Recruitment of TBK1 to cytosol‐invading Salmonella induces WIPI2‐dependent antibacterial autophagy

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti‐bacterial autophagy relies on the core autophagy machinery, cargo receptors, and “eat‐me” signals such as galectin‐8 and ubiquitin that label bacteria as autophagy cargo. Anti‐bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic. Here we show that recruitment of WIPI2, itself essential for anti‐bacterial autophagy, is dependent on the localization of catalytically active TBK1 to the vicinity of cytosolic bacteria. Experimental manipulation of TBK1 recruitment revealed that engagement of TBK1 with any of a variety of Salmonella‐associated “eat‐me” signals, including host‐derived glycans and K48‐ and K63‐linked ubiquitin chains, suffices to restrict bacterial proliferation. Promiscuity in recruiting TBK1 via independent signals may buffer TBK1 functionality from potential bacterial antagonism and thus be of evolutionary advantage to the host.     

Roles of RNA polymerase IV in gene silencing

Eukaryotes typically have three multi-subunit enzymes that decode the nuclear genome into RNA: DNA-dependent RNA polymerases I, II and III (Pol I, II and III). Remarkably, higher plants have five multi-subunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability; plus two non-essential polymerases, Pol IVa and Pol IVb, which specialize in small RNA-mediated gene silencing pathways. There are numerous examples of phenomena that require Pol IVa and/or Pol IVb, including RNA-directed DNA methylation of endogenous repetitive elements, silencing of transgenes, regulation of flowering-time genes, inducible regulation of adjacent gene pairs, and spreading of mobile silencing signals. Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function.     

The Morphological Identity of Insect Dendrites

Dendrite morphology, a neuron's anatomical fingerprint, is a neuroscientist's asset in unveiling organizational principles in the brain. However, the genetic program encoding the morphological identity of a single dendrite remains a mystery. In order to obtain a formal understanding of dendritic branching, we studied distributions of morphological parameters in a group of four individually identifiable neurons of the fly visual system. We found that parameters relating to the branching topology were similar throughout all cells. Only parameters relating to the area covered by the dendrite were cell type specific. With these areas, artificial dendrites were grown based on optimization principles minimizing the amount of wiring and maximizing synaptic democracy. Although the same branching rule was used for all cells, this yielded dendritic structures virtually indistinguishable from their real counterparts. From these principles we derived a fully-automated model-based neuron reconstruction procedure validating the artificial branching rule. In conclusion, we suggest that the genetic program implementing neuronal branching could be constant in all cells whereas the one responsible for the dendrite spanning field should be cell specific.     

Gene expression profiling of human mesenchymal stem cells chemotactically induced with CXCL12

In situ tissue engineering is a promising approach in regenerative medicine, with the possibility that adult stem or progenitor cells will be guided chemotactically to a tissue defect and subsequently differentiate into the surrounding tissue type. Mesenchymal stem cells (MSC) represent attractive candidate cells. Chemokines such as CXCL12 (SDF-1α) chemoattract MSC, but little is known about the molecular processes involved in the chemotaxis and migration of MSC. In this study, MSC recruitment by CXCL12 was investigated by genome-wide microarray analysis. The dose-dependent migration potential of bone-marrow-derived MSC toward CXCL12 was measured in an in vitro assay, with a maximum being recorded at a concentration of 1,000 nM CXCL12. Microarray analysis of MSC stimulated with CXCL12 and non-stimulated controls showed 30 differentially expressed genes (24 induced and six repressed). Pathway analysis revealed 11 differentially expressed genes involved in cellular movement and cytokine-cytokine receptor interaction, including those for migratory inducers such as the chemokines CXCL8 and CCL26, the leukocyte inhibitory factor, secretogranin II, and prostaglandin endoperoxide synthase 2. These results were confirmed by real-time polymerase chain reaction for selected genes. The obtained data provide further insights into the molecular mechanisms involved in chemotactic processes in cell migration and designate CXCL12 as a promising candidate for in situ recruitment in regenerative therapies. Stefan Stich and Marion Haag contributed equally to this work. This study was supported by the Investitionsbank Berlin and the European Regional Development Fund (grant: 10128098), Deutsche Forschungsgemeinschaft (grant: DFG SI 569/7–1), and the Bundesministerium für Bildung und Forschung (Bioinside: 13N9817).     

Toll-Like Receptor 4 Deficiency Increases Disease and Mortality after Mouse Hepatitis Virus Type 1 Infection of Susceptible C3H Mice

Severe acute respiratory syndrome (SARS) is characterized by substantial acute pulmonary inflammation with a high mortality rate. Despite the identification of SARS coronavirus (SARS-CoV) as the etiologic agent of SARS, a thorough understanding of the underlying disease pathogenesis has been hampered by the lack of a suitable animal model that recapitulates the human disease. Intranasal (i.n.) infection of A/J mice with the CoV mouse hepatitis virus strain 1 (MHV-1) induces an acute respiratory disease with a high lethality rate that shares several pathological similarities with SARS-CoV infection in humans. In this study, we examined virus replication and the character of pulmonary inflammation induced by MHV-1 infection in susceptible (A/J, C3H/HeJ, and BALB/c) and resistant (C57BL/6) strains of mice. Virus replication and distribution did not correlate with the relative susceptibilities of A/J, BALB/c, C3H/HeJ, and C57BL/6 mice. In order to further define the role of the host genetic background in influencing susceptibility to MHV-1-induced disease, we examined 14 different inbred mouse strains. BALB.B and BALB/c mice exhibited MHV-1-induced weight loss, whereas all other strains of H-2^b and H-2^d mice did not show any signs of disease following MHV-1 infection. H-2^k mice demonstrated moderate susceptibility, with C3H/HeJ mice exhibiting the most severe disease. C3H/HeJ mice harbor a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts TLR4 signaling. C3H/HeJ mice exhibit enhanced morbidity and mortality following i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.Severe acute respiratory syndrome (SARS) is a disease that was initially observed in 2002 and led to approximately 8,000 affected individuals in multiple countries with over 700 deaths (1, 24, 47, 48). The causative agent of SARS was subsequently identified as a novel coronavirus (CoV) termed SARS-CoV (8, 17, 22, 27, 32, 37). Although SARS-CoV infections following the initial outbreak in 2002 and 2003 have been limited primarily to laboratory personnel, the identification of an animal reservoir for the virus raises concern about the potential for future outbreaks (25).The pathogenesis of SARS has been difficult to study, in part because no animal model is able to fully recapitulate the morbidity and mortality observed in infected humans (35). Infection of a number of inbred mouse strains, including BALB/c, C57BL/6, and 129S, with primary human isolates of SARS-CoV results in the replication of the virus within the lung tissue without the subsequent development of readily apparent clinical disease (11, 16, 41). Infection of aged BALB/c mice results in clinically apparent disease that more closely mimics some aspects of SARS in humans (36). However, immune responses in aged mice are known to be altered (5, 15), and thus, the mechanisms that control the induction of disease may differ between adult and aged mice. Recent work has demonstrated that serial passage of SARS-CoV in mice results in a mouse adaptation that leads to more profound replication of the virus in the lung (28, 34). However, the time to death from this mouse-adapted SARS-CoV is 3 to 5 days, which is much more rapid than the time to mortality observed in fatal cases of SARS in humans.Phylogenetic analysis has revealed that SARS-CoV is most closely related to group 2 CoVs, which include the mouse hepatitis virus (MHV) family (39). Thus, information gathered by infection of mice with closely related members of the group 2 CoVs may further contribute to our understanding of SARS-CoV pathogenesis in humans. While many strains of MHV induce primarily hepatic and central nervous system diseases (6, 7, 12, 18, 21, 23, 40), a recent study demonstrated that intranasal (i.n.) infection of A/J mice with MHV type 1 (MHV-1) induces pulmonary injury that shares several pathological characteristics with SARS-CoV infection of humans (2, 3, 9, 29, 43).In the current study, we examined the relationship between MHV-1 replication in the lungs and the severity of disease in four inbred strains of mice: A/J, BALB/c, C57BL/6, and C3H/HeJ. Our results demonstrate that MHV-1 replicates to similar levels in the lung in each of these inbred strains of mice regardless of their relative levels of susceptibility, as measured by weight loss and clinical illness. Both A/J and C3H/HeJ mice exhibited enhanced weight loss and clinical illness following i.n. MHV-1 infection compared to BALB/c and C57BL/6 mice. Analysis of many different inbred mouse strains confirmed A/J and C3H/HeJ mice as the most susceptible to i.n. MHV-1 infection. Interestingly, C3H/HeJ mice harboring a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts its normal function exhibited greatly increased morbidity and mortality after i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.     

The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.     

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.     

Codon usage bias and the evolution of influenza A viruses. Codon Usage Biases of Influenza Virus

Background  

The influenza A virus is an important infectious cause of morbidity and mortality in humans and was responsible for 3 pandemics in the 20^th century. As the replication of the influenza virus is based on its host's machinery, codon usage of its viral genes might be subject to host selection pressures, especially after interspecies transmission. A better understanding of viral evolution and host adaptive responses might help control this disease.     

Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a - in agreement with overexpression studies - is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXX? type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a.