Pentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins which mediate several aspects of gene expression primarily in organelles but also in the nucleus. These proteins facilitate processing, splicing, editing, stability and translation of RNAs. While major advances in PPR research have been achieved with plant PPR proteins, the significance of non-plant PPR proteins is becoming of increasing importance. PPR proteins are classified into different subclasses based on their domain architecture, which is often a reflection of their function. This review provides an overview of the significant findings regarding the functions, evolution and applications of PPR proteins. Horizontal gene transfer appears to have played a major role in the sporadic phylogenetic distribution of different PPR subclasses in both eukaryotes and prokaryotes. Additionally, the use of synthetic biology and protein engineering to create designer PPR proteins to control gene expression in vivo is discussed. This review also highlights some of the aspects of PPR research that require more attention particularly in non-plant organisms. This includes the lack of research into the recently discovered PPR-TGM subclass, which is not only the first PPR subclass absent from plants but present in economically and clinically-relevant pathogens. Investigation into the structure and function of PPR-TGM proteins in these pathogens presents a novel opportunity for the exploitation of PPR proteins as drug targets to prevent disease.

Exposure to cigarette smoke is a risk factor for respiratory diseases. Although most research has focused on its effects on the host, cigarette smoke can also directly affect respiratory pathogens, in some cases enhancing virulence. Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia worldwide, however data on the effects of cigarette smoke on the pneumococcus are sparse. Using RNA-seq, we show that pneumococci exposed to cigarette smoke extract in a concentrated acute exposure in vitro model initiate a ‘survival’ transcriptional response including the upregulation of detoxification enzymes, efflux pumps and osmoregulator transporters, as well as the downregulation of fatty acid and D-alanyl lipoteichoic acid biosynthesis genes. Except for the downregulation of the pneumolysin gene, there were no changes in the expression of major virulence factors following exposure to cigarette smoke. Compared to unexposed pneumococci, smoke-exposed pneumococci did not exhibit any changes in viability, adherence, hydrophobicity or cell lysis susceptibility. In this study, we demonstrate that pneumococci adapt to acute noxious cigarette smoke exposure by inducing a gene expression signature that allows the bacteria to resist its harmful effects.

Objectives As part of annual cross-sectional Streptococcus pneumoniae carriage surveys in Fiji (2012–2015), we detected pneumococci in over 100 nasopharyngeal swabs that serotyped as ‘11F-like’ by microarray. We examined the genetic basis of this divergence in the 11F-like capsular polysaccharide ( cps ) locus compared to the reference 11F cps sequence. The impact of this diversity on capsule phenotype, and serotype results using genetic and serologic methods were determined. Methods Genomic DNA from representative 11F-like S. pneumoniae isolates obtained from the nasopharynx of Fijian children was extracted and subject to whole genome sequencing. Genetic and phylogenetic analyses were used to identify genetic changes in the cps locus. Capsular phenotypes were evaluated using the Quellung reaction and latex agglutination. Results Compared to published 11F sequences, the wcwC and wcrL genes of the 11F-like cps locus are phylogenetically divergent, and the gct gene contains a single nucleotide insertion within a homopolymeric region. These changes within the DNA sequence of the 11F-like cps locus have modified the antigenic properties of the capsule, such that 11F-like isolates serotype as 11A by Quellung reaction and latex agglutination. Conclusions This study demonstrates the ability of molecular serotyping by microarray to identify genetic variants of S. pneumoniae and highlights the potential for discrepant results between phenotypic and genotypic serotyping methods. We propose that 11F-like isolates are not a new serotype but rather are a novel genetic variant of serotype 11A. These findings have implications for invasive pneumococcal disease surveillance as well as studies investigating vaccine impact.

Determination of serotypes of Streptococcus pneumoniae is essential for monitoring current vaccine programmes. Since October 2017, pneumococcal serotypes in England have been derived from whole genome sequencing (WGS) data using our bioinformatic tool PneumoCaT. That tool was designed for serotype determination from pure cultures in a reference laboratory. To help determine multiple serotypes in pneumococcal carriage samples, we developed a new software tool named PneumoKITy (Pneumococcal K-mer Integrated Typing) that uses the powerful Mash k-mer screening method for pneumococcal serotyping. Mash k-mer screening is more sequence specific and much faster than the mapping method used in PneumoCaT and can determine 54 (58.1  %) of the 93 serotypes in the SSI Diagnostica phenotypical serotyping scheme to type level with the remainder called to serogroup or subgroup level (e.g., 11A/D). PneumoKITy can be run on both FastQ and assembly input, requiring up to 11× less memory and running up to 29× faster than the current version of PneumoCaT (1.2.1) on FastQ files. PneumoKITy can be used as a rapid, flexible serotype screening method which adds sensitive detection of mixed serotypes, e.g., for nasopharyngeal carriage studies where the presence of multiple serotypes is common. PneumoKITy’s ability to function from assembly file, for pure culture serotype detection, increases its speed. This speed potentially enables the software to be run using low infrastructure overhead via web-based platforms. PneumoKITy could be used as a fast initial screening method with other tools used for those serotypes that could not be fully determined to type level if necessary. PneumoKITy was found to be highly accurate and sensitive when run on a panel of FastQ files derived from mixed cultures with all serotypes in 47/51 (92.2  %) of samples being accurately detected. PneumoKITy was also able to accurately estimate the relative abundance of serotypes in the same sample. Estimates being within a mean relative abundance of 1.5 % of the expected abundance in mixtures with known concentrations. PneumoKITy was able to detect minor serotypes with expected abundance of 1 % in the known mixture serotypes. PneumoKITy is a rapid, flexible tool with wide-ranging applications outside of the pure-culture, reference laboratory serotyping remit of PneumoCaT.