Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli.

The RNA-binding protein RapZ cooperates with small RNAs (sRNAs) GlmY and GlmZ to regulate the glmS mRNA in Escherichia coli. Enzyme GlmS synthesizes glucosamine-6-phosphate (GlcN6P), initiating cell envelope biosynthesis. GlmZ activates glmS expression by base-pairing. When GlcN6P is ample, GlmZ is bound by RapZ and degraded through ribonuclease recruitment. Upon GlcN6P depletion, the decoy sRNA GlmY accumulates through a previously unknown mechanism and sequesters RapZ, suppressing GlmZ decay. This circuit ensures GlcN6P homeostasis and thereby envelope integrity. In this work, we identify RapZ as GlcN6P receptor. GlcN6P-free RapZ stimulates phosphorylation of the two-component system QseE/QseF by interaction, which in turn activates glmY expression. Elevated GlmY levels sequester RapZ into stable complexes, which prevents GlmZ decay, promoting glmS expression. Binding of GlmY also prevents RapZ from activating QseE/QseF, generating a negative feedback loop limiting the response. When GlcN6P is replenished, GlmY is released from RapZ and rapidly degraded. We reveal a multifunctional sRNA-binding protein that dynamically engages into higher-order complexes for metabolite signaling.

Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

A signaling complex of adenylate cyclase CyaC of Sinorhizobium meliloti with cAMP and the transcriptional regulators Clr and CycR.

BACKGROUND: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. RESULTS: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. CONCLUSION: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti.

Identification of Gip as a novel phage-encoded gyrase inhibitor protein of Corynebacterium glutamicum.

By targeting key regulatory hubs of their host, bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. Here, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum resulted in the identification of the gyrase-inhibiting protein Cg1978, termed Gip. Pull-down assays and surface plasmon resonance revealed a direct interaction of Gip with the gyrase subunit A (GyrA). The inhibitory activity of Gip was shown to be specific to the DNA gyrase of its bacterial host C. glutamicum. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. Furthermore, reporter assays revealed an RecA-independent induction of the cryptic CGP3 prophage, most likely caused by topological alterations. Overexpression of gip was counteracted by an increased expression of gyrAB and a reduction of topA expression at the same time, reflecting the homeostatic control of DNA topology. We postulate that the prophage-encoded Gip protein plays a role in modulating gyrase activity to enable efficient phage DNA replication. A detailed elucidation of the mechanism of action will provide novel directions for the design of drugs targeting DNA gyrase.

Transcriptional regulation of the Nε -fructoselysine metabolism in Escherichia coli by global and substrate-specific cues.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genes frlABCD remained poorly understood. Here, we used bioinformatics combined with molecular and biochemical analyses and show that fructoselysine metabolism in E. coli is tightly controlled at the transcriptional level. The global regulator CRP (CAP) as well as the alternative sigma factor σ32 (RpoH) contribute to promoter activation at high cAMP-levels and inside warm-blooded hosts, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific repressor. Our study provides profound evidence that the interplay of global and substrate-specific regulation is a perfect adaptation strategy to efficiently utilize unusual substrates within the human gut environment.

Identification of Pseudomonas asiatica subsp. bavariensis str. JM1 as the first Nε -carboxy(m)ethyllysine-degrading soil bacterium.

Thermal food processing leads to the formation of advanced glycation end products (AGE) such as Nε -carboxymethyllysine (CML). Accordingly, these non-canonical amino acids are an important part of the human diet. However, CML is only partially decomposed by our gut microbiota and up to 30% are excreted via faeces and, hence, enter the environment. In frame of this study, we isolated a soil bacterium that can grow on CML as well as its higher homologue Nε -carboxyethyllysine (CEL) as sole source of carbon. Bioinformatic analyses upon whole-genome sequencing revealed a subspecies of Pseudomonas asiatica, which we named 'bavariensis'. We performed a metabolite screening of P. asiatica subsp. bavariensis str. JM1 grown either on CML or CEL and identified N-carboxymethylaminopentanoic acid and N-carboxyethylaminopentanoic acid respectively. We further detected α-aminoadipate as intermediate in the metabolism of CML. These reaction products suggest two routes of degradation: While CEL seems to be predominantly processed from the α-C-atom, decomposition of CML can also be initiated with cleavage of the carboxymethyl group and under the release of acetate. Thus, our study provides novel insights into the metabolism of two important AGEs and how these are processed by environmental bacteria.

The Insect Pathogen Photorhabdus luminescens Protects Plants from Phytopathogenic Fusarium graminearum via Chitin Degradation.

Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of P. luminescens as a plant-growth-promoting and plant-protecting organism in agriculture. IMPORTANCE The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of P. luminescens as a plant-growth-promoting organism and biopesticide.

Two novel XRE-like transcriptional regulators control phenotypic heterogeneity in Photorhabdus luminescens cell populations.

BACKGROUND: The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria's life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. RESULTS: Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. CONCLUSION: Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

Characterization of the pleiotropic LysR-type transcription regulator LeuO of Escherichia coli.

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO. This yielded nine mutants carrying amino acid substitutions in the dimerization interface of the regulatory EBD, as shown by solving the EBD's crystal structure. Superimposing of the crystal structures of LeuO-EBD and LeuO-S120D-EBD suggests that the Ser120 to Asp substitution triggers a structural change that is related to effector-induced structural changes of LTTRs. Corresponding functional analyses demonstrated that LeuO-S120D has a higher DNA-binding affinity than wild-type LeuO. Further, a palindromic DNA-binding core-site and a consensus sequence were identified by DNase I footprinting with LeuO-S120D as well as with the dimeric DBD. The data suggest that LeuO-S120D mimics an effector-induced form of LeuO regulating a distinct set of target loci. In general, constitutive mutants and determining the DNA-binding specificity of the DBD-dimer are feasible approaches to characterize LTTRs of unknown function.

New Vocabulary for Bacterial Communication.

Quorum sensing (QS) is widely accepted as a procedure that bacteria use to converse. However, prevailing thinking places acyl homoserine lactones (AHLs) at the forefront of this communication pathway in Gram-negative bacteria. With the advent of high-throughput genomics and the subsequent influx of bacterial genomes, bioinformatics analysis has determined that the genes encoding AHL biosynthesis, originally discovered to be indispensable for QS (LuxI-like proteins and homologues), are often absent in QS-capable bacteria. Instead, the sensing protein (LuxR-like proteins) is present with an apparent inability to produce any outgoing AHL signal. Recently, several signals for these LuxR solos have been identified. Herein, advances in the field of QS are discussed, with a particular focus on recent research in the field of bacterial cell-cell communication.

The Biocontrol Agent and Insect Pathogen Photorhabdus luminescens Interacts with Plant Roots.

The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the Photorhabdus luminescens secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. P. luminescens is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The P. luminescens-EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of P. luminescens in the rhizosphere. We performed transcriptome analysis to show how P. luminescens responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that P. luminescens can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of P. luminescens with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions.IMPORTANCE Insect-pathogenic Photorhabdus luminescens bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that P. luminescens can specifically react to and interact with plant roots. Understanding the adaptation of P. luminescens in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.

Phenotypic Heterogeneity of the Insect Pathogen Photorhabdus luminescens: Insights into the Fate of Secondary Cells.

Photorhabdus luminescens is a Gram-negative bacterium that lives in symbiosis with soil nematodes and is simultaneously highly pathogenic toward insects. The bacteria exist in two phenotypically different forms, designated primary (1°) and secondary (2°) cells. Yet unknown environmental stimuli as well as global stress conditions induce phenotypic switching of up to 50% of 1° cells to 2° cells. An important difference between the two phenotypic forms is that 2° cells are unable to live in symbiosis with nematodes and are therefore believed to remain in the soil after a successful infection cycle. In this work, we performed a transcriptomic analysis to highlight and better understand the role of 2° cells and their putative ability to adapt to living in soil. We could confirm that the major phenotypic differences between the two cell forms are mediated at the transcriptional level as the corresponding genes were downregulated in 2° cells. Furthermore, 2° cells seem to be adapted to another environment as we found several differentially expressed genes involved in the cells' metabolism, motility, and chemotaxis as well as stress resistance, which are either up- or downregulated in 2° cells. As 2° cells, in contrast to 1° cells, chemotactically responded to different attractants, including plant root exudates, there is evidence for the rhizosphere being an alternative environment for the 2° cells. Since P. luminescens is biotechnologically used as a bio-insecticide, investigation of a putative interaction of 2° cells with plants is also of great interest for agriculture.IMPORTANCE The biological function and the fate of P. luminescens 2° cells were unclear. Here, we performed comparative transcriptomics of P. luminescens 1° and 2° cultures and found several genes, not only those coding for known phenotypic differences of the two cell forms, that are up- or downregulated in 2° cells compared to levels in 1° cells. Our results suggest that when 1° cells convert to 2° cells, they drastically change their way of life. Thus, 2° cells could easily adapt to an alternative environment such as the rhizosphere and live freely, independent of a host, putatively utilizing plant-derived compounds as nutrient sources. Since 2° cells are not able to reassociate with the nematodes, an alternative lifestyle in the rhizosphere would be conceivable.

Anti-Trypanosoma activity of bioactive metabolites from Photorhabdus luminescens and Xenorhabdus nematophila.

Only two drugs are currently available for the treatment of Chagas disease and their effectiveness are unsatisfactory. Photorhabdus luminescens and Xenorhabdus nematophila, two enteric bacteria highly pathogenic to a broad range of insects, have been studied as potential source for bioactive metabolites against protozoa causing neglected tropical diseases. Therefore, we tested the in vitro anti-Trypanosoma cruzi activity of secreted metabolites from these bacteria. The conditioned medium of X. nematophila and P. luminescens showed significant parasiticidal activity in a concentration-dependent manner (IC50XN = 0.34 mg/mL, IC50PL = 1.0 mg/mL). The parasiticidal compound was identified as a small molecule stable to heating and pH changes ranging from 2 to 12. Moreover, anti-Trypanosoma molecules secreted by both bacteria stimulate the trypanocidal activity of macrophages by a mechanism independent of nitric oxide. Summarizing, our studies reveal that P. luminescens and X. nematophila are potential sources of putative novel drugs against Chagas disease.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

BACKGROUND: Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR. RESULTS: Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes. CONCLUSIONS: According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

Non-canonical activation of histidine kinase KdpD by phosphotransferase protein PtsN through interaction with the transmitter domain.

The two-component system KdpD/KdpE governs K+ homeostasis by controlling synthesis of the high affinity K+ transporter KdpFABC. When sensing low environmental K+ concentrations, the dimeric kinase KdpD autophosphorylates in trans and transfers the phosphoryl-group to the response regulator KdpE, which subsequently activates kdpFABC transcription. In Escherichia coli, KdpD can also be activated by interaction with the non-phosphorylated form of the accessory protein PtsN. PtsN stimulates KdpD kinase activity thereby increasing phospho-KdpE levels. Here, we analyzed the interplay between KdpD/KdpE and PtsN. PtsN binds specifically to the catalytic DHp domain of KdpD, which is also contacted by KdpE. Accordingly, PtsN and KdpE compete for binding, providing a paradox. Low levels of non-phosphorylated PtsN stimulate, whereas high amounts reduce kdpFABC expression by blocking access of KdpE to KdpD. Ligand fishing experiments provided insight as they revealed ternary complex formation of PtsN/KdpD2 /KdpE in vivo demonstrating that PtsN and KdpE bind different protomers in the KdpD dimer. PtsN may bind one protomer to stimulate phosphorylation of the second KdpD protomer, which then phosphorylates bound KdpE. Phosphorylation of PtsN prevents its incorporation in ternary complexes. Interaction with the conserved DHp domain enables PtsN to regulate additional kinases such as PhoR.

Insulation and wiring specificity of BceR-like response regulators and their target promoters in Bacillus subtilis.

BceRS and PsdRS are paralogous two-component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, PbceA or PpsdA , resulting in a strong up-regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross-regulation has been observed between them. We therefore investigated the specificity determinants of PbceA and PpsdA that ensure the insulation of these two paralogous pathways at the RR-promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high-affinity, low-specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low-affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

Quorum Sensing and LuxR Solos in Photorhabdus.

Bacterial communication via small diffusible molecules to mediate group-coordinated behaviour is commonly referred to as 'quorum sensing'. The prototypical quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase that produces acyl-homoserine lactones (AHLs) as signals and a LuxR-type receptor that detects the AHLs to control expression of specific genes. However, many bacteria possess LuxR homologs but lack a cognate LuxI-type AHL-synthase. Those LuxR-type receptors are designated as 'LuxR orphans' or 'solos'. Entomopathogenic bacteria of the genus Photorhabdus all harbour a large number of LuxR solos, more than any other bacteria examined so far. Two novel quorum sensing systems were found to regulate cell clumping in Photorhabdus and therefore affect pathogenicity. In Photorhabdus luminescens and Photorhabdus temperata the LuxR solo PluR senses α-pyrones named 'photopyrones' instead of AHLs, which are produced by the pyrone synthase PpyS. In contrast, Photorhabdus asymbiotica, a closely related insect and human pathogen, has the PluR homolog PauR, which senses dialkylresorcinols produced by the DarABC pathway to regulate pathogenicity. All three Photorhabdus species harbour at least one LuxR solo with an intact AHL-binding motif, which might also allow sensing of exogenous AHLs. However, the majority of the LuxR solos in all Photorhabdus species have a PAS4 signal-binding domain. These receptors are assumed to detect eukaryotic compounds and are proposed to be involved in host sensing. Overall, because of the large number of LuxR solos they encode, bacteria of the genus Photorhabdus are ideal candidates to study and to identify novel bacterial communication networks.

Entomopathogenic bacteria Photorhabdus luminescens as drug source against Leishmania amazonensis.

Leishmaniasis is a widely spread and zoonotic disease with serious problems as low effectiveness of drugs, emergence of parasite resistance and severe adverse reactions. In recent years, considerable attention has been given to secondary metabolites produced by Photorhabdus luminescens, an entomopathogenic bacterium. Here, we assessed the leishmanicidal activity of P. luminescens culture fluids. Initially, promastigotes of Leishmania amazonensis were incubated with cell free conditioned medium of P. luminescens and parasite survival was monitored. Different pre-treatments of the conditioned medium revealed that the leishmanicidal activity is due to a secreted peptide smaller than 3 kDa. The Photorhabdus-derived leishmanicidal toxin (PLT) was enriched from conditioned medium and its effect on mitochondrial membrane potential of promastigotes, was determined. Moreover, the biological activity of PLT against amastigotes was evaluated. PLT inhibited the parasite growth and showed significant leishmanicidal activity against promastigote and amastigotes of L. amazonensis. PLT also caused mitochondrial dysfunction in parasites, but low toxicity to mammalian cell and human erythrocytes. Moreover, the anti-amastigote activity was independent of nitric oxide production. In summary, our results highlight that P. luminescens secretes Leishmania-toxic peptide(s) that are promising novel drugs for therapy against leishmaniasis.