Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease.

Anaerobic bacteria are responsible for half of all pulmonary infections. One such pathogen is Streptococcus pneumoniae (Spn), a leading cause of community-acquired pneumonia, bacteremia/sepsis, and meningitis. Using a panel of isogenic mutants deficient in lactate, acetyl-CoA, and ethanol fermentation, as well as pharmacological inhibition, we observed that NAD(H) redox balance during fermentation was vital for Spn energy generation, capsule production, and in vivo fitness. Redox balance disruption in fermentation pathway-specific fashion substantially enhanced susceptibility to killing in antimicrobial class-specific manner. Blocking of alcohol dehydrogenase activity with 4-methylpyrazole (fomepizole), an FDA-approved drug used as an antidote for toxic alcohol ingestion, enhanced susceptibility of multidrug-resistant Spn to erythromycin and reduced bacterial burden in the lungs of mice with pneumonia and prevented the development of invasive disease. Our results indicate fermentation enzymes are de novo targets for antibiotic development and a novel strategy to combat multidrug-resistant pathogens.

An in vivo atlas of host-pathogen transcriptomes during Streptococcus pneumoniae colonization and disease.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

Pseudomonas aeruginosa Secretes the Oxylipin Autoinducer Synthases OdsA and OdsB via the Xcp Type 2 Secretion System.

The oxylipin-dependent quorum-sensing system (ODS) of Pseudomonas aeruginosa relies on the production and sensing of two extracellular oxylipins, 10S-hydroxy-(8E)-octadecenoic acid (10-HOME) and 7S,10S-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Here, we implemented a genetic screen of P. aeruginosa strain PAO1 aimed to identify genes required for 10-HOME and 7,10-DiHOME production. Among the 14 genes identified, four encoded previously known components of the ODS and 10 encoded parts of the Xcp type II secretion system (T2SS). We subsequently created a clean xcpQ deletion mutant, which encodes the necessary outer membrane component of Xcp, and found it recapitulated the impaired functionality of the T2SS transposon mutants. Further studies showed that the ΔxcpQ mutant was unable to secrete the oxylipin synthase enzymes across the outer membrane. Specifically, immunoblotting for OdsA, which is responsible for the generation of 10-HOME from oleic acid, detected the enzyme in supernatants from wild-type PAO1 but not ΔxcpQ cultures. Likewise, chromatography of supernatants found that 10-HOME was not in supernatants collected from the ΔxcpQ mutant. Accordingly, diol synthase activity was increased in the periplasm of ΔxcpQ mutant consistent with a stoppage in its transport. Importantly, after exposure of the ΔxcpQ mutant to exogenous 10-HOME and 7,10-DiHOME, the ODS effector genes become active; thus, the sensing component of the ODS does not involve the T2SS. Finally, we observed that Xcp contributed to robust in vitro and in vivo biofilm formation in oleic acid availability- and ODS-dependent manner. Thus, T2SS-mediated transport of the oxylipin synthase enzymes to outside the bacterial cell is required for ODS functionality. IMPORTANCE We previously showed that the ODS of P. aeruginosa produces and responds to oxylipins derived from host oleic acid by enhancing biofilm formation and virulence. Here, we developed a genetic screen strategy to explore the molecular basis for oxylipins synthesis and detection. Unexpectedly, we found that the ODS autoinducer synthases cross the outer membrane using the Xcp type 2 secretion system (T2SS) of P. aeruginosa, and so the biosynthesis of oxylipins occurs extracellularly. T2SS promoted biofilm formation in the presence of oleic acid as a result of ODS activation. Our results identify two new T2SS secreted proteins in P. aeruginosa and reveal a new way by which this important opportunistic pathogen interacts with the host environment.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase.

The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU.

Engineered Superinfective Pf Phage Prevents Dissemination of Pseudomonas aeruginosa in a Mouse Burn Model.

Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa. Under stress conditions, mutations occurring in the Pf genome result in the emergence of superinfective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore, SI-Pf can even infect Pf-lysogenized P. aeruginosa. Here, we identified specific mutations located between the repressor and the excisionase genes of Pf4 phage in the P. aeruginosa PAO1 strain that resulted in the emergence of SI-Pf. Based on these findings, we genetically engineered an SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening burn wound infections caused by PAO1. In validation experiments, eSI-Pf was able to infect PAO1 grown in a lawn as well as biofilms formed in vitro on polystyrene. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 h. Despite not lowering bacterial burden in burned skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We subsequently determined that eSI-Pf induced a small-colony variant of P. aeruginosa that was unable to disseminate systemically. This attenuated phenotype was due to profound changes in virulence determinant production and altered physiology. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial-resistant P. aeruginosa infections of burn wounds. IMPORTANCE Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug-resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages can present a set of drawbacks during phage therapy, including the induction of bacterial resistance and limited bacteria-phage interactions in vivo. Here, we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., by using an engineered superinfective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.

Influenza A virus modulation of Streptococcus pneumoniae infection using ex vivo transcriptomics in a human primary lung epithelial cell model reveals differential host glycoconjugate uptake and metabolism.

Background: Streptococcus pneumoniae (Spn) is typically an asymptomatic colonizer of the nasopharynx but it also causes pneumonia and disseminated disease affecting various host anatomical sites. Transition from colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as result of influenza A virus coinfection. Methods: We investigated the pneumococcal (serotype 19F, strain EF3030) and host transcriptomes with and without influenza A virus (A/California/07 2009 pH1N1) infection at this transition. This was done using primary, differentiated Human Bronchial Epithelial Cells (nHBEC) in a transwell monolayer model at an Air-Liquid Interface (ALI), with multispecies deep RNA-seq. Results: Distinct pneumococcal gene expression profiles were observed in the presence and absence of influenza. Influenza coinfection allowed for significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways; in totality suggesting a fundamentally altered bacterial metabolic state and greater nutrient availability when coinfecting with influenza. Surprisingly, nHBEC transcriptomes were only modestly perturbed by infection with EF3030 alone in comparison to that resulting from Influenza A infection or coinfection, which had drastic alterations in thousands of genes. Influenza infected host transcriptomes suggest significant loss of ciliary function in host nHBEC cells. Conclusions: Influenza A virus infection of nHBEC promotes pneumococcal infection. One reason for this is an altered metabolic state by the bacterium, presumably due to host components made available as result of viral infection. Influenza infection had a far greater impact on the host response than did bacterial infection alone, and this included down regulation of genes involved in expressing cilia. We conclude that influenza infection promotes a pneumococcal metabolic shift allowing for transition from colonization to disseminated disease.

Biochemical characterization of the oxygenation of unsaturated fatty acids by the dioxygenase and hydroperoxide isomerase of Pseudomonas aeruginosa 42A2.

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of approximately 50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-(2)H]18:1n-9, [7R-(2)H]18:2n-6, and [8R-(2)H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.

Capsule Promotes Intracellular Survival and Vascular Endothelial Cell Translocation during Invasive Pneumococcal Disease.

The polysaccharide capsule that surrounds Streptococcus pneumoniae (Spn) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H2O2-mediated killing in vitro. Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H2O2-mediated killing in vitro, with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus. We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype's potential for invasive disease. IMPORTANCE Streptococcus pneumoniae (Spn) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn's capsule-conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo, and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Streptococcus pneumoniae Binds to Host Lactate Dehydrogenase via PspA and PspC To Enhance Virulence.

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC, also called CbpA) are major virulence factors of Streptococcus pneumoniae (Spn). These surface-exposed choline-binding proteins (CBPs) function independently to inhibit opsonization, neutralize antimicrobial factors, or serve as adhesins. PspA and PspC both carry a proline-rich domain (PRD) whose role, other than serving as a flexible connector between the N-terminal and C-terminal domains, was up to this point unknown. Herein, we demonstrate that PspA binds to lactate dehydrogenase (LDH) released from dying host cells during infection. Using recombinant versions of PspA and isogenic mutants lacking PspA or specific domains of PspA, this property was mapped to a conserved 22-amino-acid nonproline block (NPB) found within the PRD of most PspAs and PspCs. The NPB of PspA had specific affinity for LDH-A, which converts pyruvate to lactate. In a mouse model of pneumonia, preincubation of Spn carrying NPB-bearing PspA with LDH-A resulted in increased bacterial titers in the lungs. In contrast, incubation of Spn carrying a version of PspA lacking the NPB with LDH-A or incubation of wild-type Spn with enzymatically inactive LDH-A did not enhance virulence. Preincubation of NPB-bearing Spn with lactate alone enhanced virulence in a pneumonia model, indicating exogenous lactate production by Spn-bound LDH-A had an important role in pneumococcal pathogenesis. Our observations show that lung LDH, released during the infection, is an important binding target for Spn via PspA/PspC and that pneumococci utilize LDH-A derived lactate for their benefit in vivoIMPORTANCEStreptococcus pneumoniae (Spn) is the leading cause of community-acquired pneumonia. PspA and PspC are among its most important virulence factors, and these surface proteins carry the proline-rich domain (PRD), whose role was unknown until now. Herein, we show that a conserved 22-amino-acid nonproline block (NPB) found within most versions of the PRD binds to host-derived lactate dehydrogenase A (LDH-A), a metabolic enzyme which converts pyruvate to lactate. PspA-mediated binding of LDH-A increased Spn titers in the lungs and this required LDH-A enzymatic activity. Enhanced virulence was also observed when Spn was preincubated with lactate, suggesting LDH-A-derived lactate is a vital food source. Our findings define a role for the NPB of the PRD and show that Spn co-opts host enzymes for its benefit. They advance our understanding of pneumococcal pathogenesis and have key implications on the susceptibility of individuals with preexisting airway damage that results in LDH-A release.

VEJ{phi}, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes.

A novel filamentous bacteriophage, designated VEJphi, was isolated from strain MO45 of Vibrio cholerae of the O139 serogroup. A molecular characterization of the phage was carried out, which included sequencing of its whole genome, study of the genomic structure, identification of the phage receptor, and determination of the function of some of the genes, such as those encoding the major capsid protein and the single-stranded DNA-binding protein. The genome nucleotide sequence of VEJphi, which consists of 6842 bp, revealed that it is organized in modules of functionally related genes in an array that is characteristic of the genus Inovirus (filamentous phages). VEJphi is closely related to other previously described filamentous phages of V. cholerae, including VGJphi, VSK and fs1. Like these phages, VEJphi uses as a cellular receptor the type IV fimbria called the mannose-sensitive haemagglutinin (MSHA). It was also demonstrated that VEJphi, like phage VGJphi, is able to transmit the genome of phage CTXphi, and therefore the genes encoding the cholera toxin (CT), horizontally among populations of V. cholerae expressing the MSHA receptor fimbria. This suggests that the variety of phages implicated in the horizontal transmission of the CT genes could be more diverse than formerly thought.

Liquid chromatography/tandem mass spectrometric analysis of 7,10-dihydroxyoctadecenoic acid, its isotopomers, and other 7,10-dihydroxy fatty acids formed by Pseudomonas aeruginosa 42A2.

Pseudomonas aeruginosa is an opportunistic pathogen, which oxidizes oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid (7,10-(OH)(2)-18:1) of biological and industrial interest. Electrospray tandem mass spectrometric (MS/MS) analysis of hydroxylated fatty acids usually generates characteristic fragments containing the carboxylate anion and formed by alpha-cleavage at the oxidized carbon. These fragments indicate the positions of the hydroxyl group. In contrast, liquid chromatography (LC)/MS/MS analysis of 7,10-(OH)(2)-18:1 yielded a series of other ions with structural information. To study the fragmentation mechanism, we prepared (2)H- and (18)O-labeled isotopomers. We also performed MS(3) analysis of the major ions, and for comparison we generated the corresponding 7,10-dihydroxy metabolites of 16:1n-7, 18:2n-6, and 20:1n-11 with a protein extract of P. aeruginosa. The MS/MS spectra of 7,10-(OH)(2)-18:1 and its isotopomers, 7,10-(OH)(2)-16:1, and 7,10-(OH)(2)-20:1, contained a series of prominent fragments that all hold the omega end. The 8,9-double bond was not essential for this fragmentation, as 7,10-(OH)(2)-18:0, and its isotopomers, formed essentially the same fragments in the lower mass range. In contrast, 7,10-dihydroxy-8(E),12(Z)-octadecadienoic acid (7,10-(OH)(2)-18:2) fragmented by alpha-cleavage at the oxidized carbons with formation of carboxylate anions. Our results demonstrate that C(16)-C(20) fatty acids with a 7,10-dihydroxy-8(E) functionality undergo charge-driven fragmentation after charge migration to the omega-end, whereas the main ions of 7,10-(HO)(2)-18:2 retain charge at the carboxyl group.

Oxylipins mediate cell-to-cell communication in Pseudomonas aeruginosa.

Oxygenated unsaturated fatty acids, known as oxylipins, are signaling molecules commonly used for cell-to-cell communication in eukaryotes. However, a role for oxylipins in mediating communication in prokaryotes has not previously been described. Bacteria mainly communicate via quorum sensing, which involves the production and detection of diverse small molecules termed autoinducers. Here we show that oleic acid-derived oxylipins produced by Pseudomonas aeruginosa function as autoinducers of a novel quorum sensing system. We found that this system controls the cell density-dependent expression of a gene subset independently of the quorum sensing systems thus far described in this bacterium. We identified a LysR-type transcriptional regulator as the primary receptor of the oxylipin signal. The discovery of this oxylipin-dependent quorum sensing system reveals that prokaryote-derived oxylipins also mediate cell-to-cell communication in bacteria.

Pf Filamentous Phage Requires UvrD for Replication in Pseudomonas aeruginosa.

Pf is a lysogenic filamentous phage that promotes biofilm development in Pseudomonas aeruginosa. Pf replicates by a rolling circle replication system which depends on a phage-encoded initiator protein and host factors usually involved in chromosome replication. Rep, an accessory replicative DNA helicase, is crucial for replication of filamentous phages in Escherichia coli. In contrast, here we show that, instead of depending on Rep, Pf replication depends on UvrD, an accessory helicase implicated in DNA repair. In this study, we also identified the initiator protein of Pf and found that it shares similarities with that of Vibrio phages CTXφ and VGJφ, which also depend on UvrD for replication. A structural comparative analysis of the initiator proteins of most known filamentous phages described thus far suggested that UvrD, known as a nonreplicative helicase, is involved in rolling circle replication of filamentous phages in diverse bacteria genera. This report consolidates knowledge on the new role of UvrD in filamentous phage replication, a function previously thought to be exclusive of Rep helicase. IMPORTANCE Biofilm development is a key component of the ability of Pseudomonas aeruginosa to evade host immune defenses and resist multiple drugs. Induction of the filamentous phage Pf, which usually is lysogenized in clinical and environmental isolates of P. aeruginosa, plays an important role in biofilm assembly, maturation, and dispersal. Despite the clinical relevance of Pf, the molecular biology of this phage is largely unknown. In this study, we found that rolling circle replication of Pf depends on UvrD, a DNA helicase normally involved in DNA repair. We also identified the initiator protein of Pf and found that it shares structural similarity with that of Vibrio cholerae phages CTXφ and VGJφ, which also use UvrD for replication. Our results reveal that, in addition to DNA repair, UvrD plays an essential role in rolling circle replication of filamentous phages among diverse bacteria genera, adding a new, previously unrecognized function of this accessory helicase.

Oxylipins produced by Pseudomonas aeruginosa promote biofilm formation and virulence.

The oxygenation of unsaturated fatty acids by dioxygenases occurs in all kingdoms of life and produces physiologically important lipids called oxylipins. The biological roles of oxylipins have been extensively studied in animals, plants, algae and fungi, but remain largely unidentified in prokaryotes. The bacterium Pseudomonas aeruginosa displays a diol synthase activity that transforms several monounsaturated fatty acids into mono- and di-hydroxylated derivatives. Here we show that oxylipins derived from this activity inhibit flagellum-driven motility and upregulate type IV pilus-dependent twitching motility of P. aeruginosa. Consequently, these oxylipins promote bacterial organization in microcolonies, increasing the ability of P. aeruginosa to form biofilms in vitro and in vivo (in Drosophila flies). We also demonstrate that oxylipins produced by P. aeruginosa promote virulence in Drosophila flies and lettuce. Our study thus uncovers a role for prokaryotic oxylipins in the physiology and pathogenicity of bacteria.

CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications.

For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.

Integrative mobile elements exploiting Xer recombination.

Integrative mobile genetic elements directly participate in the rapid response of bacteria to environmental challenges. They generally encode their own dedicated recombination machineries. CTXφ, a filamentous bacteriophage that harbors the genes encoding cholera toxin in Vibrio cholerae provided the first notable exception to this rule: it hijacks XerC and XerD, two chromosome-encoded tyrosine recombinases for lysogenic conversion. XerC and XerD are highly conserved in bacteria because of their role in the topological maintenance of circular chromosomes and, with the advent of high throughput sequencing, numerous other integrative mobile elements exploiting them have been discovered. Here, we review our understanding of the molecular mechanisms of integration of the different integrative mobile elements exploiting Xer (IMEXs) so far described.

Functional characterization of ExFadLO, an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa.

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.

Role of MMPs in orchestrating inflammatory response in human monocytes via a TREM-1-PI3K-NF-κB pathway.

The MMPs constitute a family of endopeptidases that can cleavage extracellular proteins. They are involved in a number of events; some of these include inflammatory processes. One of its targets is the TREM-1, which has emerged as an important modulator of innate immune responses in mammals. This transmembrane glycoprotein possesses an Ig-like ectodomain readily shed by MMPs to generate sTREM-1. Whereas membrane-anchored TREM-1 amplifies inflammatory responses, sTREM-1 exhibits anti-inflammatory properties. Here we show that sustained cell surface expression of TREM-1 in human monocytes, through metalloproteinase inhibition, counteracts the well-characterized down-regulation of several proinflammatory cytokines during the ET time-frame, also known as M2 or alternative activation. In addition to the cytokines profile, other features of the ET phenotype were underdeveloped when TREM-1 was stabilized at the cell surface. These events were mediated by the signal transducers PI3Ks and Syk. We also show that sTREM-1 counteracts the proinflammatory response obtained by membrane TREM-1 stabilization but failed to induce ET on naïve human monocytes. As the sustained TREM-1 expression at the cell surface suffices to block the progress of a refractory state in human monocytes, our data indicate that TREM-1 and MMPs orchestrate an "adaptive" form of innate immunity by modulating the human monocytes response to endotoxin.

Translocated LPS might cause endotoxin tolerance in circulating monocytes of cystic fibrosis patients.

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes.