We present evidence that the ambient oxygen concentration may be an important factor in the ability of S. pneumoniae
, an acrotolerant streptococcus, to regulate the characteristics of its cell surface. Our approach took advantage of observations that the pneumococcus varies between two phenotypes previously shown to differ in their amounts of CPS (13
). Findings in the current study show that oxygen levels affect the O and T phase variants differently. There is greatly enhanced production of CPS in O variants in conditions of reduced oxygen, whereas synthesis of CPS in T variants remains comparatively low under both aerobic and anaerobic conditions. The effect of oxygen on CPS expression was shown by two different methods, quellung, which allows visualization of the capsule size, and a capture ELISA, which enables amounts of CPS to be quantified. Both of these methods rely on the use of type-specific antiserum to detect CPS. Results from experiments using either technique show that microaerophilic growth conditions (O2
levels at 17% or lower) are sufficient to induce a significant increase in CPS expression when compared to atmospheric growth conditions. A maximal effect, however, is observed in strict anaerobiasis.
Although it is not clear when and if this species would be exposed to strict anaerobic conditions, ambient oxygen levels would be diminished whenever the organism is not in its commensal state on the airway surface. In particular, the availability of oxygen is decreased in the common manifestations of pneumococcal disease such as pneumonia, empyema, and otitis media where the exudative inflammatory response occludes air spaces and limits gas exchange. Oxygen levels in the uninflamed middle ear space, for example, resemble that of venous blood, are less than a third that of the airway, and may be further reduced by the presence of effusion (8
). If an effect similar to that described in this study occurs in vivo, the reduced-availability of oxygen would select for an upregulation in CPS expression. Since even-relatively small difference in the amounts of CPS can be critical to the expression of virulence, the effect of oxygen might provide a survival advantage during infection in situations whenever O variants are represented (13
The analysis of paired isolates from human carriage and bacteremia is consistent with previous animal studies and supportive of the hypothesis that environments with lower oxygen content promote selection for the more virulent O form. It was found that in bacteremic infection of humans, which is generally a complication of pneumococcal pneumonia, most isolates from the bloodstream (or cerebrospinal fluid) are as opaque or more compared to the isolate from the same patient cultured from the nasopharynx (22
). This difference in colony morphology is indicative of increased amounts of CPS in these blood isolates. In contrast, the T variant found in 89% of the colonizing isolates in the natural host produces relatively little CPS regardless of the ambient oxygen concentration. T variants may predominate in this setting since smaller amounts of CPS may be sufficient to inhibit phagocytosis on the airway surface where there is relatively little complement and antibody, while still allowing for efficient adherence to host cells. Another consideration is that because it was necessary to carry out this study in a largely HIV-positive population due to their high rates of pneumococcal infection, these findings may or may not be relevant to immunocompetent hosts. Results from this limited study of human isolates, however, add support to the hypothesis that phenotypic variation confers on the pneumococcus the flexibility to alter its surface characteristics for the differing requirements of colonization and infection.
Since the expression of a large capsule may be detrimental to the needs of carriage but necessary to evade opsonsophagocytic clearance during infection, the amounts of CPS may need to be precisely regulated. At least two factors are described as contributing to the differences in CPS levels: the variation in opacity phenotype noted in the majority of clinical isolates and the availability of oxygen in the environment. For a pathogen such as the pneumococcus residing on the surface of the airway, the concentration of oxygen in its environment would seem to be an effective means of sensing changes in its host environment that might require adaptation. There has been little specific evidence to suggest that environmental signals, which are important, for instance, in the induction of competence, may also contribute to the pathogenesis of pneumococcal disease (19
). A recent study from our laboratory demonstrated that the expression of pyruvate oxidase (SpxB), the major factor in production of unusually high concentrations of hydrogen peroxide by the pneumococcus, is differentially regulated in O and T variants by ambient oxygen levels (20
). The generation of H2
by the pneumococcus under aerobic conditions has cytotoxic effects on host cells as well as on other bacterial species that inhabit and potentially compete for the same niche in the upper respiratory tract (5
). Oxygen has also been implicated as a factor in the regulation of natural transformation (2
). In another streptococcal species (Streptococcus pyogenes
), it is carbon dioxide rather than oxygen tension that mediates changes in the expression of virulence factors. Our findings for the pneumococcus do not suggest that carbon dioxide concentration affects the regulation of its major virulence factor, CPS. In group A streptococci, the amounts of the hyaluronic capsule are also regulated but not through signaling involving carbon dioxide concentration (18
). Instead, the amounts of the hyaluronic capsule are regulated through an unknown environmental signal transmitted through differential phosphorylation of a two-component signal transduction system (15
). In the case of the pneumococcus it is oxygen that triggers these changes and there is no evidence for a substantial effect when mutants in 11 of the 12 known two-component signal transduction systems were tested (32
; data not shown). This leaves it unclear how signals related to oxygen content in the environment are transmitted so as to affect gene expression in CPS synthesis.
One implication of this study is further evidence that the amounts of CPS are not uniform for a given strain. We have previously documented that genetic transformation tends to cause a selection bias for T variants, since the lower amount of CPS in this form appears to allow for more efficient uptake of DNA (39
). Virulence studies that depend on the analysis of mutants generated by transformation of encapsulated strains would, therefore, tend to demonstrate decreased virulence regardless of the specific mutation. In addition to the contribution of the opacity phenotype, differences in growth conditions and, in particular, oxygenation have the potential to bias studies that depend on encapsulation. Vaccine efficacy studies, for example, will increasingly depend on in vitro correlates of protection, since there is now an effective product for use in childhood that will restrict further clinical trials. The opsonophagocyctic titer of anti-CPS antibody, one of few in vitro assays that is predictive of vaccine efficacy, has been shown to be highly sensitive to variations in the amount of CPS (14
). These assays, as well as virulence studies, could be affected by differences in opacity phenotype and oxygenation during growth of test organisms.
Although the genes involved in the expression of the pneumococcal capsule have been described, the mechanism controlling the levels of CPS expression remain largely undefined. In this report, we focused on cpsD
, a gene common to the capsulation loci of all serotypes thus far examined which appears to be a negative regulator of CPS synthesis in S. pneumoniae
and other encapsulated bacteria. Homologues of CpsD are present in many other pathogens that express surface polysaccharides, such as Streptococcus agalactiae, Staphylococcus aureus, Klebsiella pneumoniae
, and Acinetobacter johnsonii
and A. lwoffii
). This family comprises one of the few known examples of bacterial proteins that are phosphorylated on tyrosine residues (11
). One of the common features of this group of bacterial tyrosine kinases is a C terminus with a tyrosine-rich repeat which has been proposed as the target of autophosphoryltation activity. For the pneumococcus, changing the tyrosine residues in the C-terminal region (YGX)4
in Cps 19fD to phenylalanine residues resulted in a loss of immunodetectable phosphotyrosine and a mutant with a mucoid colony phenotype under standard aerobic growth conditions. This led to the proposal that tyrosine phoshorylation of CpsD involving this motif increases the negative regulatory activity of the protein on CPS biosynthesis. A similar C terminus containing multiple tyrosine residues (215-CGSY
GKNKK-229) was found in CpsD of the type 6A strain analyzed in this study. An unexpected observation was that anaerobic conditions which resulted in the highest level of CPS production in this and several other isolates also had the most immunodetectable phosphotyrosine on CpsD. This suggests that although the levels of tyrosine phosphorylation correlate with differences in the amounts of CPS, its effect may not be associated with increased negative regulatory activity and lower expression of CPS. Alternatively, if tyrosine phosphorylation acts to enhance the negative regulation of CpsD as proposed, there must be other unknown upstream or downstream factors affecting the degree of phosphorylation and mediating the oxygen effect. In E. coli
the autophosphorylating enzyme Wzc is linked to a phosphotyrosine-protein phosphatase Wzb, which has no significant homologue in the pneumococcal genome. Rather, it has been suggested that CpsB functions in dephosphorylating CpsD and could be an additional factor affecting tyrosine phosphorylation (17
We also observed an absence of phosphotyrosine on CpsD in the T variants of the several strains analyzed. Although it was not technically feasible to show that this was due to decreased transcription of the cps locus in this phenotype, the lack of immunodetectable phosphotyrosine correlates with decreased expression of CpsD. These data support the conclusion that there is a downregulation of cps expression in the T variant accounting for the low levels of CPS found in T pneumococci. Thus, there appear to be at least two separate levels of regulation of CPS synthesis: one associated with the phosphorylation of two or more tyrosine residues in CpsD and the other affecting the expression of protein(s) in this locus, including CpsD. The analysis of two sets of paired clinical isolates showed that pneumococci recovered from the nasal swabs did not have the phosphotyrosine epitope regardless of the growth conditions. These data could be explained by selection for a phenotype with diminished expression of the capsulation locus when resident in the nasopharynx, a result that, together with observations on colony morphology and capsule synthesis, suggests that in contrast to invasive infection pneumococci may express relatively little CPS during human carriage.