post-test revealed that PspA-immunized mice with prior influenza receiving secondary TIGR4 infection exhibited significantly greater bacterial lung burdens than PBS-treated (P <.001), CTB-treated (P < .01) and PspA-immunized (P <.001) mice receiving primary TIGR4 challenges (Fig. 4A). As with D39 and WU2, primary infection with TIGR4 did not induce substantial albumin or LDH levels in recovered BALF, however, significant increases were observed when comparing influenza-infected mice receiving TIGR4 to mice receiving primary TIGR4 infection (Fig. 4B; all P < .001, and Fig. 4C; PBS and CTB, P <.001, PspA, P < .01). Albumin levels of PspA-immunized mice were significantly reduced when compared to PBS-treated and CTB-treated controls receiving secondary TIGR4 infection (Fig. 4B) with similar results observed for LDH levels (Fig. 4C).
Influenza infection is known to denude respiratory epithelium exposing pneumococcal ligands on the basement membrane , while bacterial hyaluronidases are believed to facilitate invasion through targeting of hyaluronic acid, a constituent of the extracellular matrix [9, 36]. We hypothesized that herein may lay an opportunity for enhanced application of pneumococcal Hyl. A strong correlation exists between clinical isolates and hyaluronidase production, specifically in pneumococcal meningitis . A serotype 19F Hyl- mutant is attenuated in a pneumonia model  and whereas a serotype 6 Hyl- mutant is attenuated in intraperitoneal infection , a D39 mutant was not . Our results show that growth of the Hyl- mutant of D39 in the lung is not attenuated in mice with or without prior influenza infection and while these results do not indicate a secondary infection contribution for Hyl, they may support suggestions that the hyaluronidase virulence role is serotype specific [38, 39].
NanA- relative fitness was not compromised in either primary or secondary pneumococcal infection. Lack of attenuation in the primary challenge was a surprise to us given NanA cleaves terminal sialic acid from host glycoconjugates decorating respiratory epithelium where this action enhances colonization [27, 40]. We suspected contribution of pneumococcal NanA to colonization would be superseded by the influenza neuraminidase in secondary bacterial challenge but that this virulence factor might then reveal some novel application, however, secondary competitive growth of the NanA- mutant failed to support this hypothesis. NanA- mutants of D39 reported little to no attenuation in intraperitoneal challenges [6, 15] in which colonization is not critical for infection. In intranasal challenges, NanA of serotype 2 strains is important for virulence in two reports [27, 28] but not in another . It has been suggested that among methodological differences, susceptibility of mouse strains employed might explain dissimilar results . This may be the case in our use of C57Bl/6 mice rather than the BALB/c employed by others. Furthermore, our results might also reflect our competitive growth model where the NanA- mutant and D39 strains were co-administered in a mixed inoculum. NanA of the wild-type D39 strain might modify the surface of the respiratory tract for both D39 and NanA- bacteria. If this is the case, the competitive growth method may serve to better identify those cis virulence factors and better potential vaccine targets, such as PspA, required of each individual bacterium.
PspA is well established as both a crucial virulence factor [12, 14–16] and a broadly protective antigen in a variety of immunization and challenge models [17–22]. Given the lower respiratory tract inflammatory response accompanying influenza infection, we hypothesized this virulence factor’s function in inhibiting complement mediated clearance would become critical in a secondary pneumococcal infection. Fitness of the PspA- mutant was reduced 47-fold relative to D39 in the absence of influenza infection in agreement with observations that PspA is required for pulmonary S. pneumoniae infection . Additionally, in support of a significant contribution of PspA to secondary pneumococcal infection, we observed an 1800-fold reduction in relative PspA- growth in mice with prior influenza infection.
PspA immunization experiments further supported a significant contribution of PspA to secondary infection. Intranasal PspA immunization significantly reduced numbers of D39, WU2 and TIGR4 in the lungs of mice both with and without prior influenza infection when compared to controls. Consistent with these results and a unique role for PspA in secondary infections, BALF levels of albumin and LDH were significantly reduced in PspA-immunized groups but not in PBS-treated or CTB-treated controls receiving D39, WU2 or TIGR4 infections following prior influenza infection. Despite substantial sequence variability, both cross-reactivity and protection have been observed between immunized sera and heterologous PspA proteins [17, 41]. The rPspA/Rx1 (family 1, clade 2 and type 25) PspA antigen used in this present study has been shown to induce immune mouse serum cross-reactive with PspA proteins present on D39, WU2 and EF3296 pneumococcal strains  where the N-terminal region of the EF3296 PspA protein is identical to that of TIGR4 . Additionally, it has been shown that immunization with the D39 type 25 PspA can provide protection against lethal challenges with the D39 and WU2 strains and perhaps some, albeit not statistically significant, protection against EF3296 . While this earlier work may support our observation of rPspA/Rx1 immunization reducing 24 h lung titers in primary and secondary infections with the D39, WU2 and TIGR4 strains, another study has specifically assessed this antigen with respect to protection against WU2 and TIGR4 . In this prior study, the JAS218 PspA/Rx1 fragment elicited protection against lethal challenge with the family 1 bearing WU2 strain but not the family 2 bearing TIGR4. The observations we present here may again reflect the different methodologies and endpoints employed in that, whereas our model is examining the ability of a PspA antigen to reduce pneumococcal infection in the respiratory compartment at a defined endpoint early in primary and secondary challenges, the aforementioned study is assessing this PspA antigen in its capacity to protect against a complex systemic disease outcome. And so while informative within the context of our model’s narrow aim, our observations may not be easily compared to studies examining protection against pneumococcal disease resulting from established infections given the different temporal, spatial, virulence and pathogenic scales under consideration. Current vaccines and vaccine candidates are assessed for their ability to protect against disease, however, our focus on a 24 h endpoint in this study reflects our belief that a vaccine developed to effectively prevent pneumococcal infection would ultimately serve to limit pneumococcal disease but may additionally serve to counteract the influenza-induced susceptibility to secondary pneumococcal colonization and infection.
PspA immunization was effective in limiting early primary and secondary infection compared to controls, however, immunization with this antigen was not sufficient to overcome influenza-induced susceptibility when comparing primary and secondary D39, WU2 and TIGR4 lung titers between PspA-immunized groups. Despite these findings, that pneumococcal growth in the influenza-infected lung can be significantly reduced by a targeted immune response regardless of virally-induced suppression of host defenses is encouraging. Furthermore, given previous observations [18, 20, 44], it is probable that combinations of different PspA families along with other pneumococcal virulence factors into a protein vaccine could, while protecting against a broad range of invasive serotypes, significantly increase protection against secondary pneumococcal infections following influenza infection.
Analyses of the 1918 Spanish influenza pandemic indicated the high mortality associated with this pandemic was likely the result of increased susceptibility to secondary bacterial pathogens [45, 46]. We strongly feel that identification of pneumococcal vaccine candidates which offer protection against both pneumococcal infection alone and the significantly greater threat of pneumococcal infection following influenza infection may serve to reduce expected increases in morbidity and mortality attributable to secondary pneumococcal infections during what many believe to be inevitable future influenza pandemic events.
We sincerely thank Dr. James C. Paton for providing the insertion-duplication pneumoniae mutants and recombinant PspA protein. We also wish to thank Drs. Susan Hollingshead, Edwin Swiatlo and Elaine Tuomanen for sharing pneumococcal strains used in this research. We additionally thank all the members of the Harmsen lab who have assisted in this work and Dr. Jovanka Voyich-Kane for assistance in manuscript development.
This work was supported in part by INBRE-BRIN grant RR16455 and NIH-COBRE grant RR020185.
Conflict of interest statement:
Quinton O. King*, no conflict.
Benfang Lei, no conflict.
Allen G. Harmsen, no conflict.
Portions of this work were presented at the Northern Rocky Mountain Conference on Infectious Disease and Environmental Health, Big Sky, MT on Sept. 27-29, 2007.