We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae , given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4 ⁺ T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone—all used without postinactivation washing—produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 μg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.

DNA strand consisting of multiple runs of guanines can adopt the non-canonical, four-stranded DNA secondary structure known as G-quadruplex or G4 DNA. G4 DNA is thought to play an important role in transcriptional and translational regulation of genes, DNA replication, genome stability, and oncogene expression in eukaryotic genomes. In other organisms including several bacterial pathogens and some plant species, the biological role of G4 DNA and G4 RNA is starting to be explored. Recent investigation showed that G4 DNA and G4 RNA are generally conserved across plant species. In silico analyses of several bacterial genomes identified the putative guanine-rich, G4 DNA-forming sequences in the promoter regions. They were particularly abundant in certain gene classes, suggesting that these highly diverse structures can be employed to regulate expression of genes involved in secondary metabolite synthesis and signal transduction. Furthermore, in the pathogen Mycobacterium tuberculosis, the distribution of G4 motifs and their potential role in the regulation of gene transcription advocate for the use of G4 ligands to develop novel antitubercular therapies. In this review, we discuss the various roles of G4 structures in bacterial DNA and the application of G4 DNA as an inhibitor or therapeutic agent to tackle the bacterial pathogens.

G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ) region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼20 kb or ∼100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed. Genome instability is not evenly distributed, but rather is highly elevated at certain genomic loci containing DNA sequences that can fold into non-canonical secondary structures. The four-stranded G-quadruplex or G4 DNA is one such DNA structure capable of instigating transcription and/or replication obstruction and subsequent genome instability. In this study, we used a reporter system to quantitatively measure the level of genome instability occurring at a G4 DNA motif integrated into the yeast genome. We showed that the disruption of Topoisomerase I function significantly elevated various types of genome instability at the highly transcribed G4 motif generating loss of heterozygosity and copy number alterations (deletions and duplications), both of which are frequently observed in cancer genomes.

G-quadruplexes (G4) are higher-order DNA and RNA secondary structures formed by G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential G4 quadruplex sequences have been identified in G-rich eukaryotic non-telomeric and telomeric genomic regions. Upon function, G4 formation is known to involve in chromatin remodelling, gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. The natural role and biological validation of G4 structures is starting to be explored, and is of particular interest for the therapeutic interventions for human diseases. However, the existence and physiological role of G4 DNA and G4 RNA in plants species have not been much investigated yet and therefore, is of great interest for the development of improved crop varieties for sustainable agriculture. In this context, several recent studies suggests that these highly diverse G4 structures in plants can be employed to regulate expression of genes involved in several pathophysiological conditions including stress response to biotic and abiotic stresses as well as DNA damage. In the current review, we summarize the recent findings regarding the emerging functional significance of G4 structures in plants and discuss their potential value in the development of improved crop varieties.

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.

Streptococci are a diverse group of bacteria, which are mostly commensals but also cause a considerable proportion of life-threatening infections. They colonize many different host niches such as the oral cavity, the respiratory, gastrointestinal, and urogenital tract. While these host compartments impose different environmental conditions, many streptococci form biofilms on mucosal membranes facilitating their prolonged survival. In response to environmental conditions or stimuli, bacteria experience profound physiologic and metabolic changes during biofilm formation. While investigating bacterial cells under planktonic and biofilm conditions, various genes have been identified that are important for the initial step of biofilm formation. Expression patterns of these genes during the transition from planktonic to biofilm growth suggest a highly regulated and complex process. Biofilms as a bacterial survival strategy allow evasion of host immunity and protection against antibiotic therapy. However, the exact mechanisms by which biofilm-associated bacteria cause disease are poorly understood. Therefore, advanced molecular techniques are employed to identify gene(s) or protein(s) as targets for the development of antibiofilm therapeutic approaches. We review our current understanding of biofilm formation in different streptococci and how biofilm production may alter virulence-associated characteristics of these species. In addition, we have summarized the role of surface proteins especially pili proteins in biofilm formation. This review will provide an overview of strategies which may be exploited for developing novel approaches against biofilm-related streptococcal infections.