Victoria Stone

Genome-wide essential gene identification in Streptococcus sanguinis

A clear perception of gene essentiality in bacterial pathogens is pivotal for identifying drug targets to combat emergence of new pathogens and antibiotic-resistant bacteria, for synthetic biology, and for understanding the origins of life. We have constructed a comprehensive set of deletion mutants and systematically identified a clearly defined set of essential genes for Streptococcus sanguinis. Our results were confirmed by growing S. sanguinis in minimal medium and by double-knockout of paralogous or isozyme genes. Careful examination revealed that these essential genes were associated with only three basic categories of biological functions: maintenance of the cell envelope, energy production, and processing of genetic information. Our finding was subsequently validated in two other pathogenic streptococcal species, Streptococcus pneumoniae and Streptococcus mutans and in two other gram-positive pathogens, Bacillus subtilis and Staphylococcus aureus. Our analysis has thus led to a simplified model that permits reliable prediction of gene essentiality.

The relationship of the lipoprotein SsaB, manganese and superoxide dismutase in Streptococcus sanguinis virulence for endocarditis

Streptococcus sanguinis colonizes teeth and is an important cause of infective endocarditis. Our prior work showed that the lipoprotein SsaB is critical for S. sanguinis virulence for endocarditis and belongs to the LraI family of conserved metal transporters. In this study, we demonstrated that an ssaB mutant accumulates less manganese and iron than its parent. A mutant lacking the manganese‐dependent superoxide dismutase, SodA, was significantly less virulent than wild‐type in a rabbit model of endocarditis, but significantly more virulent than the ssaB mutant. Neither the ssaB nor the sodA mutation affected sensitivity to phagocytic killing or efficiency of heart valve colonization. Animal virulence results for all strains could be reproduced by growing bacteria in serum under physiological levels of O2. SodA activity was reduced, but not eliminated in the ssaB mutant in serum and in rabbits. Growth of the ssaB mutant in serum was restored upon addition of Mn2+ or removal of O2. Antioxidant supplementation experiments suggested that superoxide and hydroxyl radicals were together responsible for the ssaB mutants growth defect. We conclude that manganese accumulation mediated by the SsaB transport system imparts virulence by enabling cell growth in oxygen through SodA‐dependent and independent mechanisms.

Systematic study of genes influencing cellular chain length in Streptococcus sanguinis.

Streptococcus sanguinis is a Gram-positive bacterium that is indigenous to the oral cavity. S. sanguinis, a primary colonizer of the oral cavity, serves as a tether for the attachment of other oral pathogens. The colonization of microbes on the tooth surface forms dental plaque, which can lead to the onset of periodontal disease. We examined a comprehensive mutant library to identify genes related to cellular chain length and morphology using phase-contrast microscopy. A number of hypothetical genes related to the cellular chain length were identified in this study. Genes related to the cellular chain length were analysed along with clusters of orthologous groups (COG) for gene functions. It was discovered that the highest proportion of COG functions related to cellular chain length was ‘cell division and chromosome separation’. However, different COG functions were also found to be related with altered cellular chain length. This suggested that different genes related with multiple mechanisms contribute to the cellular chain length in S. sanguinis SK36.

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

ciaR impacts biofilm formation by regulating an arginine biosynthesis pathway in Streptococcus sanguinis SK36

Streptococcus sanguinis is an early colonizer of the tooth surface and competes with oral pathogens such as Streptococcus mutans to maintain oral health. However, little is known about its mechanism of biofilm formation. Here, we show that mutation of the ciaR gene, encoding the response regulator of the CiaRH two-component system in S. sanguinis SK36, produced a fragile biofilm. Cell aggregation, gtfP gene expression and water-insoluble glucan production were all reduced, which suggested polysaccharide production was decreased in ΔciaR. RNA sequencing and qRT-PCR revealed that arginine biosynthesis genes (argR, argB, argC, argG, argH and argJ) and two arginine/histidine permease genes (SSA_1568 and SSA_1569) were upregulated in ΔciaR. In contrast to ΔciaR, most of strains constructed to contain deletions in each of these genes produced more biofilm and water-insoluble glucan than SK36. A ΔciaRΔargB double mutant was completely restored for the gtfP gene expression, glucan production and biofilm formation ability that was lost in ΔciaR, indicating that argB was essential for ciaR to regulate biofilm formation. We conclude that by promoting the expression of arginine biosynthetic genes, especially argB gene, the ciaR mutation reduced polysaccharide production, resulting in the formation of a fragile biofilm in Streptococcus sanguinis.

TetR Family Regulator brpT Modulates Biofilm Formation in Streptococcus sanguinis

Biofilms are a key component in bacterial communities providing protection and contributing to infectious diseases. However, mechanisms involved in S. sanguinis biofilm formation have not been clearly elucidated. Here, we report the identification of a novel S. sanguinis TetR repressor, brpT (Biofilm Regulatory Protein TetR), involved in biofilm formation. Deletion of brpT resulted in a significant increase in biofilm formation. Interestingly, the mutant accumulated more water soluble and water insoluble glucans in its biofilm compared to the wild-type and the complemented mutant. The brpT mutation led to an altered biofilm morphology and structure exhibiting a rougher appearance, uneven distribution with more filaments bound to the chains. RNA-sequencing revealed that gtfP, the only glucosyltransferase present in S. sanguinis, was significantly up-regulated. In agreement with these findings, we independently observed that deletion of gtfP in S. sanguinis led to reduced biofilm and low levels of water soluble and insoluble glucans. These results suggest that brpT is involved in the regulation of the gtfP-mediated exopolysaccharide synthesis and controls S. sanguinis biofilm formation. The deletion of brpT may have a potential therapeutic application in regulating S. sanguinis colonization in the oral cavity and the prevention of dental caries.

Identification of Small-Molecule Inhibitors against Meso-2, 6-Diaminopimelate Dehydrogenase from Porphyromonas gingivalis

Species-specific antimicrobial therapy has the potential to combat the increasing threat of antibiotic resistance and alteration of the human microbiome. We therefore set out to demonstrate the beginning of a pathogen-selective drug discovery method using the periodontal pathogen Porphyromonas gingivalis as a model. Through our knowledge of metabolic networks and essential genes we identified a “druggable” essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. We adopted a high-throughput virtual screen method on the ZINC chemical library to select a group of potential small-molecule inhibitors. Meso-diaminopimelate dehydrogenase from P. gingivalis was first expressed and purified in Escherichia coli then characterized for enzymatic inhibitor screening studies. Several inhibitors with similar structural scaffolds containing a sulfonamide core and aromatic substituents showed dose-dependent inhibition. These compounds were further assayed showing reasonable whole-cell activity and the inhibition mechanism was determined. We conclude that the establishment of this target and screening strategy provides a model for the future development of new antimicrobials.

Targeted antimicrobial therapy in the microbiome era

Summary Even though the oral microbiome is one of the most complex sites on the body it is an excellent model for narrow‐spectrum antimicrobial therapy. Current research indicates that disruption of the microbiome leads to a dysbiotic environment allowing for the overgrowth of pathogenic species and the onset of oral diseases. The gram‐negative colonizer, Porphyromonas gingivalis has long been considered a key player in the initiation of periodontitis and Streptococcus mutans has been linked to dental caries. With antibiotic research still on the decline, new strategies are greatly needed to combat infectious diseases. By targeting key pathogens, it may be possible to treat oral infections while allowing for the recolonization of the beneficial, healthy flora. In this review, we examine unique strategies to specifically target periodontal pathogens and address what is needed for the success of these approaches in the microbiome era.

Involvement of signal peptidase I in Streptococcus sanguinis biofilm formation

Biofilm accounts for 65–80 % of microbial infections in humans. Considerable evidence links biofilm formation by oral microbiota to oral disease and consequently systemic infections. Streptococcus sanguinis, a Gram-positive bacterium, is one of the most abundant species of the oral microbiota and it contributes to biofilm development in the oral cavity. Due to its altered biofilm formation, we investigated a biofilm mutant, ΔSSA_0351, that is deficient in type I signal peptidase (SPase) in this study. Although the growth curve of the ΔSSA_0351 mutant showed no significant difference from that of the wild-type strain SK36, biofilm assays using both microtitre plate assay and confocal laser scanning microscopy (CLSM) confirmed a sharp reduction in biofilm formation in the mutant compared to the wild-type strain and the paralogous mutant ΔSSA_0849. Scanning electron microscopy (SEM) revealed remarkable differences in the cell surface morphologies and chain length of the ΔSSA_0351 mutant compared with those of the wild-type strain. Transcriptomic and proteomic assays using RNA sequencing and mass spectrometry, respectively, were conducted on the ΔSSA_0351 mutant to evaluate the functional impact of SPase on biofilm formation. Subsequently, bioinformatics analysis revealed a number of proteins that were differentially regulated in the ΔSSA_0351 mutant, narrowing down the list of SPase substrates involved in biofilm formation to lactate dehydrogenase (SSA_1221) and a short-chain dehydrogenase (SSA_0291). With further experimentation, this list defined the link between SSA_0351-encoded SPase, cell wall biosynthesis and biofilm formation.

Diaminopimelic acid (DAP) analogs bearing isoxazoline moiety as selective inhibitors against meso-diaminopimelate dehydrogenase (m-Ddh) from Porphyromonas gingivalis

Two diastereomeric analogs (1 and 2) of diaminopimelic acid (DAP) bearing an isoxazoline moiety were synthesized and evaluated for their inhibitory activities against meso-diaminopimelate dehydrogenase (m-Ddh) from the periodontal pathogen, Porphyromonas gingivalis. Compound 2 showed promising inhibitory activity against m-Ddh with an IC50 value of 14.9µM at pH 7.8. The two compounds were further tested for their antibacterial activities against a panel of periodontal pathogens, and compound 2 was shown to be selectively potent to P. gingivalis strains W83 and ATCC 33277 with minimum inhibitory concentration (MIC) values of 773µM and 1.875mM, respectively. Molecular modeling studies revealed that the inversion of chirality at the C-5 position of these compounds was the primary reason for their different biological profiles. Based on these preliminary results, we believe that compound 2 has properties consistent with it being a lead compound for developing novel pathogen selective antibiotics to treat periodontal diseases.