Plasmin Cascade Mediates Thrombotic Events in SARS-CoV-2 Infection via Complement and Platelet-Activating Systems

Objective: Recently emerged beta-coronavirus SARS-CoV-2, has resulted in the current pandemic designated COVID-19. COVID-19 manifests as severe illness exhibiting systemic inflammatory response syndrome, acute respiratory distress syndrome (ARDS), thrombotic events, and shock, exacerbated further by co-morbidities and age. Recent clinical evidence suggests that the development of ARDS and subsequent pulmonary failure result from a complex interplay between cell types (endothelial, epithelial and immune) within the lung promoting inflammatory infiltration and a pro-coagulative state. How the complex molecular events mediated by SARS-CoV-2 in infected lung epithelial cells lead to thrombosis and pulmonary failure, is yet to be fully understood. Methods: We address these questions here, using publicly available transcriptomic data in the context of lung epithelia affected by SARS-CoV-2 and other respiratory infections, in vitro. We then extend our results to the understanding of in vivo lung, using a publicly available COVID-19 lung transcriptomic study. Results and Conclusions: Our analysis indicates that there exists a complex interplay between the fibrinolytic system particularly plasmin, and the complement and platelet-activating systems upon SARS-CoV-2 infection, with a potential for therapeutic intervention

Comparative transcriptome analyses of Pseudomonas aeruginosa

One of the hallmarks of bacterial survival is their ability to adapt rapidly to changing environmental conditions. Niche adaptation is a response to the signals received that are relayed, often to regulators that modulate gene expression. In the post-genomic era, DNA microarrays are used to study the dynamics of gene expression on a global scale. Numerous studies have used Pseudomonas aeruginosa--a Gram-negative environmental and opportunistic human pathogenic bacterium--as the model organism in whole-genome transcriptome analysis. This paper reviews the transcriptome studies that have led to immense advances in our understanding of the biology of this intractable human pathogen. Comparative analysis of 23 P. aeruginosa transcriptome studies has led to the identification of a unique set of genes that are signal specific and a core set that is differentially regulated. The 303 genes in the core set are involved in bacterial homeostasis, making them attractive therapeutic targets

Identification of Novel Genomic Islands in Liverpool Epidemic Strain of Pseudomonas aeruginosa Using Segmentation and Clustering

This article utilizes a recursive segmentation and cluster procedure presented as a genome-mining tool, GEMINI, to decipher genomic islands and understand their contributions to the evolution of virulence and antibiotic resistance in Pseudomonas aeruginosa

Adapting Flint for Calculating Bacterial Replication Rates in Microbiomes

We extend Flint, a Spark-based metagenomic profiling tool, to efficiently measure bacterial growth rates for large data sets. The tool bPTR for bacterial growth rate measurement from metagenomic samples [Brown et al., Nat Biotech, 2016] was adapted and integrated into Flint’s MapReduce framework in order to take advantage of Flint\u27s efficient read alignments and mapping, thus enabling the creation of bacterial abundance profiles that are enhanced with growth-rate information.To show the viability of our method we analyzed whole metagenome sequence data from a longitudinal study of sampled preterm infants [Gibson et al., Nat Micro, 2016], computing the abundance profile enhanced with growth rate information. The conclusions shed light on the new perspective obtained on antibiotics treatments and antibiotic resistance by looking at replication rates

A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is a metabolically versatile bacterium that is found in a wide range of biotic and abiotic habitats. It is a major human opportunistic pathogen causing numerous acute and chronic infections. The critical traits contributing to the pathogenic potential of P. aeruginosa are the production of a myriad of virulence factors, formation of biofilms and antibiotic resistance. Expression of these traits is under stringent regulation, and it responds to largely unidentified environmental signals. This review is focused on providing a global picture of virulence gene regulation in P. aeruginosa. In addition to key regulatory pathways that control the transition from acute to chronic infection phenotypes, some regulators have been identified that modulate multiple virulence mechanisms. Despite of a propensity for chaotic behaviour, no chaotic motifs were readily observed in the P. aeruginosa virulence regulatory network. Having a ‘birds-eye’ view of the regulatory cascades provides the forum opportunities to pose questions, formulate hypotheses and evaluate theories in elucidating P. aeruginosa pathogenesis. Understanding the mechanisms involved in making P. aeruginosa a successful pathogen is essential in helping devise control strategies

The effect of mucA allele on biofilm architecture and the biofilm-related proteomes

In this study, a unique mucA mutation (designated mucA56) was introduced, which was characterized by deletion of bases 166-333, encoding MucA56 protein with the deletion of the trans-membrane region, which then was proved to be cytoplasmic with phoA-mucA fusion method. PAOmucA56 was constructed with homologous recombination; two PAO1 derivatives PAOmucA22 (PDO300) and PAOmucA56 displayed mucoid phenotype on pseudomonas isolation agar (PIA) agar, but PDO300 produced more alginate than PAOmucA56. Scanning confocal laser microscopy was used to observe the biofilm structures of the three strains during various biofilm development stages. PDO300 developed biofilm with low substratum coverage and high structural heterogeneity, while PAOmucA56 and PAO1 formed uniform biofilm with complete substratum coverage. The proteomes of crude protein extracts of biofilm cells revealed that there are 17 candidate proteins differentially expressed between the two kinds of biofilm, which were proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism and so on. We might conclude that alginate production may affect biofilm architecture, and proteins involved in protein synthesis, MucA degradation, energy metabolism, carbon catabolism and amino acid metabolism might play a role in biofilm development alternatively

Causal Inference in Microbiomes Using Intervention Calculus

Inferring causal effects is critically important in biomedical research as it allows us to move from the typical paradigm of associational studies to causal inference, and can impact treatments and therapeutics. Association patterns can be coincidental and may lead to wrong inferences in complex systems. Microbiomes are highly complex, diverse, and dynamic environments. Microbes are key players in health and diseases. Hence knowledge of genuine causal relationships among the entities in a microbiome, and the impact of internal and external factors on microbial abundance and interactions are essential for understanding disease mechanisms and making treatment recommendations. In this paper, we investigate fundamental causal inference techniques to measure the causal effects of various entities in a microbiome. In particular, we show how to use these techniques on microbiome datasets to study the rise and impact of antibiotic-resistance in microbiomes. Our main contributions include the following. We introduce a novel pipeline for microbiome studies, new ideas for experimental design under weaker assumptions, and data augmentation by context embedding. Our pipeline is robust, different from traditional approaches, and able to predict interventional effects without any controlled experiments. Our work shows the advantages of causal inference in identifying potential pathogenic, beneficial, and antibiotic-resistant bacteria. We validate our results using results that were previously published

Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

<p>Abstract</p> <p>Background</p> <p>In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link.</p> <p>Results</p> <p><it>Pseudomonas aeruginosa </it>PAO1 has two <it>ampG </it>paralogs, <it>PA4218 </it>(<it>ampP</it>) and <it>PA4393 </it>(<it>ampG</it>). Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates <it>ampP </it>and <it>ampG </it>encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both <it>ampP </it>and <it>ampG </it>are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of <it>ampG </it>affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either <it>ampP </it>or <it>ampG</it>. Both <it>ampG </it>and <it>ampP </it>are the second members of two independent two-gene operons. Analysis of the <it>ampG </it>and <it>ampP </it>operon expression using β-galactosidase transcriptional fusions showed that in PAO1, <it>ampG </it>operon expression is β-lactam and <it>ampR</it>-independent, while <it>ampP </it>operon expression is β-lactam and <it>ampR</it>-dependent. β-lactam-dependent expression of the <it>ampP </it>operon and independent expression of the <it>ampG </it>operon is also dependent upon <it>ampP</it>.</p> <p>Conclusions</p> <p>In <it>P. aeruginosa</it>, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an <it>ampP </it>and <it>ampG </it>dependent pathway, and at high concentrations where although both <it>ampP </it>and <it>ampG </it>play a role, <it>ampG </it>may be of greater importance. Both <it>ampP </it>and <it>ampG </it>are required for maximum induction. Similar to <it>ampC</it>, <it>ampP </it>expression is inducible in an <it>ampR</it>-dependent manner. Importantly, <it>ampP </it>expression is autoregulated and <it>ampP </it>also regulates expression of <it>ampG</it>. Both AmpG and AmpP have topologies consistent with functions in transport. Together, these data suggest that the mechanism of β-lactam resistance of <it>P. aeruginosa </it>is distinct from well characterized systems in Enterobacteriaceae and involves a highly complicated interaction between these putative permeases and known Amp proteins.</p

Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for alpha-Ketoglutarate Utilization

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (sigma(54)). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a beta-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO Delta mifS, PAO Delta mifR and PAO Delta mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO Delta mifS and PAO Delta mifR revealed that these mutants were unable to utilize C-5-dicarboxylate alpha-ketoglutarate (alpha-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except alpha-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in alpha-KG transport. The inability of the mutants to utilize alpha-KG was rescued by expressing PA5530, encoding C-5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, alpha-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing a-KG and regulating its transport and subsequentmetabolism