Linking the environment, DAF-7/TGFß signaling and LAG-2/DSL ligand expression in the germline stem cell niche.

The developmental accumulation of proliferative germ cells in the C. elegans hermaphrodite is sensitive to the organismal environment. Previously, we found that the TGFß signaling pathway links the environment and proliferative germ cell accumulation. Neuronal DAF-7/TGFß causes a DAF-1/TGFßR signaling cascade in the gonadal distal tip cell (DTC), the germline stem cell niche, where it negatively regulates a DAF-3 SMAD and DAF-5 Sno-Ski. LAG-2, a founding DSL ligand family member, is produced in the DTC and activates the GLP-1/Notch receptor on adjacent germ cells to maintain germline stem cell fate. Here, we show that DAF-7/TGFß signaling promotes expression of lag-2 in the DTC in a daf-3-dependent manner. Using ChIP and one-hybrid assays, we find evidence for direct interaction between DAF-3 and the lag-2 promoter. We further identify a 25 bp DAF-3 binding element required for the DTC lag-2 reporter response to the environment and to DAF-7/TGFß signaling. Our results implicate DAF-3 repressor complex activity as a key molecular mechanism whereby the environment influences DSL ligand expression in the niche to modulate developmental expansion of the germline stem cell pool.

Increased airway glucose increases airway bacterial load in hyperglycaemia.

Diabetes is associated with increased frequency of hospitalization due to bacterial lung infection. We hypothesize that increased airway glucose caused by hyperglycaemia leads to increased bacterial loads. In critical care patients, we observed that respiratory tract bacterial colonisation is significantly more likely when blood glucose is high. We engineered mutants in genes affecting glucose uptake and metabolism (oprB, gltK, gtrS and glk) in Pseudomonas aeruginosa, strain PAO1. These mutants displayed attenuated growth in minimal medium supplemented with glucose as the sole carbon source. The effect of glucose on growth in vivo was tested using streptozocin-induced, hyperglycaemic mice, which have significantly greater airway glucose. Bacterial burden in hyperglycaemic animals was greater than control animals when infected with wild type but not mutant PAO1. Metformin pre-treatment of hyperglycaemic animals reduced both airway glucose and bacterial load. These data support airway glucose as a critical determinant of increased bacterial load during diabetes.

Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane ß-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.

The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

The pathogenicity island encoded PvrSR/RcsCB regulatory network controls biofilm formation and dispersal in Pseudomonas aeruginosa†PA14.

Pseudomonas aeruginosa biofilm formation is linked to persistent infections in humans. Biofilm formation is facilitated by extracellular appendages, some of which are assembled by the Chaperone Usher Pathway (Cup). The cupD gene cluster is located on the PAPI-1 pathogenicity island of strain PA14 and has probably been acquired together with four genes encoding two-component signal transduction proteins. We have previously showed that the RcsB response regulator activates expression of the cupD genes, which leads to the production of CupD fimbriae and increased attachment. Here we show that RcsB activity is tightly modulated by two sensors, RcsC and PvrS. While PvrS acts as a kinase that enhances RcsB activity, RcsC has a dual function, first as a phosphorelay, and second as a phosphatase. We found that, under certain growth conditions, overexpression of RcsB readily induces biofilm dispersal. Microarray analysis shows that RcsB positively controls expression of pvrR that encodes the phosphodiesterase required for this dispersal process. Finally, in addition to the PAPI-1 encoded cupD genes, RcsB controls several genes on the core genome, some of which encode orphan response regulators. We thus discovered that RcsB is central to a large regulatory network that fine-tunes the switch between biofilm formation and dispersal.

The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system.

In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1- to H3-T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1-T6SS and absent from the H2- and H3-T6SSs. We demonstrate that HsiE1 interacts with a predicted N-terminal α-helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath-like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ-like component are often devoid of the predicted N-terminal helical region, which suggests co-evolution. We observe that a synthetic peptide corresponding to the N-terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non-essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.

DLK1 promotes neurogenesis of human and mouse pluripotent stem cell-derived neural progenitors via modulating Notch and BMP signalling.

A better understanding of the control of stem cell maintenance and differentiation fate choice is fundamental to effectively realising the potential of human pluripotent stem cells in disease modelling, drug screening and cell therapy. Dlk1 is a Notch related transmembrane protein that has been reportedly expressed in several neurogenic regions in the developing brain. In this study, we investigated the ability of Dlk1 in modulating the maintenance and differentiation of human and mouse ESC-derived neural progenitors. We found that DLK1, either employed as an extrinsic factor, or via transgene expression, consistently promoted the generation of neurons in both the mouse and human ESC-derived neural progenitors. DLK1 exerts this function by inducing cell cycle exit of the progenitors, as evidenced by an increase in the number of young neurons retaining BrdU labelling and cells expressing the cycling inhibitor P57Kip2. DLK1 antagonised the cell proliferation activity of Notch ligands Delta 1 and Jagged and inhibited Hes1-mediated Notch signaling as demonstrated by a luciferase reporter assay. Interestingly, we found that DLK1 promotes the neurogenic potential of human neural progenitor cells via suppression of Smad activation when they are challenged with BMP. Together, our data demonstrate for the first time a regulatory role for DLK1 in human and mouse neural progenitor differentiation and establish an interaction between DLK1 and Hes1-mediated Notch signaling in these cells. Furthermore, this study identifies DLK1 as a novel modulator of BMP/Smad signalling.