Indoor swimming pool environments and self-reported irritative and respiratory symptoms among lifeguards.

A web survey was conducted among 870 lifeguards (current and former) to assess the relationship between exposure to indoor swimming pool environments and respiratory health. Associations between respiratory symptoms and asthma with varying lengths of occupational exposure were assessed by multiple logistic regression. Lifeguards exposed more than 500 hours in the previous 12 months experienced more cough (adjustedOR = 2.54, IC95 % = 1.51-4.25), throat (aOR = 2.47, IC95 % = 1.44-4.24) and eye irritation (aOR = 4.34, IC95 % = 2.52-7.50) during this period than non-exposed lifeguards. Upper and lower respiratory symptoms while on duty were related to duration of lifetime exposure (> 500 days vs. ≤ 50 days: Upper aOR = 5.84, IC95 % = 3.60-9.50; Lower aOR = 2.53, IC95 % = 1.58-4.06). Physician-diagnosed asthma was high among lifeguards (23 %). Highly exposed asthmatic lifeguards (> 500 hours) over the previous 12 months had a significantly higher risk (aOR = 3.74, IC95 % = 1.39-10.02) of suffering from asthma attack(s) than non-exposed asthmatic subjects. Exposure to indoor swimming pool environments is related to respiratory symptoms among lifeguards.

Catecholate siderophore esterases Fes, IroD and IroE are required for salmochelins secretion following utilization, but only IroD contributes to virulence of extra-intestinal pathogenic Escherichia coli.

Salmochelins are glucosylated forms of enterobactin (enterochelin) and contribute to the virulence of Salmonella enterica and some extra-intestinal pathogenic Escherichia coli (ExPEC). Fes, IroD and IroE esterases degrade salmochelins and enterobactin to release iron. We investigated the apparently redundant role of these esterases in virulence and in salmochelin production and utilization of the ExPEC strain χ7122. The ΔiroD, ΔfesΔiroD and ΔfesΔiroDΔiroE mutants displayed attenuated virulence phenotypes in an avian systemic infection model. Growth of ΔfesΔiroD and ΔfesΔiroDΔiroE mutants was severely reduced in the presence of conalbumin, and although enterobactin was produced, no salmochelins were detected in the culture supernatants of these mutants. Elimination of catecholate synthesis via an entA deletion in a ΔfesΔiroDΔiroE restored growth in the presence of conalbumin, but only partially restored the virulence of the strain. Salmochelin production was reestablished by reintroducing active esterases. Intracellular accumulation of cyclic mono-glucosylated enterobactin was observed in the triple mutant ΔfesΔiroDΔiroE, and deletion of fepC, required for catecholate import into the cytoplasm, restored salmochelin detection in supernatants. These results suggest that in the absence of esterases, cyclic salmochelins are synthesized and secreted, but remain cell-bound after internalization indicating that esterase-mediated degradation is required for re-secretion of catecholate siderophore molecules following their utilization.

Identification of anti-virulence compounds that disrupt quorum-sensing regulated acute and persistent pathogenicity.

Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS) virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS) and structure-activity relationship (SAR) analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR); inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections.

The stringent response modulates 4-hydroxy-2-alkylquinoline biosynthesis and quorum-sensing hierarchy in Pseudomonas aeruginosa.

As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C4-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.

The small RNA RyhB contributes to siderophore production and virulence of uropathogenic Escherichia coli.

In Escherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a ΔryhB mutant and a Δfur ΔryhB double mutant showed significantly reduced bladder colonization. The Δfur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the ΔryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Biodegradation of endocrine disruptors in solid-liquid two-phase partitioning systems by enrichment cultures.

Naturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such as Sphingomonadales. One Rhodococcus isolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents.

A small RNA promotes siderophore production through transcriptional and metabolic remodeling.

Siderophores are essential factors for iron (Fe) acquisition in bacteria during colonization and infection of eukaryotic hosts, which restrain iron access through iron-binding protein, such as lactoferrin and transferrin. The synthesis of siderophores by Escherichia coli is considered to be fully regulated at the transcriptional level by the Fe-responsive transcriptional repressor Fur. Here we characterized two different pathways that promote the production of the siderophore enterobactin via the action of the small RNA RyhB. First, RyhB is required for normal expression of an important enterobactin biosynthesis polycistron, entCEBAH. Second, RyhB directly represses the translation of cysE, which encodes a serine acetyltransferase that uses serine as a substrate for cysteine biosynthesis. Reduction of CysE activity by RyhB allows serine to be used as building blocks for enterobactin synthesis through the nonribosomal peptide synthesis pathway. Thus, RyhB plays an essential role in siderophore production and may modulate bacterial virulence through optimization of siderophore production.

Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain χ7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (ΔentDΔiuc), ΔentS and ΔentSΔiroC export mutants were attenuated in tissues and blood, whereas the ΔiroC export mutant was only attenuated in blood. Interestingly, the ΔentD mutant, producing only aerobactin, retained full virulence, and loss of entD in the ΔentSΔiroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (ΔentD and ΔiroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.

The Acheta domesticus densovirus, isolated from the European house cricket, has evolved an expression strategy unique among parvoviruses.

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses.

Homeostatic interplay between bacterial cell-cell signaling and iron in virulence.

Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.

A liquid chromatography - mass spectrometry method to measure ¹³C-isotope enrichment for DNA stable-isotope probing.

DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of ¹³C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the ¹³C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [¹³C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C8 column in isocratic elution mode and the detection and quantification of ¹³C-labeled thymine by QMS. To mimic a DNA-SIP assay, a DNA mixture was made using ¹³C-labeled E. coli DNA with DNA extracted from five bacterial species. The HPLC-MS method was able to measure the correct proportion of ¹³C-DNA in the mix. This method can then be used as an alternative to the use of isotope ratio mass spectrometry in DNA-SIP assays.

Red death in Caenorhabditis elegans caused by Pseudomonas aeruginosa PAO1.

During host injury, Pseudomonas aeruginosa can be cued to express a lethal phenotype within the intestinal tract reservoir-a hostile, nutrient scarce environment depleted of inorganic phosphate. Here we determined if phosphate depletion activates a lethal phenotype in P. aeruginosa during intestinal colonization. To test this, we allowed Caenorhabditis elegans to feed on lawns of P. aeruginosa PAO1 grown on high and low phosphate media. Phosphate depletion caused PAO1 to kill 60% of nematodes whereas no worms died on high phosphate media. Unexpectedly, intense redness was observed in digestive tubes of worms before death. Using a combination of transcriptome analyses, mutants, and reporter constructs, we identified 3 global virulence systems that were involved in the "red death" response of P. aeruginosa during phosphate depletion; they included phosphate signaling (PhoB), the MvfR-PQS pathway of quorum sensing, and the pyoverdin iron acquisition system. Activation of all 3 systems was required to form a red colored PQS+Fe(3+) complex which conferred a lethal phenotype in this model. When pyoverdin production was inhibited in P. aeruginosa by providing excess iron, red death was attenuated in C. elegans and mortality was decreased in mice intestinally inoculated with P. aeruginosa. Introduction of the red colored PQS+Fe(3+) complex into the digestive tube of C. elegans or mouse intestine caused mortality associated with epithelial disruption and apoptosis. In summary, red death in C. elegans reveals a triangulated response between PhoB, MvfR-PQS, and pyoverdin in response to phosphate depletion that activates a lethal phenotype in P. aeruginosa.

Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.

Klebsiella pneumoniae is a pathogen of increasing concern because of multidrug resistance, especially due to K. pneumoniae carbapenemases (KPCs). K. pneumoniae must acquire iron to replicate, and it utilizes iron-scavenging siderophores, such as enterobactin (Ent). The innate immune protein lipocalin 2 (Lcn2) is able to specifically bind Ent and disrupt iron acquisition. To determine whether K. pneumoniae must produce Lcn2-resistant siderophores to cause disease, we examined siderophore production by clinical isolates (n = 129) from respiratory, urine, blood, and stool samples and by defined siderophore mutants through genotyping and liquid chromatography-mass spectrometry. Three categories of K. pneumoniae isolates were identified: enterobactin positive (Ent(+)) (81%), enterobactin and yersiniabactin positive (Ent(+) Ybt(+)) (17%), and enterobactin and salmochelin (glycosylated Ent) positive (Ent(+) gly-Ent(+)) with or without Ybt (2%). Ent(+) Ybt(+) strains were significantly overrepresented among respiratory tract isolates (P = 0.0068) and ß-lactam-resistant isolates (P = 0.0019), including the epidemic KPC-producing clone multilocus sequence type 258 (ST258). In ex vivo growth assays, gly-Ent but not Ybt allowed evasion of Lcn2 in human serum, whereas siderophores were dispensable for growth in human urine. In a murine pneumonia model, an Ent(+) strain was an opportunistic pathogen that was completely inhibited by Lcn2 but caused severe, disseminated disease in Lcn2(-/-) mice. In contrast, an Ent(+) Ybt(+) strain was a frank respiratory pathogen, causing pneumonia despite Lcn2. However, Lcn2 retained partial protection against disseminated disease. In summary, Ybt is a virulence factor that is prevalent among KPC-producing K. pneumoniae isolates and promotes respiratory tract infections through evasion of Lcn2.

Quantitative analysis of the relative transcript levels of four chlorophenol reductive dehalogenase genes in Desulfitobacterium hafniense PCP-1 exposed to chlorophenols.

Relative to those of unexposed cultures, the transcript levels of the four CprA-type reductive dehalogenase genes (cprA2, cprA3, cprA4, and cprA5) in Desulfitobacterium hafniense PCP-1 were measured in cultures exposed to chlorophenols. In 2,4,6-trichlorophenol-amended cultures, cprA2 and cprA3 were upregulated, as was cprA5, but concomitantly with the appearance of 2,4-dichlorophenol (DCP). In 3,5-DCP-amended cultures, only cprA5 was upregulated. In pentachlorophenol-amended cultures grown for 12 h, cprA2 and cprA3 were upregulated but not cprA5. cprA4 was not upregulated significantly in cultures containing any tested chlorophenols.

Isolation of estrogen-degrading bacteria from an activated sludge bioreactor treating swine waste, including a strain that converts estrone to ß-estradiol.

An estrogen-degrading bacterial consortium from a swine wastewater biotreatment was enriched in the presence of low concentrations (1 mg/L) of estrone (E1), 17ß-estradiol (ßE2), and equol (EQO) as sole carbon sources. The consortium removed 99% ± 1% of these three estrogens in 48 h. Estrogen removal occurred even in the presence of an ammonia monooxygenase inhibitor, suggesting that nitrifiers are not involved. Five strains showing estrogen-metabolizing activity were isolated from the consortium on mineral agar medium with estrogens as sole carbon source. They are related to four genera ( Methylobacterium (strain MI6.1R), Ochrobactrum (strains MI6.1B and MI9.3), Pseudomonas (strain MI14.1), and Mycobacterium (strain MI21.2)) distributed among three classes (Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria). Depending on the culture medium, strains MI6.1B, MI9.3, MI14.1, and MI21.2 partially transform ßE2 into E1, whereas Methylobacterium sp. strain MI6.1R reduces E1 into ßE2 under aerobic conditions, in contrast with the usually observed conversion of ßE2 into E1. Since ßE2 is a more potent endocrine disruptor than E1, it means that the presence of Methylobacterium sp. strain MI6.1R (or other bacteria with the same E1-reducing activity) in a treatment could transiently increase the estrogenicity of the effluent. MI6.1R can also reduce the ketone group of 16-ketoestradiol, a hydroxylated analog of E1. All ßE2 and E1 transformation activities were constitutive, and many of them are favoured in a rich medium than a medium containing no other carbon source. None of the isolated strains could degrade EQO.

HHQ and PQS, two Pseudomonas aeruginosa quorum-sensing molecules, down-regulate the innate immune responses through the nuclear factor-kappaB pathway.

To explore whether bacterial secreted 4-hydroxy-2-alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild-type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor-alpha and interleukin-6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild-type did not. The addition of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild-type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor-kappaB (NF-kappaB) binding to its binding sites and the expression of NF-kappaB target genes, and PQS delayed inhibitor of kappaB degradation, indicating that the effect of HHQ and PQS was mediated through the NF-kappaB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

Identification and characterization of a novel CprA reductive dehalogenase specific to highly chlorinated phenols from Desulfitobacterium hafniense strain PCP-1.

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K(m) value for PCP was 46.7 µM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.

Global gene expression analysis on the target genes of PQS and HHQ in J774A.1 monocyte/macrophage cells.

We have previously shown that PQS and HHQ, two quorum sensing molecules, can down-regulate host the innate immune responses and that this is mediated through the NF-kappaB pathway. In this study, to search for a comprehensive set of genes regulated by these quorum sensing molecules, we performed a global gene expression analysis using DNA microarray in J774A.1 monocyte/macrophage cells line. The expression of these genes was confirmed by RT-PCR. We found that PQS and HHQ down-regulated the expression of genes involved in immune responses and transcription as well as other functions, some of which are downstream of NF-kappaB pathway consistent with our previous results. PQS and HHQ inhibited LPS-induced morphological change and nitric oxide production, suggesting that they inhibit macrophage activation. However, PQS and HHQ did not affect apoptosis, suggesting that their effects on immune system are not from general alteration of cell function. This study provides insight how the quorum sensing molecules influence host cells.