A dual role for proline iminopeptidase in the regulation of bacterial motility and host immunity

During plant–pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant‐associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella‐mediated bacterial motility by decreasing intracellular bis‐(3′,5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c‐di‐GMP levels. We also found that PIP is a type III secretion system‐dependent effector capable of eliciting a hypersensitive response in non‐host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip‐overexpressing plants relative to wild‐type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant–microbe interactions, i.e. it affects intracellular c‐di‐GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost‐efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.     

Parasitoid genus‐specific manipulation of orb‐web host spiders (Araneae,Araneidae)

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Host activity and wasp experience affect parasitoid wasp foraging behaviour and oviposition on nematode‐infected larvae of the forestry pest Hylobius abietis

1. Entomopathogenic nematodes (EPN) are currently being used as introduced biological control agents against the larvae of the native European forestry pest Hylobius abietis L. which develop under the bark of stumps and roots of newly dead conifer trees. 2. The potential for resource competition between gregarious ectoparasitoid Bracon hylobii Ratz and EPN by recording oviposition and related behaviours of B. hylobii females on EPN‐infected H. abietis larvae was investigated. Wasps did not parasitise EPN‐infected host larvae that were dead when presented, but naÏve and experienced wasps parasitised live EPN‐infected hosts. NaÏve wasps parasitised live EPN‐infected hosts significantly less frequently than healthy hosts only when the infected larvae were close to death (i.e. died during 24‐h trial). Parasitism by experienced wasps was unaffected by host infection. 3. Wasp probing and oviposition were positively associated with the amount of host movement. Preventing H. abietis larvae from chewing on bark significantly reduced parasitism by naÏve, but not experienced wasps. 4. The number of eggs per clutch was not affected by bark chewing or EPN‐infection of H. abietis larvae. 5. NaÏve and experienced B. hylobii parasitised two abnormal hosts (larvae of coleopteran Rhagium bifasciatum Fabricius and lepidopteran Galleria mellonella L.), both of which moved and chewed on bark during trials. 6. It was concluded that B. hylobii can use vibrational cues generated by host movement and feeding to locate hosts at short range and accepts unsuitable (EPN‐infected or abnormal) hosts as long as these create such cues. The implications for competition between B. hylobii and EPN and possible ways of minimising it when applying EPN are discussed.     

Arginine–pyrimidine conjugates with therapeutic and prophylactic activity in lethal bacterial infections

Arginine–pyrimidine conjugates represent a novel class of compounds that exhibits therapeutic and prophylactic activity in lethal infections by Gram-positive and Gram-negative bacteria without showing antibacterial activity in vitro.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K_D) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K_D of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain–domain motion in EI can contribute as large as ∼−3.2 kcal/mol toward PEP binding.     

Host preference of a temperate mistletoe: Disproportional infection on three co‐occurring host species influenced by differential success

The mistletoe Tristerix verticillatus (Loranthaceae) parasitizes within a small area of the Yerba Loca Nature Sanctuary near Santiago, Chile, three co‐occurring hosts: Schinus montanus (Anacardiaceae), Fabiana imbricata (Solanaceae) and Berberis montana (Berberidaceae). Previous studies suggest that T. verticillatus may be favoured when parasitizing S. montanus relative to the other two host species. We hypothesize that infection of S. montanus is not proportional to its local abundance or appearance, that S. montanus is more intensively parasitized than other available hosts, and that host provenance is a determinant of the fate of the infecting seed. We compare the incidence of infection of T. verticillatus in relation to local availability and appearance variables, and the intensity of infection of T. verticillatus, on the three co‐occurring host species. We then test the effects of host provenance on mistletoe seed establishment success with a seed cross inoculation experiment varying the donor and receptor hosts. Finally, we test whether there are differences in establishment success between manually processed seeds and seeds defecated by the avian disperser Mimus thenca (Passeriformes: Mimidae). Our results show that the three hosts have an aggregated spatial distribution. Schinus montanus was parasitized at a higher rate than expected by its local availability and appearance, and inoculated seeds showed differential development depending on the origin of the seeds: seeds from T. verticillatus parasitizing S. montanus inoculated to S. montanus twigs showed higher germination and lower mortality than seeds from T. verticillatus parasitizing F. imbricata inoculated to S. montanus twigs. Furthermore, seeds defecated by the avian disperser, M. thenca, had higher adherence and reduced mortality when compared to manually processed seeds. The disproportional host infection found is discussed in terms of the differential establishment of mistletoe seeds, morphological characteristics of hosts and the behaviour of dispersing birds.     

Role of distinct type‐IV‐secretion systems and secreted effector sets in host adaptation by pathogenic Bartonella species

The α‐proteobacterial genus Bartonella comprises a large number of facultative intracellular pathogens that share a common lifestyle hallmarked by hemotrophic infection and arthropod transmission. Speciation in the four deep‐branching lineages (L1–L4) occurred by host adaptation facilitating the establishment of long lasting bacteraemia in specific mammalian reservoir host(s). Two distinct type‐IV‐secretion systems (T4SSs) acquired horizontally by different Bartonella lineages mediate essential host interactions during infection and represent key innovations for host adaptation. The Trw‐T4SS confined to the species‐rich L4 mediates host‐specific erythrocyte infection and likely has functionally replaced flagella as ancestral virulence factors implicated in erythrocyte colonisation by bartonellae of the other lineages. The VirB/VirD4‐T4SS translocates Bartonella effector proteins (Bep) into various host cell types to modulate diverse cellular and innate immune functions involved in systemic spreading of bacteria following intradermal inoculation. Independent acquisition of the virB/virD4/bep locus by L1, L3, and L4 was likely driven by arthropod vectors associated with intradermal inoculation of bacteria rather than facilitating direct access to blood. Subsequently, adaptation to colonise specific niches in the new host has shaped the evolution of complex species‐specific Bep repertoires. This diversification of the virulence factor repertoire of Bartonella spp. represents a remarkable example for parallel evolution of host adaptation.     

Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin‐B1 RhoGTPase binding domain with Rnd1 and active Rac1

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin‐B1 and mapped its binding interface with several Rho‐GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin‐B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin‐B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin‐B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation.     

A novel Meloidogyne graminicola effector,MgMO237, interacts with multiple host defence‐related proteins to manipulate plant basal immunity and promote parasitism

Plant‐parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up‐regulated in parasitic third‐/fourth‐stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two‐hybrid and co‐immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3‐β‐glucan synthase component (OsGSC), cysteine‐rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis‐related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence‐related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence‐related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes.     

Structural characterization of the heme‐based oxygen sensor,AfGcHK, its interactions with the cognate response regulator,and their combined mechanism of action in a bacterial two‐component signaling system

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109‐5 forms a two‐component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N‐terminal sensor domain causes the C‐terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX‐MS studies on the AfGcHK:RR complex also showed that the N‐side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β‐strand B2 area of the RR protein's Rec1 domain, and that the C‐side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β‐strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375–1389. © 2016 Wiley Periodicals, Inc.     

Hermansky–Pudlak syndrome: pigmentary and non‐pigmentary defects and their pathogenesis

Hermansky–Pudlak syndrome (HPS) is an autosomal recessive and genetically heterogeneous disorder characterized by oculocutaneous albinism, bleeding tendency, and ceroid deposition, which likely leads to deleterious lesions in lungs, heart, and other organs. Currently, nine genes have been identified as causative for HPS in humans. Their pathological effects are attributable to the disrupted biogenesis of lysosome‐related organelles (LROs) existing in multiple cell types or tissues, causing the pigmentory and non‐pigmentory defects. This review focuses on the functional aspects of HPS genes in regulating LRO biogenesis and signal transduction. The understanding of these mechanisms expands our knowledge about the involvement of lysosomal trafficking in the targeting of cargoes for constitutive transport, degradation, and secretion. This opens an avenue to the pathogenesis of lysosomal trafficking disorders at the cellular and developmental levels.     

Do‐or‐die life cycles and diverse post‐infection resistance mechanisms limit the evolution of parasite host ranges

In light of the dynamic nature of parasite host ranges and documented potential for rapid host shifts, the observed high host specificity of most parasites remains an ecological paradox. Different variants of host‐use trade‐offs have become a mainstay of theoretical explanations of the prevalence of host specialism, but empirical evidence for such trade‐offs is rare. We propose an alternative theory based on basic features of the parasite life cycle: host selection and subsequent intrahost replication. We introduce a new concept of effective burst size that accounts for the fact that successful host selection does not guarantee intrahost replication. Our theory makes a general prediction that a parasite will expand its host range if its effective burst size is positive. An in silico model of bacteria‐phage coevolution verifies our predictions and demonstrates that the tendency for relatively narrow host ranges in parasites can be explained even in the absence of trade‐offs.     

Time‐resolved dissection of the molecular crosstalk driving Fusarium head blight in wheat provides new insights into host susceptibility determinism

Fungal plant diseases are controlled by a complex molecular dialogue that involves pathogen effectors able to manipulate plant susceptibility factors at the earliest stages of the interaction. By probing the wheat–Fusarium graminearum pathosystem, we profiled the coregulations of the fungal and plant proteins shaping the molecular responses of a 96‐hr‐long infection's dynamics. Although no symptoms were yet detectable, fungal biomass swiftly increased along with an extremely diverse set of secreted proteins and candidate effectors supposed to target key plant organelles. Some showed to be early accumulated during the interaction or already present in spores, otherwise stored in germinating spores and detectable in an in vitro F. graminearum exudate. Wheat responses were swiftly set up and were evidenced before any visible symptom. Significant wheat protein abundance changes co‐occurred along with the accumulation of putative secreted fungal proteins and predicted effectors. Regulated wheat proteins were closely connected to basal cellular processes occurring during spikelet ontogeny, and particular coregulation patterns were evidenced between chloroplast proteins and fungal proteins harbouring a predicted chloroplast transit peptide. The described plant and fungal coordinated responses provide a resourceful set of data and expand our understanding of the wheat–F. graminearum interaction.