The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

Abstract

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus.

The Pneumococcal Type 1 Pilus Genes Are Thermoregulated and Are Repressed by a Member of the Snf2 Protein Family

“…However, several years later, with the rise of replacement strains not covered by the vaccine, we showed that the frequency of colonizing strains carrying pilus type 1 genes returned to pre-2000 levels, strongly suggesting that the presence of these genes confers an important fitness advantage on the organism (11). Complicating the picture further, our group (12) and another (13) showed that pilus expression in vitro is bistable among a clonal population, such that at any one time, some cells of a pilus-containing clone express the pilus (so-called high pilus expressing [HPE]), whereas in others, pilus expression is either very low or virtually undetectable (low pilus expressing [LPE]). We showed that this regulation is subject to a positive-feedback loop by the regulator RlrA (12).…”

Toll-like Receptor (TLR) 2 Mediates Inflammatory Responses to Oligomerized RrgA Pneumococcal Pilus Type 1 Protein

“…It is also striking that the prevalence of the pilus across different geographic areas and various pneumococcal clones is similar (23,24). Complicating the picture further, our group and others recently showed that pilus expression is bistable among a clonal population (25,26), such that only roughly 30% of cells derived from one clone express the pilus at any one time. This regulation appears to be negatively regulated by RrgA (25) and positively regulated by the RlrA-positive feedback loop (27).…”

“…We and others showed that individual clones of piliated pneumococci can have very different pilus expression patterns, such that the comparison of a wild type strain with one carrying a modification in the rrgA gene should include an examination of the pattern of pilus expression (25,26). To control for these issues, we confirmed by Western blot and flow cytometry studies that a pneumococcal mutant carrying the rrgA gene with a deletion of the corresponding sequence of the P3 (T4⌬P3) expresses the pilus in the same pattern as the wild type strain (Fig.…”

RrgB321, a Fusion Protein of the Three Variants of the Pneumococcal Pilus Backbone RrgB, Is ProtectiveIn Vivoand Elicits Opsonic Antibodies

“…In addition, the observation that the RrgB321-mediated killing never reached 100%, in contrast to what was observed with antisera against conjugated polysaccharides, is supported by the recent report of pneumococcal pilus biphasic expression (9). In fact, two bacterial phenotypes that express or do not express the pilus are present in PI-1-positive strains.…”