Streptococcus pneumoniae (the pneumococcus) is the world’s foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or ‘phases’) that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.

Summary: Two main patterns of gene expression of Streptococcus pneumoniae were observed during infection in the host by quantitative real time RT‐PCR; one was characteristic of bacteria in blood and one of bacteria in tissue, such as brain and lung. Gene expression in blood was characterized by increased expression of pneumolysin, pspA and hrcA, while pneumococci in tissue infection showed increased expression of neuraminidases, metalloproteinases, oxidative stress and competence genes. In vitro situations with similar expression patterns were detected in liquid culture and in a newly developed pneumococcal model of biofilm respectively. The biofilm model was dependent on addition of synthetic competence stimulating peptide (CSP) and no biofilm was formed by CSP receptor mutants. As one of the differentially expressed gene sets in vivo were the competence genes, we exploited competence‐specific tools to intervene on pneumococcal virulence during infection. Induction of the competence system by the quorum‐sensing peptide, CSP, not only induced biofilm formation in vitro, but also increased virulence in pneumonia in vivo. In contrast, a mutant for the ComD receptor, which did not form biofilm, also showed reduced virulence in pneumonia. These results were opposite to those found in a bacteraemic sepsis model of infection, where the competence system was downregulated. When pneumococci in the different physiological states were used directly for challenge, sessile cells grown in a biofilm were more effective in inducing meningitis and pneumonia, while planktonic cells from liquid culture were more effective in inducing sepsis. Our data enable us, using in vivo gene expression and in vivo modulation of virulence, to postulate the distinction – from the pneumococcal point of view – between two main types of disease. During bacteraemic sepsis pneumococci resemble planktonic growth, while during tissue infection, such as pneumonia or meningitis, pneumococci are in a biofilm‐like state.

The polysaccharide capsule of Streptococcus pneumoniae is the dominant surface structure of the organism and plays a critical role in virulence, principally by interfering with host opsonophagocytic clearance mechanisms. The capsule is the target of current pneumococcal vaccines, but there are 98 currently recognised polysaccharide serotypes and protection is strictly serotype-specific. Widespread use of these vaccines is driving changes in serotype prevalence in both carriage and disease. This chapter summarises current knowledge on the role of the capsule and its regulation in pathogenesis, the mechanisms of capsule synthesis, the genetic basis for serotype differences, and provides insights into how so many structurally distinct capsular serotypes have evolved. Such knowledge will inform ongoing refinement of pneumococcal vaccination strategies.

The correlation between carbohydrate availability, pneumococcal biofilm formation, nasopharyngeal colonization, and invasion of the host has been investigated. Of a series of sugars, only sialic acid (i.e., N-acetylneuraminic acid) enhanced pneumococcal biofilm formation in vitro, at concentrations similar to those of free sialic acid in human saliva. In a murine model of pneumococcal carriage, intranasal inoculation of sialic acid significantly increased pneumococcal counts in the nasopharynx and instigated translocation of pneumococci to the lungs. Competition of both sialic acid–dependent phenotypes was found to be successful when evaluated using the neuraminidase inhibitors DANA (i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir, and oseltamivir. The association between levels of free sialic acid on mucosae, pneumococcal colonization, and development of invasive disease shows how a host-derived molecule can influence a colonizing microbe and also highlights a molecular mechanism that explains the epidemiologic correlation between respiratory infections due to neuraminidase-bearing viruses and bacterial pneumonia. The data provide a new paradigm for the role of a host compound in infectious diseases and point to new treatment strategies

The thiol-containing tripeptide glutathione is an important cellular constituent of many eukaryotic and prokaryotic cells. In addition to its disulfide reductase activity, glutathione is known to protect cells from many forms of physiological stress. This report represents the first investigation into the role of glutathione in the Gram-positive pathogenStreptococcus pneumoniae. We demonstrate that pneumococci import extracellular glutathione using the ABC transporter substrate binding protein GshT. Mutation ofgshTand the gene encoding glutathione reductase (gor) increases pneumococcal sensitivity to the superoxide generating compound paraquat, illustrating the importance of glutathione utilization in pneumococcal oxidative stress resistance. In addition, thegshTandgormutant strains are hypersensitive to challenge with the divalent metal ions copper, cadmium, and zinc. The importance of glutathione utilization in pneumococcal colonization and invasion of the host is demonstrated by the attenuated phenotype of thegshTmutant strain in a mouse model of infection.

During infection, Streptococcus pneumoniae exists mainly in sessile biofilms rather than in planktonic form, except during sepsis. The capacity to form biofilms is believed to be important for nasopharyngeal colonization as well as disease pathogenesis, but relatively little is known about the regulation of this process. Here, we investigated the effect of exogenous iron [Fe(III)] as well as the role of luxS (encoding S-ribosylhomocysteine lyase) on biofilm formation by S. pneumoniae D39. Fe(III) strongly enhanced biofilm formation at concentrations of ≥50 μM, while Fe(III) chelation with deferoxamine was inhibitory. Importantly, Fe(III) also upregulated the expression of luxS in wild-type D39. A luxS -deficient mutant (D39 luxS ) failed to form a biofilm, even with Fe(III) supplementation, whereas a derivative overexpressing luxS (D39 luxS +) exhibited enhanced biofilm formation capacity and could form a biofilm without added Fe(III). D39 luxS exhibited reduced expression of the major Fe(III) transporter PiuA, and the cellular [Fe(III)] was significantly lower than that in D39; in contrast, D39 luxS + had a significantly higher cellular [Fe(III)] than the wild type. The release of extracellular DNA, which is an important component of the biofilm matrix, also was directly related to luxS expression. Similarly, genetic competence, as measured by transformation frequency as well as the expression of competence genes comD , comX , comW , cglA , and dltA and the murein hydrolase cbpD , which is associated with fratricide-dependent DNA release, all were directly related to luxS expression levels and were further upregulated by Fe(III). Moreover, mutagenesis of cbpD blocked biofilm formation. We propose that competence, fratricide, and biofilm formation are closely linked in pneumococci, and that luxS is a central regulator of these processes. We also propose that the stimulatory effects of Fe(III) on all of these parameters are due to the upregulation of luxS expression, and that LuxS provides for a positive Fe(III)-dependent amplification loop by increasing iron uptake.