Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.     

Does distant homology with Evf reveal a lipid binding site in Bacillus thuringiensis cytolytic toxins?

The Cry and Cyt classes of insecticidal toxins derived from the sporulating bacterium Bacillus thuringiensis are valuable substitutes for synthetic pesticides in agricultural contexts. Crystal structures and many biochemical data have provided insights into their molecular mechanisms, generally thought to involve oligomerization and pore formation, but have not localised the site on Cyt toxins responsible for selective binding of phospholipids containing unsaturated fatty acids. Here, distant homology between the structure of Cyt toxins and Erwinia virulence factor (Evf) is demonstrated which, along with sequence conservation analysis, allows a putative lipid binding site to be localised in the toxins.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Response to Dangl: Complex harmonies of the host–pathogen interaction

In the accompanying Research Focus article that summarises our recent research article [1], Jeffery Dangl likens the interplay of the intracellular pathogen Salmonella enterica serovar Typhimurium with the host resistance protein Nramp1 to the response of a congregation to the teachings of a preacher. In principle, this is a fair interpretation of what we believe to be occurring on the molecular level between host and pathogen within the limited scope of our experiments. However, as with most biological systems, we believe that this analogy might be too simplistic. As Dangl points out, Nramp1 ‘might have a multitude of messages that it sends to different vacuolar inhabitants’. This cannot exclude the fact that the effects of Nramp1 on any single pathogen are pleiotropic and that we focused solely on one aspect of Nramp1 function in our studies, that is, divalent cation limitation. Therefore, we suggest that the interplay between innate host defences and pathogens is far more complex than simple call-and-response. Instead, we propose that it is more like a musical round, where one instrument makes the initial statement of the tune followed by the response of a second instrument, building over successive entrances to a complex orchestral whole. As a case in point, we wish to draw the reader's attention to the complex influences of both Nramp1 and S. enterica Typhimurium on the generation of toxic oxygen and nitrogen intermediates by the host during infection.     

Galleria melonella as an experimental in vivo host model for the fish-pathogenic oomycete Saprolegnia parasitica

Oomycetes are eukaryotic pathogens infecting animals and plants. Amongst them Saprolegnia parasitica is a fish pathogenic oomycete causing devastating losses in the aquaculture industry. To secure fish supply, new drugs are in high demand and since fish experiments are time consuming, expensive and involve animal welfare issues the search for adequate model systems is essential. Galleria mellonella serves as a heterologous host model for bacterial and fungal infections. This study extends the use of G. mellonella for studying infections with oomycetes. Saprolegniales are highly pathogenic to the insects while in contrast, the plant pathogen Phytophthora infestans showed no pathogenicity. Melanisation of hyphae below the cuticle allowed direct macroscopic monitoring of disease progression. However, the melanin response is not systemic as for other pathogens but instead is very local. The mortality of the larvae is dose-dependent and can be induced by cysts or regenerating protoplasts as an alternative source of inoculation.     

Inhibition of a malaria host–pathogen interaction by a computationally designed inhibitor

Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.     

Effects of fat body on α-ecdysone induced morphogenesis in cultured wing disks of the wax moth, Galleria mellonella

Fat body promoted ecdysone induced morphogenesis in Galleria wing disks cultured in vitro. Medium preincubated with fat body and α-ecdysone from any of the first 5 days of the final larval instar enhanced tracheal migration and elongation in the disks. Disks from the third, fourth, and fifth days of the final larval instar responded equally well to the fat body and α-ecdysone. We suggest a physiological rôle for Galleria fat body as an intermediary in the stimulation of wing disk development by α-ecdysone.     

Gene suppression in a tolerant tomato–vascular pathogen interaction

A plant can respond to the threat of a pathogen through resistance defenses or through tolerance. Resistance has been widely studied in many host pathogen systems but little is known about genetic changes which underlie a tolerant interaction. A recently developed model system for a tolerant tomato (Lycopersicon esculentum Mill) interaction with a fungal wilt pathogen, Verticillium dahliae Kleb, is examined with respect to changes in gene expression and compared to a susceptible infection. The results indicate that genetic changes can be dramatically different and some genes that are strongly elevated in the susceptible interaction are actually down-regulated in tolerance. Similar levels of fungal DNA and an up-regulation of many pathogenesis related genes indicate that in both types of interaction the presence of fungus is clearly recognized by the plant but other changes correlate with the absence of symptoms in the tolerant interaction. For example, a gene encoding a known 14-3-3 regulatory protein and a number of genes normally affected by this protein are down-regulated. Furthermore, genes which may contribute to foliar necrosis and cell death in the susceptible interaction also appear to be suppressed in the tolerant interaction, raising the possibility that the wilt symptoms, chlorosis and necrosis which are observed in the susceptible interaction, are actually programmed to further limit the growth of the fungal pathogen, and protect the general tomato population.     

Effects of temperature on the host–pathogen interaction in Ascochyta blight (Ascochyta lentis) of lentil

The effects of temperature regimes on the radial growth rate of different isolates of Ascochyta lentis and pathogen virulence and host susceptibility were studied in the laboratory and growth chamber using different pathogen isolates, and lentil genotypes with varying levels of resistance to Ascochyta blight. The growth rate of most isolates increased as temperature increased up to 20°C and declined thereafter. In experiment 1, the highest disease severities were observed on cvs. Laird and Eston and the lowest on ILL5588. Mean disease severities were similar from 10 to 20°C but substantially lower at 25°C for all genotypes except ILL5588. In experiment 2, no significant differences were observed between the two mating types, or in their interactions with genotypes and temperatures. The interactions of genotypes with temperature and with isolates indicated that the relative susceptibility of lentil genotypes depended on temperature and on the isolates of A. lentis. These findings indicated that when temperature changes during epidemic development in the field, different isolates could predominate in the pathogen population at different times.     

When does pathogen evolution maximize the basic reproductive number in well-mixed host–pathogen systems?

Pathogen evolution towards the largest basic reproductive number, $\mathcal R _0$ , has been observed in many theoretical models, but this conclusion does not hold universally. Previous studies of host–pathogen systems have defined general conditions under which $\mathcal R _0$ maximization occurs in terms of $\mathcal R _0$ itself. However, it is unclear what constraints these conditions impose on the functional forms of pathogen related processes (e.g. transmission, recover, or mortality) and how those constraints relate to the characteristics of natural systems. Here we focus on well-mixed SIR-type host–pathogen systems and, via a synthesis of results from the literature, we present a set of sufficient mathematical conditions under which evolution maximizes $\mathcal R _0$ . Our conditions are in terms of the functional responses of the system and yield three general biological constraints on when $\mathcal R _0$ maximization will occur. First, there are no genotype-by-environment interactions. Second, the pathogen utilizes a single transmission pathway (i.e. either horizontal, vertical, or vector transmission). Third, when mortality is density dependent: (i) there is a single infectious class that individuals cannot recover from, (ii) mortality in the infectious class is entirely density dependent, and (iii) the rates of recovery, infection progression, and mortality in the exposed classes are independent of the pathogen trait. We discuss how this approach identifies the biological mechanisms that increase the dimension of the environmental feedback and prevent $\mathcal R _0$ maximization.     

The plant pathogen Rhodococcus fascians. History,disease symptomatology,host range,pathogenesis and plant–pathogen interaction

Since 1927, outbreaks of the leafy gall disease caused by Rhodococcus fascians on annual and perennial herbaceous plants have put the ornamental plant industry at risk. More recently, Pistachio Bushy top syndrome (PBTS), a serious disease emerging in the Western United States, has been declared as a natural disaster by the U.S. Department of Agriculture. Recent developments indicate that the taxonomic position of R. fascians should be re-evaluated and circumscribed from other members of the genus. Due to the broad host range of this bacterium, economically important crops are in a vulnerable position especially with new members within the genus becoming able to interact synergistically with R. fascians to cause symptom development. The virulence strategies employed by R. fascians and the involvement of cytokinins (CKs) in its pathogenicity have been the subject of much controversy in the last years. Additionally, the detection of virulent strains of R. fascians from symptomatic tissues has been problematic for many researchers. This review focuses on plant–pathogen interaction in context with more recent findings on the association of CKs in virulence of R. fascians. Current knowledge on the worldwide distribution of the pathogen, a historical recap of its discovery and the economic impact of the disease caused by R. fascians are also described here.     

Out of the ESCPE room: Emerging roles of endosomal SNX-BARs in receptor transport and host–pathogen interaction

Several functions of the human cell, such as sensing nutrients, cell movement and interaction with the surrounding environment, depend on a myriad of transmembrane proteins and their associated proteins and lipids (collectively termed “cargoes”). To successfully perform their tasks, cargo must be sorted and delivered to the right place, at the right time, and in the right amount. To achieve this, eukaryotic cells have evolved a highly organized sorting platform, the endosomal network. Here, a variety of specialized multiprotein complexes sort cargo into itineraries leading to either their degradation or their recycling to various organelles for further rounds of reuse. A key sorting complex is the Endosomal SNX-BAR Sorting Complex for Promoting Exit (ESCPE-1) that promotes the recycling of an array of cargos to the plasma membrane and/or the trans-Golgi network. ESCPE-1 recognizes a hydrophobic-based sorting motif in numerous cargoes and orchestrates their packaging into tubular carriers that pinch off from the endosome and travel to the target organelle. A wide range of pathogens mimic this sorting motif to hijack ESCPE-1 transport to promote their invasion and survival within infected cells. In other instances, ESCPE-1 exerts restrictive functions against pathogens by limiting their replication and infection. In this review, we discuss ESCPE-1 assembly and functions, with a particular focus on recent advances in the understanding of its role in membrane trafficking, cellular homeostasis and host–pathogen interaction.     

Heat shock protein 70 is required for tabtoxinine-β-lactam-induced cell death in Nicotiana benthamiana

Tabtoxinine-β-lactam (TβL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TβL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions. To better understand the mechanisms involved in TβL-induced cell death, we studied its regulation in Nicotiana benthamiana. TβL-induced lesions, similar to those in controls, could be observed in SGT1-, RAR1- and Hsp90-silenced plants. In contrast, Hsp70-silenced plants showed suppression of lesion formation. Expression of hin1, a marker gene for the hypersensitive response (HR), which is a characteristic of programmed cell death in plants, was strongly induced in controls by TβL treatment but only slightly in Hsp70-silenced plants. However, in these TβL-treated Hsp70-silenced plants, the amount of ammonium ions was considerably increased. Furthermore, the silencing of Hsp70 also suppressed l-methionine sulfoximine-induced cell death and hin1 expression and caused the over-accumulation of ammonium ions. When inoculated directly with P. syringae pv. tabaci, Hsp70-silenced plants showed only reduced symptoms. Our results suggest that the TβL-induced pathway to cell death in N. benthamiana is at least partially similar to HR response, and that Hsp70 might play an essential role in these events.