Secreted Phosphatase and Deoxyribonuclease Are Required by Pseudomonas aeruginosa To Defend against Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are produced by neutrophils as an innate immune defense mechanism to trap and kill microbial pathogens. NETs are comprised of ejected chromatin that forms a lattice structure enmeshed with numerous antimicrobial proteins. In addition to forming the structural backbone of NETs, extracellular DNA (eDNA) has membrane-disrupting antimicrobial activity that contributes to NET killing. Many pathogens produce secreted extracellular DNases to evade the antimicrobial activity of NETs. Pseudomonas aeruginosa encodes an operon of two secreted enzymes, a predicted alkaline phosphatase and a DNase. The DNase (eddB) degrades eDNA to use as a nutrient source. Here we report that both eDNA and NETs are potent inducers of this DNase-phosphatase operon. Furthermore, the secreted DNase contributes to degrading NET DNA and defends P. aeruginosa against NET-mediated killing. We demonstrate that EddA has both alkaline phosphatase and phosphodiesterase (PDase) activities and also protects against the antimicrobial activity of NETs. Although the phosphatase does not cause DNA degradation similar to that of the DNase, its protective function is likely a result of removing the cation-chelating phosphates from the eDNA phosphodiester backbone. Therefore, both the DNase and PDase contribute to defense against NET killing of P. aeruginosa, highlighting the role of DNA-manipulating enzymes in targeting the eDNA in neutrophil extracellular traps.

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

Hyperbiofilm phenotype of Pseudomonas aeruginosa defective for the PlcB and PlcN secreted phospholipases.

Biofilms are dense communities of bacteria enmeshed in a protective extracellular matrix composed mainly of exopolysaccharides, extracellular DNA, proteins, and outer membrane vesicles (OMVs). Given the role of biofilms in antibiotic-tolerant and chronic infections, novel strategies are needed to block, disperse, or degrade biofilms. Enzymes that degrade the biofilm matrix are a promising new therapy. We screened mutants in many of the enzymes secreted by the type II secretion system (T2SS) and determined that the T2SS, and specifically phospholipases, play a role in biofilm formation. Mutations in the xcp secretion system and in the plcB and plcN phospholipases all resulted in hyperbiofilm phenotypes. PlcB has activity against many phospholipids, including the common bacterial membrane lipid phosphatidylethanolamine, and may degrade cell membrane debris or OMVs in the biofilm matrix. Exogenous phospholipase was shown to reduce aggregation and biofilm formation, suggesting its potential role as a novel enzymatic treatment to dissolve biofilms.

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa.

Biofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2- to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

Extracellular DNA-induced antimicrobial peptide resistance in Salmonella enterica serovar Typhimurium.

BACKGROUND: The Salmonella enterica serovar Typhimurium PhoPQ two component system (TCS) is activated by low Mg2+ levels, low pH and by antimicrobial peptides (AP). Under Mg2+ limitation, the PhoPQ system induces pmrD expression, which post-translationally activates the PmrAB TCS. PhoPQ and PmrAB control many genes required for intracellular survival and pathogenesis. These include the polymyxin resistance (pmr) operon, which is required for aminoarabinose modification of LPS and protecting the outer membrane from antimicrobial peptide disruption and killing. Extracellular DNA is a ubiquitous polymer in the matrix of biofilms and accumulates in some infection sites. Extracellular DNA chelates cations and thus activates the Pseudomonas aeruginosa PhoPQ/PmrAB systems, leading to expression of the orthologous arn (pmr) operon. RESULTS: Here we show that extracellular DNA induces expression of the S. Typhimurium pmr antimicrobial peptide resistance operon in a PhoPQ and PmrAB-dependent manner. Induction of the pmr genes by DNA was blocked when present with excess Mg2+. Exogenous DNA led to increased resistance of planktonic cultures to aminoglycosides, antimicrobial peptides (AP) and ciprofloxacin, but only AP resistance was PhoPQ/PmrAB-dependent. Extracellular DNA was shown to be a matrix component of S. Typhimurium biofilms cultivated in flow chambers and on glass surfaces. A pmrH-gfp fusion was highly expressed in flow chamber biofilms cultivated in medium with repressing levels of 10 mM Mg2+ and co-localized with eDNA. Expression of pmrH-lux was monitored in plastic peg biofilms and shown to require PhoPQ and PmrAB. Biofilms had higher levels of pmrH expression compared to planktonic cultures. We propose that DNA accumulation in biofilms contributes to the increased pmrH-lux expression in biofilms. CONCLUSIONS: The Salmonella PhoPQ/PmrAB systems and antimicrobial peptide resistance are activated by the cation chelating properties of extracellular DNA. DNA-induced AP resistance may allow immune evasion and increased survival of S. Typhimurium biofilms formed during extracellular growth stages of an infection or outside the host.

Pseudomonas aeruginosa produces an extracellular deoxyribonuclease that is required for utilization of DNA as a nutrient source.

Pseudomonas aeruginosa is an opportunistic pathogen that occupies a wide variety of environmental niches. Extracellular DNA is ubiquitous in various environments and is a rich source of carbon, nitrogen and phosphate. Here we show that P. aeruginosa is capable of using DNA as a nutrient source. Under phosphate-limiting conditions, or when DNA is supplied as a source of phosphate, expression of PA3909 is induced. PA3909 encodes an extracellular deoxyribonuclease (DNase), which is required for degradation of DNA and utilization of DNA as a source of carbon, nitrogen and phosphate. Stabilization of PA3909 by the addition of excess Mg(2+) and Ca(2+) was required for DNase activity in culture supernatants. Extracellular DNase activity was seen in multiple P. aeruginosa strains and isolates from cystic fibrosis patients. The primary Xcp type II secretion system but not the Hxc type II secretion system is required for DNase activity and the ability to use DNA as a source of nutrients. This study identifies an extracellular DNase produced by P. aeruginosa that enables degradation of extracellular DNA into an accessible source of carbon, nitrogen and phosphate. DNase production by P. aeruginosa also has important implications for virulence and biofilm formation.

Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms.

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552-PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on beta-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation.

Extracellular DNA controls expression of Pseudomonas aeruginosa genes involved in nutrient utilization, metal homeostasis, acid pH tolerance and virulence.

Introduction. Pseudomonas aeruginosa grows in extracellular DNA (eDNA)-enriched biofilms and infection sites. eDNA is generally considered to be a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify growth media and serve as a nutrient source.Aim. We wanted to determine the genome-wide influence on the transcriptome of planktonic P. aeruginosa PAO1 grown in the presence of eDNA.Methodology. RNA-seq analysis was performed to determine the genome-wide effects on gene expression of PAO1 grown with eDNA. Transcriptional lux fusions were used to confirm eDNA regulation and to validate phenotypes associated with growth in eDNA.Results. The transcriptome of eDNA-regulated genes included 89 induced and 76 repressed genes (FDR<0.05). A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and eDNA/acidic pH promoted an acid tolerance response in P. aeruginosa. The cyoABCDE terminal oxidase is induced by both eDNA and pH 5.5, and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence in the acute infection model of fruit flies.Conclusion. The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance and nutrient utilization; phenotypes that contribute to long-term survival.

Feeding behaviour of Caenorhabditis elegans is an indicator of Pseudomonas aeruginosa PAO1 virulence.

Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secretion (T3S) system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced virulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.

Identification of bacterial contaminants in sinus irrigation bottles from chronic rhinosinusitis patients.

OBJECTIVE: To determine if sinus irrigation bottles from patients with chronic rhinosinusitis (CRS) harbour bacterial contaminants. DESIGN: Patients with symptoms of CRS who showed no mucopurulent infection and had no history of surgery were enrolled in the study. Patients were instructed on the proper use and cleaning of sinus irrigation bottles and were asked to return their rinse bottle during follow-up visits. METHODS: Bacterial contaminants were cultured from the inner surface of the sinus irrigation bottles obtained from patients. Genomic deoxyribonucleic acid (DNA) was isolated from purified colonies and used to polymerase chain reaction (PCR) amplify the 16S ribosomal ribonucleic acid (rRNA) genes. PCR products were sequenced and analyzed in the Human Oral Microbiome Database (HOMD) for genus and species identification based on 16S ribosomal DNA (rDNA) sequence comparisons. MAIN OUTCOME MEASURES: The outcomes included the recovery of bacterial contaminants and their subsequent identification. RESULTS: In total, 142 bacterial isolates were cultured and identified. The organisms included known oral flora bacteria, as well as pathogens of the upper respiratory tract and sinuses. Thirty-two different bacterial species were identified from 11 patients. There was no correlation between the length of bottle use and the degree of contamination. CONCLUSION: This study highlights the risk of bacterial contamination of sinus irrigation bottles and the potential for patient reinoculation.