Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

AIM: (i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. METHODOLOGY: Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. RESULTS: Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. CONCLUSIONS: The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Salivary Proteomic Profiling Identifies Role of Neutrophil Extracellular Traps Formation in Pregnancy Gingivitis.

Pregnancy gingivitis peaks during mid-pregnancy and resolves transiently towards the postpartum period. However, the role of maternal immune response in orchestrating gingival inflammation has not yet been fully understood. Hence, in this study, we examined the salivary protein profile during the three trimesters of pregnancy, in context to pregnancy gingivitis, employing iTRAQ-based quantitative proteomics. Unstimulated saliva was collected from 10 subjects in each trimester of pregnancy and postpartum period. Samples were analysed using iTRAQ analysis and ELISA and SEM was performed to validate results. Neutrophil mediated immune response was overrepresented in all three trimesters of pregnancy, despite the decrease in phagocytic responses during the second and third trimesters. ELISA showed a significantly higher Neutrophil Extracellular Traps (NETs) formation in the third trimester of pregnancy coinciding with the resolution of pregnancy gingivitis. The NETs-associated proteins (neutrophil elastase and myeloperoxidase) showed a positive correlation with estrogen hormones, which was also highest during the third trimester. Sex hormone-driven NETs formation could be the mainstay of defence that contributes to the remission of pregnancy gingivitis. This study has provided a new insight into the role of immune-modulation in pregnancy gingivitis, which will aid development of new therapeutics for managing pregnancy gingivitis in future.

Multi-omics tools for studying microbial biofilms: current perspectives and future directions.

The advent of omics technologies has greatly improved our understanding of microbial biology, particularly in the last two decades. The field of microbial biofilms is, however, relatively new, consolidated in the 1980s. The morphogenic switching by microbes from planktonic to biofilm phenotype confers numerous survival advantages such as resistance to desiccation, antibiotics, biocides, ultraviolet radiation, and host immune responses, thereby complicating treatment strategies for pathogenic microorganisms. Hence, understanding the mechanisms governing the biofilm phenotype can result in efficient treatment strategies directed specifically against molecular markers mediating this process. The application of omics technologies for studying microbial biofilms is relatively less explored and holds great promise in furthering our understanding of biofilm biology. In this review, we provide an overview of the application of omics tools such as transcriptomics, proteomics, and metabolomics as well as multi-omics approaches for studying microbial biofilms in the current literature. We also highlight how the use of omics tools directed at various stages of the biological information flow, from genes to metabolites, can be integrated via multi-omics platforms to provide a holistic view of biofilm biology. Following this, we propose a future artificial intelligence-based multi-omics platform that can predict the pathways associated with different biofilm phenotypes.

Oral microbiome-systemic link studies: perspectives on current limitations and future artificial intelligence-based approaches.

In the past decade, there has been a tremendous increase in studies on the link between oral microbiome and systemic diseases. However, variations in study design and confounding variables across studies often lead to inconsistent observations. In this narrative review, we have discussed the potential influence of study design and confounding variables on the current sequencing-based oral microbiome-systemic disease link studies. The current limitations of oral microbiome-systemic link studies on type 2 diabetes mellitus, rheumatoid arthritis, pregnancy, atherosclerosis, and pancreatic cancer are discussed in this review, followed by our perspective on how artificial intelligence (AI), particularly machine learning and deep learning approaches, can be employed for predicting systemic disease and host metadata from the oral microbiome. The application of AI for predicting systemic disease as well as host metadata requires the establishment of a global database repository with microbiome sequences and annotated host metadata. However, this task requires collective efforts from researchers working in the field of oral microbiome to establish more comprehensive datasets with appropriate host metadata. Development of AI-based models by incorporating consistent host metadata will allow prediction of systemic diseases with higher accuracies, bringing considerable clinical benefits.

Quantitative Proteomics of Strong and Weak Biofilm Formers of Enterococcus faecalis Reveals Novel Regulators of Biofilm Formation.

Enterococcus faecalis is a bacterial pathogen associated with both endodontic and systemic infections. The biofilm formation ability of E. faecalis plays a key role in its virulence and drug resistance attributes. The formation of E. faecalis biofilms on implanted medical devices often results in treatment failure. In the present study, we report protein markers associated with the biofilm formation ability of E. faecalis using iTRAQ-based quantitative proteomics approach. In order to elucidate the biofilm-associated protein markers, we investigated the proteome of strong and weak biofilm-forming E. faecalis clinical isolates in comparison with standard American Type Culture Collection (ATCC) control strains. Comparison of E. faecalis strong and weak biofilm-forming clinical isolates with ATCC control strains showed that proteins associated with shikimate kinase pathway and sulfate transport were up-regulated in the strong biofilm former, while proteins associated with secondary metabolites, cofactor biosynthesis, and tetrahydrofolate biosynthesis were down-regulated. In the weak biofilm former, proteins associated with nucleoside and nucleotide biosynthesis were up-regulated, whereas proteins associated with sulfate and sugar transport were down-regulated. Further pathway and gene ontology analyses revealed that the major differences in biofilm formation arise from differences in metabolic activity levels of the strong and weak biofilm formers, with higher levels of metabolic activity observed in the weak biofilm former. The differences in metabolic activity could therefore be a major determinant of the biofilm ability of E. faecalis The new markers identified from this study can be further characterized in order to understand their exact role in E. faecalis biofilm formation ability. This, in turn, can lead to numerous therapeutic benefits in the treatment of this oral and systemic pathogen. The data has been deposited to the ProteomeXchange with identifier PXD006542.

Use of Haploid Model of Candida albicans to Uncover Mechanism of Action of a Novel Antifungal Agent.

Antifungal agents for the treatment of Candida albicans infections are limited. We recently discovered a novel antifungal small molecule, SM21, with promising in vivo activity. Herein, we employed the newly developed C. albicans haploid toolbox to uncover the mechanism of action of SM21. Comprehensive RNA-Seq analyses of the haploid susceptible GZY803 strain revealed significant gene expression changes related to mitochondria when exposed to SM21. Mitochondrial structure visualization and measurement of ATP generation, reactive oxygen species (ROS) levels, and the antioxidant potential of SM21-treated and untreated GZY803, mitochondrial structure defective haploid mutant (dnm1Δ), and wild-type diploid SC5314 strains confirmed defects in mitochondria. Exploiting the advantage of C. albicans haploids as a single ploidy model, we further exposed GZY803 to repetitive treatments of SM21 in order to generate resistant mutants. Three colonies designated S3, S5 and S6, which displayed resistance to SM21, were isolated. All resistant strains exhibited enhanced transcriptomic responses for peptide and protein metabolism and secreted aspartate proteases (SAPs) activity under SM21 treatment compared to the parent strain GZY803. Consistently, supplementing the resistant strains, GZY803, and SC5314 with peptone, a form of digested peptides, decreased susceptibility to SM21. The present study demonstrates the usefulness of haploid C. albicans model in antifungal drug discovery. The findings will be invaluable to develop SM21 as a novel antifungal agent, which will benefit millions of patients suffering from Candida infections.

Transcriptomics Analysis Reveals Putative Genes Involved in Biofilm Formation and Biofilm-associated Drug Resistance of Enterococcus faecalis.

INTRODUCTION: Enterococcus faecalis is a gram-positive bacterium associated with endodontic infections and is capable of forming biofilms that can confer drug resistance to the bacterium, resulting in treatment failure. Current knowledge on E. faecalis drug resistance is of a limited and conflicting nature. The present study examined the genetic basis of E. faecalis biofilm formation and drug resistance using a RNA sequencing (RNA-Seq)-based transcriptome approach. METHODS: Eighteen clinical isolates of E. faecalis were screened for their biofilm formation abilities using the crystal violet assay, colony counting, and confocal imaging. Selected isolates were then evaluated for antibiotic susceptibility in planktonic and biofilm growth modes followed by RNA-Seq analysis of E. faecalis planktonic, biofilm, and vancomycin-treated biofilm samples and Kyoto Encyclopedia of Genes and Genomes mapping in order to identify genes associated with biofilm formation and drug resistance of E. faecalis. RESULTS: All 18 clinical isolates retained biofilm formation ability and were classified as strong, weak, or laboratory American Type Culture Collection strainlike biofilm formers. Interestingly, both the strong and weak biofilm-forming isolates were uniformly resistant to ampicillin and vancomycin at the treated concentrations (256-4096 µg/mL). RNA-Seq analysis of these isolates identified a total of 163 and 101 differentially regulated genes in planktonic versus biofilm and vancomycin-treated biofilm versus biofilm comparisons, respectively, with significant differences in arsenic resistance operon genes arsR and arsD, sporulation regulatory gene paiA, ABC drug transporter classes, and penicillin-binding proteins. CONCLUSIONS: The present transcriptomic study revealed putative genes associated with E. faecalis biofilm formation and drug resistance, which will provide a foundation for improved therapeutic strategies against E. faecalis infections in the future.

Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1.

Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal.

Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1.

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors--surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.