Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

Introduction: Nonencapsulated Streptococcus pneumoniae (NESp) are isolated worldwide. Due to the lack of capsule in NESp strains the current vaccines, that target the pneumococcal capsule are ineffective. Some NESp contain the oligopeptide transporters AliC and AliD which are required for virulence through unknown mechanisms. AliC and AliD have been previously shown to reduce rates of phagocytosis and alter the transcriptome and proteome of MNZ41. We hypothesize that oligopeptide regulated genes are responsible for reduced phagocytosis and increased survival through resistance to reactive oxygen species (ROS).Methods: To test this a mutant library of AliC and AliD regulated genes was used in in vitro and in vivo models. ROS resistance was tested through quantifying bacterial counts after exposure to hydrogen peroxide (H₂O₂). A modified surface killing assay was also used to calculate resistance to phagocytosis of our mutant library. A Galleria mellonella larvae model of infection was used to determine survival curve analyses.Results: Two mutant genes in our library, ∆lytFN1 (CDT04) and ∆mgtC (CDT05), displayed greater sensitivity to H₂O₂ killing and phagocytosis compared to wildtype MNZ41. Deletion of AliD in an AliD-expressing encapsulated strain reduced virulence.Conclusion: This research demonstrates that proteins encoded by genes regulated by AliC and AliD alter susceptibility to host-derived mechanisms for bacterial clearance and increases bacterial survival in response to ROS.

BACKGROUND: Surveillance of pneumococcus is reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance (AMR), and are increasingly implicated in disease. Disease-associated NESp commonly carry the virulence genes _pspK,_ or _aliC_ and _aliD_ in their _cps_ locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. METHODS: We conducted bioinformatic analysis on sequences from 30 NESp and 23 other mitis-group streptococcal sequences and developed a multiplex-qPCR, targeting _pspK_, _aliD_ and two regions of _aliC_. The assay was validated using 11 previously characterised, and 5 uncharacterised NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva, and isolated and characterised suspected NESp colonies, with confirmation by whole genome sequencing. RESULTS: Bioinformatic analyses demonstrated that previously published primers for _aliC_ and _aliD_ had low pneumococcal-specificity but indicated that targeting two regions of _aliC_ would improve species-specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of _aliC_ and _aliD_, even in samples negative for pneumococcal genes _lytA_ and _piaB_. Isolated colonies which were _aliC_ and _aliD_ positive could be differentiated as non-pneumococcal streptococci using our assay. CONCLUSION: Our multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp, and their impact upon pneumococcal carriage, disease, and AMR.

Pneumococcal surveillance studies are reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance, and are increasingly implicated in disease. Disease-associated NESp commonly carries the virulence genes pspK, or aliC and aliD in their cps locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for the classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. We conducted bioinformatic analysis on sequences from 402 NESp and 45 other mitis-group streptococci and developed a multiplex-qPCR, targeting pspK, aliD and two regions of aliC. The assay was validated using 16 previously identified NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva and isolated and characterized suspected NESp colonies, with confirmation by whole genome sequencing. Bioinformatic analyses demonstrated that previously published primers for aliC and aliD had low pneumococcal specificity but indicated that targeting two regions of aliC would improve species specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of aliC and aliD, even in samples negative for pneumococcal genes lytA and piaB. Isolated colonies which were aliC and aliD positive could be differentiated as non-pneumococcal streptococci using our assay. Our multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp and its impact on pneumococcal carriage and disease.

Background: Surveillance of pneumococcus is reporting increasing prevalence of non-encapsulated pneumococci (NESp). NESp are an important reservoir for genetic exchange among streptococci, including for antimicrobial resistance (AMR), and are increasingly implicated in disease. Disease-associated NESp commonly carry the virulence genes pspK, or aliC and aliD in their cps locus instead of capsule genes. While molecular methods targeting the cps region are widely used for serotyping encapsulated strains, there are few assays available for classification of NESp, meaning it is not widely undertaken. Therefore, we exploited these genes as targets for a novel qPCR assay for detecting and classifying NESp strains with improved efficiency and specificity. Methods: We conducted bioinformatic analysis on sequences from 30 NESp and 23 other mitis-group streptococcal sequences and developed a multiplex-qPCR, targeting pspK , aliD and two regions of aliC . The assay was validated using 11 previously characterised, and 5 uncharacterised NESp isolates. We then applied the assay to DNA extracted from culture-enriched saliva, and isolated and characterised suspected NESp colonies, with confirmation by whole genome sequencing. Results: Bioinformatic analyses demonstrated that previously published primers for aliC and aliD had low pneumococcal-specificity but indicated that targeting two regions of aliC would improve species-specificity, without compromising sensitivity. Our novel multiplex assay accurately typed all isolates. When screening saliva, we found a high prevalence of aliC and aliD , even in samples negative for pneumococcal genes lytA and piaB . Isolated colonies which were aliC and aliD positive could be differentiated as non-pneumococcal streptococci using our assay. Conclusion: Our multiplex-qPCR assay can be used to efficiently screen even highly polymicrobial samples, such as saliva, for NESp genes, to detect and differentiate potentially pathogenic NESp clades from closely related mitis-group streptococci. This will allow for a better understanding of the true prevalence of NESp, and their impact upon pneumococcal carriage, disease, and AMR.

Streptococcus pneumoniae is a leading cause of respiratory infections and is often treated with β-lactam antibiotics despite frequent resistance. The β-lactam resistance is through mutations in penicillin-binding proteins (PBPs), but the impact of PBP regulation remains poorly understood. Here, we investigate the role of gene regulation by the oligopeptide-binding protein aliD on β-lactam susceptibility. aliD-expressing strains exhibit elevated minimum inhibitory concentrations (MICs) to the β-lactam antibiotics amoxicillin and cefdinir compared to isogenic aliD mutants. In contrast, aliD had minimal effects on susceptibility to vancomycin, suggesting a mechanism specific to β-lactam antibiotics. We demonstrate that aliD increases clpL expression with or without antibiotics present. Inducible expression of clpL resulted in stepwise increases in amoxicillin MIC, demonstrating that elevated clpL expression directly contributes to decreased β-lactam susceptibility. Furthermore, we show that increased clpL expression enhances PBP2x surface exposure in a dose-dependent manner. Additionally, aliD-expressing strains exhibited significantly increased cell wall cross-linking relative to aliD mutants. Together, these findings identify a novel regulatory pathway in which aliD enhances clpL expression, promotes PBP2x surface exposure, and increases peptidoglycan cross-linking, ultimately reducing β-lactam susceptibility. This work expands current understanding of pneumococcal antibiotic resistance and suggests that peptide sensing through oligopeptide transport systems may influence antibiotic susceptibility in host-specific environments.