Requirement for capsule in colonization by Streptococcus pneumoniae.

Nasopharyngeal colonization is a necessary first step in the pathogenesis of Streptococcus pneumoniae. Using isolates containing defined mutations in the S. pneumoniae capsule locus, we found that expression of the capsular polysaccharide is essential for colonization by the type 2 strain D39 and the type 3 strains A66 and WU2. Nonencapsulated derivatives of each of these strains were unable to colonize BALB/cByJ mice. Similarly, type 3 mutants that produced < 6% of the parental amounts of capsule could not colonize. Capsule production equivalent to that of the parent strain was not required for efficient colonization, however, as type 3 mutants producing approximately 20% of the parental amounts of capsule colonized as effectively as the parent. This 80% reduction in capsule level had only a minimal effect on intraperitoneal virulence but caused a significant reduction in virulence via the intravenous route. In the X-linked immunodeficient CBA/N mouse, the type 3 mutant producing ~20% of the parental amount of capsule (AM188) was diminished in its ability to cause invasive disease and death following intranasal inoculation. Following intravenous or intraperitoneal challenge, however, only extended survival times were observed. Our results demonstrate an additional role for capsule in the pathogenesis of S. pneumoniae and show that isolates producing reduced levels of capsule can remain highly virulent.

Essential role for cellular phosphoglucomutase in virulence of type 3 Streptococcus pneumoniae.

Synthesis of the Streptococcus pneumoniae type 3 capsule requires the pathway glucose-6-phosphate (Glc-6-P) --> Glc-1-P --> UDP-Glc --> UDP-glucuronic acid (UDP-GlcUA) --> (GlcUA-Glc)(n). The UDP-Glc dehydrogenase and synthase necessary for the latter two steps, and essential for capsule production, are encoded by genes (cps3D and cps3S, respectively) located in the type 3 capsule locus. The phosphoglucomutase (PGM) and Glc-1-P uridylyltransferase activities necessary for the first two steps are derived largely through the actions of cellular enzymes. Homologues of these enzymes, encoded by cps3M and cps3U in the type 3 locus, are not required for capsule production. Here, we show that cps3M and cps3U also are not required for mouse virulence. In contrast, nonencapsulated isolates containing defined mutations in cps3D and cps3S were avirulent, as were reduced-capsule isolates containing mutations in pgm. Insertion mutants that lacked PGM activity were avirulent in both immunologically normal (BALB/cByJ) and immunodeficient (CBA/N) mice. In contrast, a mutant (JY1060) with reduced PGM activity was avirulent in the former but had only modestly reduced virulence in the latter. The high virulence in CBA/N mice was not due to the lack of antibodies to phosphocholine but reflected a growth environment distinct from that found in BALB/cByJ mice. The reduced PGM activity of JY1060 resulted in enhanced binding of complement and antibodies to surface antigens. However, decomplementation of BALB/cByJ mice did not enhance the virulence of this mutant. Suppressor mutations, only some of which resulted in increased capsule production, increased the virulence of JY1060 in BALB/cByJ mice. The results suggest that PGM plays a critical role in pneumococcal virulence by affecting multiple cellular pathways.

Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots.

Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5' TC sites in the eight base palindrome 5' CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in the primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis.

The influence of single base changes on UV mutational activity at two translocated hotspots.

Mutation hotspots have been a staple of mutation spectra since the introduction of fine structure mutation mapping almost 40 years ago. It has been well established that sequence context is an important determinant of mutational activity at mutagen induced hotspots and coldspots. However, our understanding of the sequence effectors of base substitution hotspots is quite limited. This is because manipulation of the sequence about a hotspot site in a marker gene is restricted by the need to maintain a functional marker. In this work, we describe a generalizable system for studying sequence context effects on mutagenesis. We have prepared a variant of the supF tRNA gene (a marker used by us in previous studies) in which an eight-base palindrome, the site of two UV hotspots in the interior of the gene, was copied into the acceptor stem and pre-tRNA region. The variant tRNA was active. The UV mutation spectrum of this variant showed that the new copy of the palindrome generated two hotspots which were as intense as the original sites in the interior of the gene. Variant genes were constructed with all possible bases at the first position in the palindrome in the pre-tRNA sequence, which does not affect tRNA function. The mutation analysis showed that activity at one of the hotspots could be reduced or enhanced by the changes, while activity at the other site was not significantly affected. The base changes did not influence the frequency of cyclobutane dimer or (6-4) photoproduct formation at the two hotspot sites. Thus, the changes in mutational activity were due to the influence of sequence context on the efficiency of mutation formation at the sites of UV lesions.