Auranofin inhibits virulence pathways in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Repurposing the anticancer drug cisplatin with the aim of developing novel Pseudomonas aeruginosa infection control agents.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

The MapZ-Mediated Methylation of Chemoreceptors Contributes to Pathogenicity of Pseudomonas aeruginosa.

The pathogenic bacterium Pseudomonas aeruginosa is notorious for causing acute and chronic infections in humans. The ability to infect host by P. aeruginosa is dependent on a complex cellular signaling network, which includes a large number of chemosensory signaling pathways that rely on the methyl-accepting chemotaxis proteins (MCPs). We previously found that the second messenger c-di-GMP-binding adaptor MapZ modulates the methylation of an amino acid-detecting MCP by directly interacting with a chemotaxis methyltransferase CheR1. The current study further expands our understanding of the role of MapZ in regulating chemosensory pathways by demonstrating that MapZ suppresses the methylation of multiple MCPs in P. aeruginosa PAO1. The MCPs under the control of MapZ include five MCPs (Aer, CtpH, CptM, PctA, and PctB) for detecting oxygen/energy, inorganic phosphate, malate and amino acids, and three MCPs (PA1251, PA1608, and PA2867) for detecting unknown chemoattractant or chemorepellent. Chemotaxis assays showed that overexpression of MapZ hampered the taxis of P. aeruginosa toward chemoattractants and scratch-wounded human cells. Mouse infection experiments demonstrated that a dysfunction in MapZ regulation had a profound negative impact on the dissemination of P. aeruginosa and resulted in attenuated bacterial virulence. Together, the results imply that by controlling the methylation of various MCPs via the adaptor protein MapZ, c-di-GMP exerts a profound influence on chemotactic responses and bacterial pathogenesis.

Selective labelling and eradication of antibiotic-tolerant bacterial populations in Pseudomonas aeruginosa biofilms.

Drug resistance and tolerance greatly diminish the therapeutic potential of antibiotics against pathogens. Antibiotic tolerance by bacterial biofilms often leads to persistent infections, but its mechanisms are unclear. Here we use a proteomics approach, pulsed stable isotope labelling with amino acids (pulsed-SILAC), to quantify newly expressed proteins in colistin-tolerant subpopulations of Pseudomonas aeruginosa biofilms (colistin is a 'last-resort' antibiotic against multidrug-resistant Gram-negative pathogens). Migration is essential for the formation of colistin-tolerant biofilm subpopulations, with colistin-tolerant cells using type IV pili to migrate onto the top of the colistin-killed biofilm. The colistin-tolerant cells employ quorum sensing (QS) to initiate the formation of new colistin-tolerant subpopulations, highlighting multicellular behaviour in antibiotic tolerance development. The macrolide erythromycin, which has been previously shown to inhibit the motility and QS of P. aeruginosa, boosts biofilm eradication by colistin. Our work provides insights on the mechanisms underlying the formation of antibiotic-tolerant populations in bacterial biofilms and indicates research avenues for designing more efficient treatments against biofilm-associated infections.

Complete Genome Sequence and Transcriptomic Analysis of the Novel Pathogen Elizabethkingia anophelis in Response to Oxidative Stress.

Elizabethkingia anophelis is an emerging pathogen that can cause life-threatening infections in neonates, severely immunocompromised and postoperative patients. The lack of genomic information on E. anophelis hinders our understanding of its mechanisms of pathogenesis. Here, we report the first complete genome sequence of E. anophelis NUHP1 and assess its response to oxidative stress. Elizabethkingia anophelis NUHP1 has a circular genome of 4,369,828 base pairs and 4,141 predicted coding sequences. Sequence analysis indicates that E. anophelis has well-developed systems for scavenging iron and stress response. Many putative virulence factors and antibiotic resistance genes were identified, underscoring potential host-pathogen interactions and antibiotic resistance. RNA-sequencing-based transcriptome profiling indicates that expressions of genes involved in synthesis of an yersiniabactin-like iron siderophore and heme utilization are highly induced as a protective mechanism toward oxidative stress caused by hydrogen peroxide treatment. Chrome azurol sulfonate assay verified that siderophore production of E. anophelis is increased in the presence of oxidative stress. We further showed that hemoglobin facilitates the growth, hydrogen peroxide tolerance, cell attachment, and biofilm formation of E. anophelis NUHP1. Our study suggests that siderophore production and heme uptake pathways might play essential roles in stress response and virulence of the emerging pathogen E. anophelis.