Transcriptomic Analysis of Aggregatibacter actinomycetemcomitans Core and Accessory Genes in Different Growth Conditions

Aggregatibacter actinomycetemcomitans genome can be divided into an accessory gene pool (found in some but not all strains) and a core gene pool (found in all strains). The functions of the accessory genes (genomic islands and non-island accessory genes) are largely unknown. We hypothesize that accessory genes confer critical functions for A. actinomycetemcomitans in vivo. This study examined the expression patterns of accessory and core genes of A. actinomycetemcomitans in distinct growth conditions. We found similar expression patterns of island and non-island accessory genes, which were generally lower than the core genes in all growth conditions. The median expression levels of genomic islands were 29%–37% of the core genes in enriched medium but elevated to as high as 63% of the core genes in nutrient-limited media. Several putative virulence genes, including the cytolethal distending toxin operon, were found to be activated in nutrient-limited conditions. In conclusion, genomic islands and non-island accessory genes exhibited distinct patterns of expression from the core genes and may play a role in the survival of A. actinomycetemcomitans in nutrient-limited environments.</p

Dormancy-like Phenotype of <i>Aggregatibacter actinomycetemcomitans</i>: Survival during Famine

Microbes frequently experience nutrient deprivations in the natural environment and may enter dormancy. Aggregatibacter actinomycetemcomitans is known to establish long-term infections in humans. This study examined the dormancy-like phenotype of an A. actinomycetemcomitans strain D7S-1 and its isogenic smooth-colony mutant D7SS. A tissue culture medium RPMI-1640 was nutrient-deficient (ND) and unable to support A. actinomycetemcomitans growth. RPMI-1640 amended with bases was nutrient-limited (NL) and supported limited growth of A. actinomycetemcomitans less than the nutrient-enriched (NE) laboratory medium did. Strain D7S-1, after an initial 2-log reduction in viability, maintained viability from day 4 to day 15 in the NL medium. Strain D7SS, after 1-log reduction in viability, maintained viability from day 3 to day 5. In contrast, bacteria in the NE medium were either non-recoverable (D7S-1; >6-log reduction) or continued to lose viability (D7SS; 3-log reduction) on day 5 and beyond. Scanning and transmission electron microscopy showed that A. actinomycetemcomitans in the NL medium formed robust biofilms similar to those in the NE medium but with evidence of stress. A. actinomycetemcomitans in the ND medium revealed scant biofilms and extensive cellular damage. We concluded that A. actinomycetemcomitans grown in the NL medium exhibited a dormancy-like phenotype characterized by minimum growth, prolonged viability, and distinct cellular morphology

A photoacoustic-fluorescent imaging probe for proteolytic gingipains expressed by Porphyromonas gingivalis

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. This probe harnesses the intramolecular dimerization of peptide-linked cyanine dyes to induce fluorescence and photoacoustic off-states. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU/mL bacteria in vitro. RgpB activity was imaged in the subgingival pocket of porcine jaws with 25 nM sensitivity. The diagnostic efficacy of the probe was evaluated in gingival crevicular fluid (GCF) samples from subjects with (n = 14) and without (n = 6) periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson’s r = 0.71). The highest activity was seen in subjects with the most severe disease. Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, demonstrating relevance and potential utility for animal models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer’s disease

Schematic of the inverted repeat region upstream of the K-antigen capsule operon.

<p>The K-antigen capsule is encoded by a series of genes in an operon (<i>PG0106</i> – <i>PG0120</i>). Two genes flanking this operon, <i>PG0104</i> and <i>PG0121</i>, are predicted to encode DNA binding proteins due to their high similarity to the known DNA binding proteins. They are oriented in the same direction as the capsule operon, and they are being co-transcribed to yield multiple transcripts, including one large polycistronic message encoding the entire region from <i>PG0104</i> – <i>PG0121</i>. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093266#pone.0093266-AlbertiSegui1" target="_blank">[28]</a>.</p

EMSA of HU PG0121 protein and Homoduplex I and Heteroduplex I DNA substrates.

<p>2(Panel A) and Heteroduplex I (Panel B) DNA substrates were incubated with the indicated concentrations of purified HU PG0121 protein, locations of shifted bands are indicated with arrows. The calculated equilibrium dissociation constant (K_d) values for the binding of HU PG0121 to the Homoduplex and Heteroduplex DNA substrates were 84.0±18.9 nM and 67.5±14.2 nM, respectively.</p

Sequences of oligonucleotides for DNA substrates used in this study.

<p>Sequences of oligonucleotides for DNA substrates used in this study.</p

Datasheet1_Machine learning enabled design features of antimicrobial peptides selectively targeting peri-implant disease progression.docx

Peri-implantitis is a complex infectious disease that manifests as progressive loss of alveolar bone around the dental implants and hyper-inflammation associated with microbial dysbiosis. Using antibiotics in treating peri-implantitis is controversial because of antibiotic resistance threats, the non-selective suppression of pathogens and commensals within the microbial community, and potentially serious systemic sequelae. Therefore, conventional treatment for peri-implantitis comprises mechanical debridement by nonsurgical or surgical approaches with adjunct local microbicidal agents. Consequently, current treatment options may not prevent relapses, as the pathogens either remain unaffected or quickly re-emerge after treatment. Successful mitigation of disease progression in peri-implantitis requires a specific mode of treatment capable of targeting keystone pathogens and restoring bacterial community balance toward commensal species. Antimicrobial peptides (AMPs) hold promise as alternative therapeutics through their bacterial specificity and targeted inhibitory activity. However, peptide sequence space exhibits complex relationships such as sparse vector encoding of sequences, including combinatorial and discrete functions describing peptide antimicrobial activity. In this paper, we generated a transparent Machine Learning (ML) model that identifies sequence-function relationships based on rough set theory using simple summaries of the hydropathic features of AMPs. Comparing the hydropathic features of peptides according to their differential activity for different classes of bacteria empowered predictability of antimicrobial targeting. Enriching the sequence diversity by a genetic algorithm, we generated numerous candidate AMPs designed for selectively targeting pathogens and predicted their activity using classifying rough sets. Empirical growth inhibition data is iteratively fed back into our ML training to generate new peptides, resulting in increasingly more rigorous rules for which peptides match targeted inhibition levels for specific bacterial strains. The subsequent top scoring candidates were empirically tested for their inhibition against keystone and accessory peri-implantitis pathogens as well as an oral commensal bacterium. A novel peptide, VL-13, was confirmed to be selectively active against a keystone pathogen. Considering the continually increasing number of oral implants placed each year and the complexity of the disease progression, prevalence of peri-implant diseases continues to rise. Our approach offers transparent ML-enabled paths towards developing antimicrobial peptide-based therapies targeting the changes in the microbial communities that can beneficially impact disease progression.</p

Permutations of the 2-nucleotide loop structure along the inverted repeat sequence.

<p>Panel A contains the 2; Panel B, 25 nM HU PG0121; Panel C, 1 nM <i>E. coli</i> HU. Lanes are labelled with structures shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093266#pone-0093266-g006" target="_blank">Figure 6</a>. The location of the shifted bands was marked with a bracket.</p

HU PG0121 protects the structure of HJ DNA during thermal denaturation.

<p>EMSA was performed in which 25(B) was then heated to 55°C for 10 minutes, while the control group (A) remained at room temperature. Arrows indicate the location of the various DNA-protein complexes.</p

HU PG0121 protein stabilizes the HJ structure.

<p>Labeled *Y-DNA controls are shown in Panel A with *Y-DNA alone *Y-DNA mixed with unlabeled Y-DNA, 100 nM PG0121, BSA and ComE. Panel B shows *Y-DNA controls with <i>E</i> coli HU protein and a positive control branch migration assay with 100 to 1000 nM <i>E. coli</i> HU protein. Branch migration assays with increasing protein concentrations from 100 to 1000 nM for PG0121 (Panel C), BSA (Panel D) and <i>S. mutans</i> ComE (Panel E). Arrows indicate the branch migration intermediates, the asterisks indicate the location of the branch migration product bands, and the filled circles indicate the location of the substrate bands.</p