Department of Molecular Biosciences, Adelaide University, Adelaide, South Australia 5005, Australia1
Author for correspondence: James C. Paton. Tel: +61 8 83035929. Fax: +61 8 83033262. e-mail: james.paton{at}adelaide.edu.au
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ABSTRACT |
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Keywords: pneumococcus, pneumolysin, surface proteins, mRNA, quantitative RT-PCR
Abbreviations: PS, polysaccharide
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INTRODUCTION |
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The polysaccharide (PS) capsule is strongly anti-phagocytic and is a sine qua non of pneumococcal virulence (Austrian, 1981 ). There are 90 recognized PS serotypes; synthesis of the serotype-specific capsular PS is encoded by distinct clusters of up to 20 tightly linked genes transcribed as single operons (Paton & Morona, 2000
). Antibodies to the PS are highly protective, and the global impact of pneumococcal disease has led to the development of polyvalent PS and PSprotein conjugate vaccine formulations. Although appreciable levels of success have been achieved with these vaccination strategies, the problems of serotype specificity of protection, geographical and temporal variations in serotype distribution, cost of vaccine formulations, and the possibility of nasopharyngeal replacement of vaccine serotypes by non-vaccine serotypes in vaccinated individuals still exist (Obaro, 2000
). For this reason, we and others have been investigating the capacity of pneumococcal virulence proteins, administered either singly or in combination, to elicit non-serotype-dependent protection against pneumococcal disease (Alexander et al., 1994
; Briles et al., 2000a
, b
; Ogunniyi et al., 2000
).
The pneumococcal virulence proteins which have received the greatest attention to date are the thiol-activated toxin pneumolysin (Ply) (for reviews see Boulnois et al., 1991 ; Boulnois, 1992
; Paton, 1996
; Paton et al., 1993
), two choline-binding surface proteins called pneumococcal surface protein A (PspA) (Yother & Briles, 1992
) and choline-binding protein A (CbpA) (also referred to as PspC or SpsA) (Brooks-Walter et al., 1999
; Hammerschmidt et al., 1997
; Rosenow et al., 1997
), and a metal-binding lipoprotein called pneumococcal surface antigen A (PsaA) (Berry & Paton, 1996
; Dintilhac et al., 1997
; Paton, 1998
). These proteins possess a range of biological activities, indicating that they act at different stages of the pathogenic process. Ply, for instance, has both direct cytotoxic and complement activation properties, mediated by different domains within the toxin (Boulnois et al., 1991
). Apart from directly damaging respiratory epithelium (Feldman et al., 1990
), the cytotoxic property accounts for inhibition of specific and non-specific immune responses (Ferrante et al., 1984
; Paton & Ferrante, 1983
), as well as stimulation of the release of inflammatory cytokines from host cells (Houldsworth et al., 1994
). Direct activation of the classical complement pathway is the result of binding of Ply to the Fc region of IgG, which also contributes to inflammation and depletes serum opsonic activity (Mitchell et al., 1991
; Paton et al., 1984
). On the other hand, PspA interferes with complement activation and slows the clearance of pneumococci from the blood of infected mice (McDaniel et al., 1987
; Tu et al., 1999
). Hammerschmidt et al. (1999)
have shown that PspA binds lactoferrin, and so it may also function by scavenging iron in the nasopharynx. CbpA is structurally related to PspA, and mediates adherence to cytokine-activated lung cells, as well as playing a major role in colonization of the nasopharynx in an infant rat model (Rosenow et al., 1997
). It has also been shown to specifically bind the secretory component of human secretory immunoglubulin A (Hammerschmidt et al., 2000
). PsaA forms part of an ABC-type manganese permease complex (Dintilhac et al., 1997
) and mutations in psaA have pleiotropic effects on various pneumococcal functions, including adherence, autolysis and virulence (Berry & Paton, 1996
; Claverys et al., 1999
; Novak et al., 1998
).
Mutagenesis studies have shown that Ply, PspA, CbpA and PsaA contribute to virulence in a variety of animal models of pneumococcal disease (Berry et al., 1989 , 1999
; Berry & Paton, 1996
; McDaniel et al., 1987
; Rosenow et al., 1997
). Moreover, mutagenesis of certain combinations of virulence factor genes results in additive attenuation, implying that their products function independently in virulence (Berry & Paton, 2000
). However, it is not currently known whether any of these genes are coordinately regulated, or whether they are specifically up-regulated in vivo. Moreover, evidence for in vivo expression is largely confined to indirect serological studies (Lankinen et al., 1999
; Orihuela et al., 2000
; Rapola et al., 2000
; Virolainen et al., 2000
). In this study, we provide direct molecular evidence for in vivo expression of important pneumococcal virulence factors. The levels of mRNA transcript for each gene have been measured using relative quantitative RT-PCR analysis of total bacterial RNA isolated from the blood of mice at various times after intraperitoneal infection. This has permitted what is to our knowledge the first comparison of the relative kinetics of in vivo expression of proven pneumococcal virulence factors.
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METHODS |
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Infection of mice and harvesting of bacteria for RNA isolation.
Two groups of twenty-four 68-week-old male BALB/c mice were infected intraperitoneally with approximately 1x103 c.f.u. of D39. Mice were exsanguinated after 12 h or 24 h by cardiac puncture under deep anaesthesia, and blood obtained from each group of mice was pooled into sterile heparinized tubes on ice. The bacteria were harvested from the blood by initial centrifugation at 825 g at 4 °C for 5 min to pellet erythrocytes and leucocytes. The resulting supernatant, consisting of a mixture of plasma, bacteria and thrombocytes, was then centrifuged at 15500 g at 4 °C for 5 min to pellet the bacterial cells. The supernatant was discarded and RNA was extracted from the bacterial pellet. The same procedure was used to harvest bacteria grown in fresh whole blood obtained from naïve mice.
RNA isolation.
Total RNA was extracted from in vitro- and in vivo-harvested bacteria by a modification of the method of Mortier-Barrière et al. (1998) . The bacterial pellet was resuspended completely in 300 µl prewarmed (65 °C) acid-phenol and incubated for 5 min at 65 °C. To this, 300 µl prewarmed NAES buffer (50 mM sodium acetate pH 5·1, 10 mM EDTA, 1% SDS) was added, and the mixture was incubated for another 5 min at 65 °C, with intermittent mixing. The mixture was cooled on ice for 1 min and the phases were separated by centrifugation at 15500 g for 1 min. The aqueous phase was re-extracted twice with acid-phenol followed by two further extractions with chloroform. Sodium acetate was added to the resulting supernatant to a final concentration of 300 mM and RNA was then precipitated with 2 vols ethanol at -20 °C overnight. The resulting RNA pellet was washed briefly in 70% ethanol and then resuspended in 50 µl nuclease-free water. Recombinant RNasin ribonuclease inhibitor (Promega N251A) was added to a final concentration of 1 U µl-1 and the RNA was then treated with 0·5 U µl-1 RQ1 RNase-free DNase (Promega M610A) at 37 °C for 30 min. The RNA preparation was then treated with RQ1 DNase stop buffer (Promega M198A) to inactivate the DNase. The purity of the RNA preparation was confirmed by subjecting it to one-step RT-PCR with or without reverse transcriptase, using gene-specific primers (see Table 1
). An aliquot of the total RNA sample was also electrophoresed on a 1% TAE-agarose gel to check for integrity, and then stored at -70 °C until required.
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The linear range of amplification for each RNA preparation was determined separately under these conditions by removing replicate tubes from the thermal cycler at cycle 10 and at every subsequent fourth cycle. The reaction products were resolved by electrophoresis on a 2% TAE-agarose gel (Agarose LE, Roche Molecular Biochemicals) and stained with Sybr Gold (Molecular Probes). They were then scanned at a resolution of 50 µm at medium sample intensity on a Bio-Rad FX Molecular Imager connected to an external laser and quantified using Quantity One software. The cycle number was plotted against the log of signal intensity to determine the linear range of amplification for each mRNA species. Amplicon intensity in a given RNA extract was determined at a cycle number within the linear range for each species. Between RNA extracts, intensities of individual mRNA species were corrected with reference to that obtained for the internal 16S rRNA control.
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RESULTS |
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Quantitative comparison of virulence factor mRNA levels
Relative quantitative RT-PCR was employed to compare the abundance of cps2A (the first gene in the type 2 PS biosynthesis locus), ply, pspA, cbpA and psaA mRNAs in total RNA extracts from pneumococci grown in serum broth, or isolated from the blood of mice 12 or 24 h post-infection (see Methods). This involved quantitation of RT-PCR products after 10, 14, 18, 22, 26, and 30 cycles, and for each assay, determining the period during which the rate of RT-PCR product accumulation was directly related to RNA concentration. By way of illustration, agarose gel analyses for the virulence gene and 16S rRNA RT-PCR products from serum broth, and 12 h and 24 h post-infection RNA preparations are shown in Fig. 1. mRNA levels were then expressed relative to that of 16S rRNA; 16S rRNA was selected as an internal control because rRNAs are known to be maintained at a constant level. The various primer pairs for the virulence genes and rRNA were designed to amplify cDNA fragments of comparable size to ensure similar amplification kinetics. This precludes performing RT-PCR for virulence gene transcripts and rRNA in the same tube. However, reaction tubes were prepared from aliquots of the same master mix to which the different primer pairs were added last. They were then amplified in the same PCR run and electrophoresed on the same gel.
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DISCUSSION |
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In the present study, we have measured and compared the levels of mRNA for four well-characterized virulence protein genes (ply, pspA, psaA and cbpA), as well as the first gene in the type 2 PS biosynthesis operon (cps2A) in pneumococci grown in broth culture or isolated from the blood of mice at different times after intraperitoneal infection. This was achieved using relative quantitative RT-PCR, with 16S rRNA as an internal standard. Twelve hours after intraperitoneal infection, the mRNA species of ply, pspA and psaA were up-regulated, unequivocally demonstrating an altered pattern of gene expression in vivo vs in vitro. However, the pattern was not constant for the virulence genes during the course of infection. At 24 h, expression of ply and, significantly, pspA, was again increased, but expression of the other virulence factors was not greatly dissimilar to 12 h levels. These findings clearly show that these virulence genes are not coordinately expressed, under the control of a single global regulatory element.
Interpretation of the significance of differences in virulence-associated gene expression between in vitro and in vivo conditions is complicated, particularly since marked variation in gene expression between cells grown in vitro in THY versus serum broth were observed in the present study. Expression levels in the latter medium were used as a baseline for comparison with expression in blood in vivo, which seems appropriate given that virulence gene expression in pneumococci grown in serum broth was similar to that observed for bacteria grown in whole mouse blood in vitro. Thus, up-regulation of virulence gene expression in pneumococci in the blood of infected mice appears to be largely attributable to host factors as opposed to the nutritional parameters of blood. While the possibility that initial intraperitoneal growth may have triggered some of the changes in expression observed in mouse blood in vivo, the levels of ply and pspA mRNA are markedly greater in blood at 24 h compared with blood at 12 h post-infection.
Given what is known of the functions of the various virulence factors, it is perhaps not surprising that their genes are upregulated in vivo, particularly during the early stages of infection. None of these functions would be expected to confer any advantage on the pneumococcus during in vitro culture. Ply is known to be important for preventing the establishment of early non-specific host immune responses capable of limiting net exponential growth of pneumococci in the blood (Benton et al., 1995 ). Thus, high initial levels of expression of ply may be critical. PsaA is a metal-binding lipoprotein with specificity for Mn2+ and possibly also Zn2+ (Dintilhac et al., 1997
; Lawrence et al., 1998
) and so sustained expression of psaA in vivo is consistent with an ongoing need to scavenge these metal ions from the blood.
The pattern of pspA expression was an interesting finding of this study. It was the least abundant mRNA species in vitro, but was up-regulated threefold at 12 h after intraperitoneal infection. One of the known functions of PspA is inhibition of complement-mediated clearance of pneumococci from the blood, by interference with deposition of C3b on the bacterial surface (Tu et al., 1999 ). This property would be expected to be advantageous throughout the infectious process. Indeed, levels of PspA are reported to be higher in opaque variants of pneumococci, a phenotype associated with systemic virulence (Kim & Weiser, 1998
). Remarkably, pspA mRNA levels increased approximately 36-fold at 24 h post-infection relative to levels detected in serum broth. Conversely, cbpA mRNA levels were similar to in vitro levels at both 12 h and 24 h post-infection. CbpAs primary function is believed to be that of an adhesin with an important role in nasopharyngeal colonization, and effects of mutagenesis of cbpA on systemic virulence were reported to be minimal (Gosink et al., 2000
; Ring et al., 1998
; Rosenow et al., 1997
). CbpA has recently been shown to interact with the human polymeric immunoglobulin receptor, thereby facilitating invasion of the mucosa (Zhang et al., 2000
). However, we have shown that a S. pneumoniae cbpA/ply double mutant has massively attenuated systemic virulence compared with either cbpA or ply single mutants (Berry & Paton, 2000
). These latter findings are consistent with a role for CbpA in the post-adherence/invasion stages of the pathogenic process. Moreover, CbpA has also recently been shown to be capable of binding to C3 (Smith & Hostetter, 2000
), and Brooks-Walter et al. (1999)
have suggested that CbpA and PspA could perform analogous functions in vivo. This is supported by their observation that mutagenesis of pspA has a much lesser impact on systemic virulence in S. pneumoniae strains which contain cbpA than in those which lack it, implying a degree of functional complementation. Thus, the poor expression of cbpA observed in the present study could be offset by the marked increase in expression of pspA.
The precise manner in which the PS contributes to virulence is not fully understood, although it is known to have strong anti-phagocytic properties in non-immune hosts (Paton & Morona, 2000 ). The capsular PS of the majority of serotypes is highly charged at physiological pH and this may directly interfere with interactions with phagocytes. Pneumococcal cell wall teichoic acid is capable of activating the alternative complement pathway. In addition, antibodies to this and other cell surface constituents (e.g. surface proteins), which are found in most adult human sera, may result in activation of the classical complement pathway, as does interaction of the teichoic acid with C-reactive protein. However, the PS forms an inert shield, which appears to prevent interaction of either the Fc region of IgG or iC3b fixed to deeper cell surface structures from interacting with receptors on phagocytic cells (Musher, 1992
). Thus, in systemic challenge models such as that used in the present study, maximal expression of PS might be expected to be beneficial to the pneumococcus at all stages of the infection. However, studies in nasopharyngeal colonization models suggest that high encapsulation might interfere with the interaction between other surface molecules (e.g. PspA and CbpA) and the host epithelium (Kim & Weiser, 1998
; Talbot et al., 1996
).
We believe this to be the first study in which pneumococcal virulence gene mRNAs have been directly quantified from bacteria present in the blood of infected mice. The only previous study addressing in vivo pneumococcal virulence gene expression examined mRNA levels in type 3 pneumococci grown in sealed dialysis bags implanted in the murine peritoneal cavity (Orihuela et al., 2000 ). However, this is a suboptimal in vivo surrogate, because it prevents interactions between pathogen and host cells, or host molecules larger than the dialysis bag pore size (25 kDa cutoff). Using Northern blotting, Orihuela et al. (2000)
detected a 2·8-fold stimulation in ply expression at 8 h, compared with bacteria grown in vitro, but intraperitoneal up-regulation of pspA expression was not detected. Expression of a gene from the type 3 PS biosynthesis locus (cap3A) was also increased 2·2-fold, although we observed slightly higher levels in the present study for cps2A. We have now demonstrated that cps2A and three of the key virulence protein genes, ply, pspA and psaA, are up-regulated in vivo. Moreover, there are marked differences in the patterns of expression of the various genes at different times post-infection, although little is known of the mechanism whereby this occurs. Jakubovics et al. (2000)
have recently described a Streptococcus gordonii gene scaR, which encodes a metallorepressor-like protein that regulates the expression of a homologue of PsaA (the Sca Mn2+ permease). A homologue of this gene, designated psaR, is known to be present in the genome of S. pneumoniae, but to date a role in regulation of the psa operon has not been demonstrated. Establishing that certain key virulence genes respond to host signals is a major step forward; identifying these as markers will assist in studies of the molecular genetics of virulence gene regulation in this important human pathogen. However, the finding that the relative expression of these virulence genes differed between organisms grown in vitro in serum broth, and those grown in vivo, or in different in vitro media (whole blood or THY broth) suggests that the expression of these genes is highly complex and multifactorial.
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ACKNOWLEDGEMENTS |
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Received 13 February 2002;
revised 25 March 2002;
accepted 27 March 2002.