The size of bacterial genomes, and consequently the amount of genes that a bacterium may harbour, varies greatly amongst species. Commonly, the larger amount of genes that an organism has, the larger number of functions that it can perform in order to survive in their competitive ecological niches. In this rat race, an exception is found in those bacteria that have evolved alternative strategies to avoid this intense competition. Endosymbionts have some of the smallest bacterial genomes found to date. These bacteria have evolved mechanisms to inhabit inside eukaryotic cells or in specific eukaryotic tissues, ecological niches that are restricted to most and where competition no longer exerts a strong selective pressure. Endosymbionts are commonly found in arthropods (including insects) and in the case of nutritional endosymbionts, they play necessary roles by synthesizing compounds like vitamins and amino acids that are essential for the survival of insects with a restricted diet (sap and blood-feeding insects). Free from competition from other microbes, these endosymbionts no longer require many of the genes that were essential before their symbiotic association, since the host produces most of the essential compounds required for their survival. As a result, these bacteria undergo with time a process called genome minimization, where non-essential genes are lost without an impact on their survival. Consequently, their genome size is reduced to harbour a minimal amount of core genes required for survival and symbiosis establishment. This process irreversibly binds host and endosymbiont making them essential to each other’s survival. This binding can only be removed by two natural processes: 1) when the symbiont is replaced by another symbiont capable of providing the same functions and more successful in colonization, and 2) when the genes essential for the insect survival are horizontally transferred to its genome, no longer requiring the endosymbiont maintenance.
Candidatus Tremblaya princeps (PCIT) is an endosymbiont of the mealybug Planococcus citri the smallest genome reported to date (139 kb) coding for around 120 genes. This unusual genome size could in part be explained by a second endosymbiont, Candidatus Moranella endobia, present in Planococcus citri. Several mechanisms could explain how Tremblaya has been able to continue functioning in its host: a) Tremblaya genes may have been transferred to the host genome allowing their products to still be acquired by the bacterium; b) the host could be producing all the products necessary for Tremblaya’s survival; c) other bacteria could have transferred genes to the host genome, which result in the host providing essential products to Tremblaya; and d) Tremblaya may be acquiring essential products from Moranella. While the pathways for the biosynthesis of the ten essential amino acids are incomplete in all three members of this tripartite, when considered as a group they possess all of them, suggesting that each organisms is essential for the survival of the other two.
In this manuscript, Husnik and co-workers perform comparative genomics and transcriptomics to establish the mechanism of this symbiosis and to understand how this endosymbiont-host triad functions to allow such reduced endosymbiont genomes. The complete genome of the Tremblaya endosymbiont (PAVE) from the mealybug species Phenacoccus avenae was sequenced. This species has only Tremblaya, thus not requiring Moranella. The results revealed that Tremblaya PAVE encodes all the amino acid genes found in the Tremblaya PCIT- Moranella complex, thus explaining why Moranella is not needed in the PAVE-Ph.avenae symbiosis to fulfil the amino acid needs.
Both histidine and lysine pathways are incomplete in both Tremblaya genomes, suggesting those as good candidates to be complemented by genes acquired by horizontal gene transfer (HGT) to the insect genome. 22 expressed genes from bacterial origin were found in the Pl.citri genome as a result of HGT. Some of these genes complement the lysine pathway while others are involved in nutrient biosynthesis and bacterial cell-wall maintenance. Another expressed gene, cysK, was found in this insect genome, which could complement the biosynthesis of the non-essential amino acid cysteine and/or methionine in Moranella. In addition, genes complementing vitamin, peptidoglycan biosynthesis and peptidoglycan recycling were also in the list of expressed insect genes from HGT origin. The missing genes in other amino acid pathways are likely to be complemented by genes of insect origin. The presence of a large number of HGT derived genes in the genome of mealybugs also suggests that cell lysis is the mechanism responsible for the exchange of gene products between Moranella and Tremblaya.
Phylogenetic analyses of these genes from HGT origin reveal that their potential donors were mostly facultative symbionts (Wolbachia sp., Rickettsia sp., Cardinium sp., Sodalis sp., Serratia symbiotica and Arsenophonus sp.), while others cluster with bacteria not known to be facultative symbionts (Pantoea sp, Pseudomonas sp. and Ralstonia sp.). All these bacteria are not known to be part of the symbiotic complex of mealybugs, thus these HGTs may have been the result of ancient interactions. In contrast to what was expected, Tremblaya does not seem to have contributed by HGTs to the insect genome.
From this work, the authors conclude that the acquisition of Moranella is indeed the force leading to the very extreme genome reduction that occurred in Tremblaya PCIT. In addition, they provide an interesting discussion on whether Tremblaya PCIT should be considered a bacterium or an organelle. This differentiation is often based on the fact that organelles no longer retain genes involved in DNA replication, transcription and translation. While the size of Tremblaya PCIT genome is consistent with that found in the genome of organelles and they also lack DNA replication, transcription and translation genes; its dependency of Moranella and the insect host, questions this classification.
The data presented in this manuscript also suggests that this symbiosis results from the interaction between six distinct organisms: Tremblaya, Moranella, the mealybug and three bacterial groups that provided genes by HGT.
In this manuscript, Harumoto and Lemaitre reveal the mechanism of a bacterial endosymbiont to manipulate the reproduction of its insect host. Bacterial endosymbionts have evolved multiple strategies to manipulate reproduction in their arthropod hosts in order to ensure transmission. Most of these manipulations favour female production since males are dead ends for bacterial transmission. Male-killing strategies have evolved independently in various bacterial taxa including Spiroplasma, Rickettsia and Wolbachia; and their genetic basis remains a mystery despite being first described in the 1950s in the Spiroplasma-Drosophila symbiosis. As in the case of other reproductive-manipulator symbionts, the major reason for this mechanism to remain unsolved lies in the inability to culture this bacterium and apply molecular methods. In this work, Harumoto and Lemaitre report a spontaneous Spiroplasma mutant that has reduced ability (50%) to induce male-killing. Having assessed that this reduction is not due to host differences, the authors sequenced the whole genome of this mutant strain, which was named Spiroplasma MSRO-H99 and compared it to its parental strain (MSRO-SE). The results revealed an 828bp deletion in a gene coding for a protein of 1,065 amino acids with ankyrin repeats and the OTU (ovarian tumour) deubiquitinase domain. This protein, which is present in a plasmid, was subsequently named SpAID and was further analysed. When the full-length SpAID was cloned and overexpressed in the genome of Drosophila melanogaster, it induced lethality of the male offspring. Subsequently, the authors verified that the male-killing effects observed in transgenic flies were identical to those observed in flies infected with Spiroplasma MSRO-SE. In both cases, strong apoptosis and neural defects were observed in male embryos while no defects were present in female embryos. In Drosophila (XX female, XY males), the X chromosome of males is 2-fold expressed to equal the expression levels originating in the two copies that females have. This regulation is controlled by an RNA-protein system called the male-specific lethal (MSL) complex. Previous reports indicated that this complex is indeed the target of Spiroplasma to induce male-killing (Cheng et al, 2016, Curr Biol.; and Harumoto et al, 2016, Nat Commun.). Spiroplasma is unable to kill males lacking MSL components while it induces death in females artificially modified to express the MSL complex.
Mutations in the ankyrin repeats of SpAID led to male-killing abolishment while mutations in the OTU domain led to a delay in the stage at which males are killed (pupal stage vs 2nd instar larval stage). Both domains were thus considered essential for the male-killing effects of SpAID. The authors also suggest a mode-of-action model in which the OTU domain promotes nuclear localization, while the ankyrin repeats are linked to interactions with the MSL complex or subsequent histone modifications. The authors make a final concluding statement where they point to the presence of deubiquitinase domains in both SpAID and the recently reported cytoplasmic incompatibility-inducing genes from Wolbachia (Beckmann and Fallon, 2013, Insect Biochem Mol Biol.; Beckmann et al., 2017, Nat Microbiol.; LePage DP, et al., 2017, Nature). This exciting report by Harumoto and Lemaitre provide the first genetic basis of a male-killing mechanism used by a bacterial endosymbiont to control the reproduction of its insect host. These findings set the basis for the development of novel strategies to control vector-borne diseases and agricultural pests by targeting the insect vector.
This is an excellent review published in 2018, which covers all aspects being exploited to date to fight vector-borne diseases:
Current situation: The prevalence of malaria has decreased by 50% since 2000 in contrast with the uprise of dengue observed since the same period. These changes are partly due to urban development and improvement in householding facilities in tropical areas. The rise of dengue can be explained but the better adaptability to urban environments of its vector, the mosquito Aedes aegypti. Despite malaria affecting globally 30 times more people than dengue, malaria is mostly a burden in Africa, where effective drugs as artemisinin are not available to the whole population. In contrast, no effective antivirals exist for dengue, despite the current advances, and epidemics remain unpredictable and causing serious burdens. In addition, other viral infections as Chikungunya, Zika and yellow fever are responsible for regular outbreaks in endemic areas and little is known about the global impact of these infections. The impact of these epidemics has led to a great deal of attention being given to the development of effective strategies to control vector-borne diseases that include vaccine development, the use of insect nets and vector control strategies. In the cases where vaccines are not available, strategies to vector control are focused on limiting vector exposure to induce a collapse of the population of infected vectors that would lead to disease eradication. In the case of arboviruses (as dengue, zika, yellow fever, West Nile and Japanese encephalitis), the virus induces an immune response that prevents further infections with the same serotype in their lifetime. In the case of dengue, 4 serotypes exist, implying that an individual can be affected by dengue 4 times in its lifetime, with secondary infections being the most severe ones. In this context, reducing exposure to the virus also brings a serious negative side effect, since infections at a later age are prone to more severe, even fatal outcomes. When comparing malaria and dengue outbreaks and the number of people being infected in each outbreak, the author determines that complete eradication of these diseases can only be achieved by an 80% exposure reduction in the case of dengue and over 99% reduction in the case of malaria. However, these predictions do not take into consideration the heterogeneity of the disease, in particular the hotspots with high transmission rates.
The problems related to each disease and the status of current strategies: While malaria eradication approaches have been pretty successful in the last decade, dengue infections are still on the rise despite control measures. A major determinant in these outcomes relies on the ecology of the different vectors. Anopheles gambiae, the major vector of malaria, bites at night (where better housing prevents exposure) while A. aegypti, the major vector of dengue, bites during the day (where housing has no effect). In addition, A. aegypti is more adapted to urban landscapes and in contrast to malaria, little efforts have been made to obtain data and to develop effective measures on the incidence and control of dengue besides reducing insect populations. Current malaria and dengue vaccines (CYD-TDV and RTS,S respectively) have limited efficacy, especially in areas with high prevalence of disease, and in the case of dengue, the effects may be very different while immunizing seropositive and seronegative patients. In the case of the dengue vaccine, adverse effects have been observed in seronegative patients when infected by dengue transmitted mosquitoes. Immunization mimics the effects of a first infection leading to a severe response when vaccinated patients are infected by the first time through mosquitoes. This led to a WHO recommendation to vaccinate only seropositive patients. Additional vaccines are currently under development and are likely to be available in the next decade.
Novel approaches under development: The development of alternatives to vaccines is also undergoing with promising results in various areas that include novel insecticide development and delivery strategies. An additional target is the use of bacterial endosymbionts as Wolbachia, which alter the reproductive strategies and consequently fitness of insect populations. One of the most promising reproductive phenotypes induced by Wolbachia is cytoplasmic incompatibility, which allows only reproduction between males and females infected with a similar Wolbachia strain. This approach offers the possibility of replacing populations of disease-infected mosquitoes by disease-free ones. In addition, certain strains of Wolbachia as wMelPop, shorten the life span of mosquitoes, reducing their infective capabilities, while other strains as wMel protect the insects from certain arboviruses as dengue. This last approach has been shown to be very successful in small cities and is now undergoing in larger cities as Yogyakarta (Indonesia), Medellin (Colombia) and Rio de Janeiro (Brazil). The possibilities of using this approach to fight malaria and to implement gene-delivery technologies (as homing endonucleases and CRISP-Cas9) are being explored. Major challenges faced by these approaches are public acceptance/regulatory approvals and the need for stakeholder engagement. In addition, it is possible that upon application, Wolbachia and their insect hosts may develop mechanisms of resistance that prevent population extinction.
Conclusions: Altogether, old and new approaches to eradicate these diseases are promising but are facing multiple challenges and may require of multiple parallel strategies that include technological, economical and effective monitoring approaches to be fully effective. This review perfectly summarizes the current situation, the problems being faced in fighting these vector-borne diseases and the future challenges that are to be taken in order to achieve this highly desired goal.
Wolbachia are intracellular bacteria infecting arthropods including two-thirds of insect species. The success of Wolbachia to be transmitted to the next generation is linked to its ability to promote the maternal germline through an array of reproductive manipulations that include feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI). CI blocks the production of viable progeny unless the mating pair is infected with the same Wolbachia strain. The mechanism behind CI had been unsolved despite years of research on the topic. In this manuscript, Beckmann and coworkers elucidate the genetic mechanism behind CI by revealing the role of a Wolbachia deubiquitylating enzyme (DUB) in this process. The small polypeptide ubiquitin binds to multiple proteins modifying their activity/function and its removal is performed by DUBs. The DUB-enzyme, CidB, cleaves ubiquitin from substrates and is strongly associated with its operonic partner CidA. In Drosophila melanogaster CI could be observed after transgenic expression of cidB-cidA. In yeast, CidB induces toxicity as a result of DUB activity, but this toxicity is abolished by CidA coexpression. This mechanism reveals that CI is induced by secretion of interacting Wolbachia proteins into the germline and provides new targets for the development of insect control drugs. In CI, paternal chromatin is unable to condense properly in the first cell cycle, leading to embryo lethality. This lethality can be rescued when both parents are infected with the same Wolbachia strain in what has been described as a toxin-antidote system. The authors identified the cidB-cidA operon while looking for proteins in the Wolbachia wPip strain that would associate with Wolbachia-modified mosquito spermatozoids. CI systems may have evolved differently in various host-symbiont partnerships, thus CI rescue can only be performed when both male and female contain Wolbachia strains with homologous CI systems. In addition, CI-inducing strains may possess various CI-inducing systems with different duplication numbers, which lead to a strain specificity. In Wolbachia strain wPip, two homologous operons coexist: cidB-cidA and cinB-cinA. When coexpressed in artificial systems, each protein revealed a highly specific affinity for its cognate partner. Both CinB and CidB (but not their partners) expression in the yeast Saccharomyces cerevisiae induced temperature-dependent growth inhibition. This phenotype could be rescued by coexpression of their CidA or CinA partners. When the cidB-cidA operon was transgenically inserted into a D. melanogaster strain lacking Wolbachia, CI was observed in transgenic male crosses with wt females, but females expressing the operon did not induce CI. This result indicated that cidB-cidA only induces embryonic lethality when inherited by males. When these transgenic males were crossed with transgenic females expressing CidA or the cidB-cidA operon, CI rescue did not take place. The cytological and embryonic CI-like effects observed in transgenic flies were similar to those observed in wild type flies infected with Wolbachia. The location of the cidB-cidA operon within a WO prophage also suggests that CI-abilities could be transmitted to other bacteria via viral cell lysis. This excellent manuscript provides the first genetic evidence for the mechanism responsible for the induction of CI since its discovery in Wolbachia in 1971.
Wolbachia-induced parthenogenesis is less common than CI and has been found in some species of mites, hymenopterans and thrips. In these species, males drive from unfertilized eggs (haploids) and females develop from fertilized eggs (diploids). Wolbachia-induced parthenogenesis leads to the development of diploid females from unfertilized eggs. These females, unlike haploid males, are able to transmit Wolbachia to their progeny, thus explaining the advantage for Wolbachia in inducing this parthenogenesis. Parthenogenesis originates also from an early disruption in the cell cycle during embryogenesis. In some cases, anaphase is abortive at the first embryonic division leading to the development of a single diploid nucleus. In other species, the first mitosis occurs without major problems, but the diploid females occur from the fusion of two haploid nuclei. In a third case, Wolbachia-induced parthenogenesis results from an alteration in meiosis, resulting in diploid gametes. Feminization occurs in isopods and some insects through different mechanisms and results in the development of females from genetic males. This phenotype is thought to result from an alteration of the gender-determination system of the insect. Male-killing occurs in Coleoptera, Diptera, Lepidoptera and Pseudoscorpiones where Wolbachia is responsible for the killing of the male progeny during embryogenesis. Similarly to feminization and parthenogenesis, this phenotype results in an increased amount of females, who are responsible for Wolbachia transmission. In that particular case, the killing of the male progeny increases the food available for the remaining females securing their survival. In the lepidopteran Ostrinia scapulalis, tetracycline treatment to kill Wolbachia transforms all-female broods into all-male broods. This phenomenon suggests that the gender ratio of this moth is also targeted by another gender-distorting mechanism independent of Wolbachia. In addition, this result also evidences that in the absence of Wolbachia, females die during larval development, while in the presence of Wolbachia genetic males are feminized and die during larval development (lethal feminization). In some cases, male-killing may spread through the whole population altering the previous reproductive strategy of their hosts. Some Wolbachia strains have been found to induce different phenotypes when infecting different hosts. This data also suggests that the genetic mechanisms to induce different phenotypes may be present in a single strain, and the different hosts may either be unaffected or have acquired resistance mechanisms. As previously stated, in nematodes, Wolbachia seems to exert a mutualistic relationship. Antibiotic treatments in filarial nematodes affect moulting and reproduction in these worms. As a result, antibiotic depletion of Wolbachia is a useful treatment for filariasis, resulting in slow death of the adult worms and female sterilization. Wolbachia has been found to influence the inflammatory response during infections due to the presence of Wolbachia antigens as the surface protein Wsp. Although little is known about the relationship between Wolbachia and filarial nematodes, in the parasitoid wasp Asobara tabida, antibiotic depletion of Wolbachia leads to a failure in ovarian development, which could potentially infer a mutualistic relationship. Although not observed in other related wasps, another study suggests that Wolbachia downregulate apoptotic processes in the ovaries, thus, Wolbachia removal leads to ovarian cells’ death. In D.melanogaster, Wolbachia induces protection against RNA viruses (Teixeira et al., (2008), PLoS Biol; Chrostek et al., (2013) PLoS Genet). Wolbachia is considered the one the history greatest pandemics affecting at least 10^6 species of insects. The mechanisms responsible for the movement of Wolbachia between species are unclear, although reports have shown evidence of Wolbachia remaining viable for a good period after their host death. That viability may allow the spread amongst taxa parasitizing a common host. On the other hand, Wolbachia may disappear from a host as a consequence of the appearance of resistance events or may be displaced by the acquisition of a different strain. One of the most interesting effects of Wolbachia with major ecological impacts is the possibility of playing or having played a major role in host speciation. In this context, phenotypes as CI may have driven the segregation of a single species into distinct populations that after several generations possess enough genetic divergence to be considered different species with incompatible backgrounds. In addition, lateral gene transfer between Wolbachia and their invertebrate hosts is common. This could provide additional gene functions to the host and play additional roles in speciation. Besides all the interesting features of Wolbachia biology, a major interest lies in the practical applications of this symbiont. Wolbachia is an excellent target for the treatment of filarial infections and the search for novel anti-Wolbachia drugs is undergoing. Additionally, Wolbachia-induced CI is an excellent target for the development of novel drugs to target vector-borne diseases and insect pests. Efforts in this area consist of replacing insect populations infected with vector-borne diseases by disease-free populations. Alternatively, the replacement of insect populations by others, containing Wolbachia strains providing resistance to viral infections is an excellent approach that has already proven extremely successful (Walker et al., (2011), Nature; Hoffmann et al., (2011), Nature). Altogether, this fantastic review, although now being not so up-to-date, provides a fantastic background on the biology of Wolbachia and the applicability of this symbiont to understand biological processes and resolve problems derived from vector-borne diseases.
This fantastic review from Werren, Baldo and Clark is a must-read article for people interested in insect symbiosis. The authors perfectly summarize all the information published to date on the effects of the bacterial symbiont Wolbachia in the biology of their hosts, with a keen interest in the reproductive manipulations that they produce in them. All aspects of insect symbiosis are covered in this manuscript, from basic definitions to the most complex interactions specifically taking place in the different hosts. Symbiosis was initially defined by Anton de Bary in 1879 as the living together of two distinct organisms. This interaction can be classified into three main types: mutualism (when both organisms benefit from the interaction), commensalism (when one of the organisms benefits from the interaction) or parasitism (when one of the organisms is harmed by the presence of the other). Although this classification has been maintained to date, the complexity of these interactions goes beyond that, and requires a careful examination of all aspects of the symbiosis in all biological functions, at different time points during the symbiosis lifetime and at different generations (since symbioses are dynamic interactions that may evolve under changing conditions). The bacterial symbiont Wolbachia provides an excellent opportunity to study these interactions since is considered the most prevalent symbiont in invertebrates. Wolbachia are members of the order Rickettsiales, a group of intracellular bacteria that establish all three (mutualistic, commensal and parasitic) interactions with their hosts. Currently, Wolbachia are classified into 8 different supergroups (A-H), but this classification may be further revised as more information is gathered on these bacteria. Supergroups C and D are commonly found in filarial nematodes (roundworms transmitted by insect hosts that cause disease in animals including humans), while the remaining 6 groups are found in arthropods with a higher prevalence of supergroups A and B. While filarial- Wolbachia associations are considered mutualist, in arthropods Wolbachia associations are generally considered as parasitic since generally, they behave as reproductive parasites in their hosts. The advances in molecular typing methods in the 1990s switched the view on Wolbachia as a rare bacterial genus, and revealed its presence in 10^6 insect species, making it the most abundant intracellular bacteria found so far. An interesting feature of Wolbachia is the large amount of reproductive phenotypes it induces in their different hosts: feminization (genetic males appear as females), parthenogenesis (reproduction occurs from unfertilized eggs without the need of males), male-killing (killing of the male progeny), and cytoplasmic incompatibility (where only mating between insects infected with the same Wolbachia are able to produce viable progeny). All these phenotypes promote the transfer of Wolbachia to the next generation, selecting for an abundance of females that are generally the only ones capable of transmitting Wolbachia (an exception to this rule has been reported in the article published by Watanabe et al., (2014), PNAS, that I previously commented in this platform, but even in this report, female-transmission was shown to be more effective than male transmission). The major questions surrounding the success of Wolbachia lie on the molecular mechanisms and gene determinants of symbiosis and reproductive parasitism. Since the publication of this review, significant advances have been made on these matters (Beckmann et al., (2017) Nat Microbiol; Lepage et al.,(2017), Nature), but these questions still remain due to the unculturability of Wolbachia in regular laboratory media, which would allow the use of molecular microbiology techniques to elucidate the genes responsible for these functions. At the time of this review, 2 completed genomes of Wolbachia had been solved: the CI-inducing Wolbachia strain wMel from the fruit fly Drosophila melanogaster and the mutualistic wBm strain from the filarial nematode Brugia malayi. To date (March 2020) 54 completed genomes have been solved and are available in public databases. The main characteristic of these genomes is their small size and a large number of mobile repetitive elements including ankyrin domains (ANK linked to type IV secretion effectors involved in pathogenesis in Legionella pneumophila), common in eukaryotes but rare in prokaryotes. Another significant trait in these genomes is the presence of multiple viral elements, including the lambda bacteriophage WO, shown to be linked to cytoplasmic incompatibility in a later publication (Lepage et al., (2017), Nature). Interestingly, the amount of repetitive elements is lower in filarial Wolbachia, and phage DNA is absent. Three additional (surface) proteins Wsp, WspA and WspB, associated with host-pathogen interactions have been found in Wolbachia genomes and are studied for their potential role in immunological responses in their hosts. A great deal of the success of Wolbachia lies in its ability to associate with the host cells, where it tightly associates with centrosomes and microtubules, localizing at almost equal numbers to each spindle pole to ensure equal bacterial loads in each cell. During oogenesis, Wolbachia is localized at specific regions of the germline, in close interaction with the molecular motors dynein and kinesin to ensure passage to the new generation. The unculturability of Wolbachia has been the major problem to both, link genotypes and phenotypes, and to provide a proper classification of study of strain diversity. Sequencing of Wolbachia genomes is performed by co-extraction of the symbiont DNA together with that of the host and other symbionts and microbes present. In addition, in some cases multiple Wolbachia strains co-inhabit the same host, making it harder to obtain high-quality DNA sequences that can be linked to one single strain. Additionally, the presence of multiple recombination events in Wolbachia also adds difficulties to obtain single genomes. The discovery of extensive recombination events amongst Wolbachia strains also adds more difficulties to classification and single genome sequencing attempts. Novel sequencing and genotyping approaches have led to a tremendous increase in the number of Wolbachia strains sequenced since the publication of this review. One of the most interesting features of Wolbachia is their ability to induce diverse reproductive manipulations in their hosts, the most common of them being cytoplasmic incompatibility (CI). CI results in the incompatibility between spermatozoid and eggs when both progenitors have different Wolbachia types. The defects induced by Wolbachia in the spermatozoid are rescued in embryos infected by the same Wolbachia type. Despite recent advances (Beckmann et al., (2017) Nat Microbiol; Lepage et al.,(2017), Nature), little is known about the molecular mechanisms of Wolbachia-induced CI. CI-resulting incompatible crosses lead to asynchrony of male and female pronuclei at the early stages of mitosis, where female chromatids are properly condensed at the first metaphase stage, while male chromosomes are found in a semi-condensed state. At the anaphase, female chromosomes separate normally but male pronuclei are stretched towards the centrosome poles or lay entirely excluded. In haplodiploid organisms, this leads to the development of haploid development, while in diploid organisms it leads to embryonic lethality. Different Wolbachia strains may have different CI (modification and rescue) mechanisms, thus resulting in double incompatibility mechanisms rather than CI rescue.
In this fantastic research article, Hehemann and coworkers were interested in finding enzymes capable of degrading the sulphated polysaccharides present in marine algae but absent in terrestrial plants. Some of these algae as Porphyra (nori), Ulva (sea lettuce) or Undaria (wakame) are edible algae often used in various recipes in Asian countries. While looking at the genome of the marine Bacteroidetes Zobellia galactanivorans, the authors identified five glycoside hydrolases: Zg1017, Zg2600, Zg3376, Zg3628 and Zg3640 remotely related to β-agarases and κ-carrageenases but lacking the required residues to act on agarose or κ-carrageenan. All these sequences contain the catalytic signature EXDXXE typical of glycoside hydrolase family 16 (GH16).
Homologous expression of these five genes in Escherichia coli revealed activity on extracts from the agarophytic red algae Gelidium, Gracilaria and Porphyra. Two of these proteins, Zg2600 and Zg1017, we shown to be active on the β-1,4-glycosidic bond of the disaccharide α-L-galactopyranose-6-sulphate (L6S) with the 3-linked β-D-galactopyranose units (G). These proteins were the first two β-porphyranases to be described and were subsequently named PorA ad PorB. The genes encoding the remaining homologues found in Z. galactanivorans (Zg3376, Zg3628 and Zg3640) were named porC, porD and porE, respectively. The crystal structures of PorB and an inactive mutant of PorA were solved and the structure of PorA was elucidated in complex with a porphyran tetrasaccharide substrate allowing the identification of key residues in porphyran binding. While looking for additional porphyranases in sequence databases, the authors found six homologues allowing the establishment of a new GH16 subfamily. Five out of six homologues were found in marine bacterial genomes and one homologue, Bp1689, in the genome of the human gut bacterium Bacteroides plebeius. All isolated species of this bacterium have been exclusively isolated from the microbiota of Japanese individuals and none of the additional 24 Bacteroides sequenced genomes contain homologues to β-porphyranase or β-agarase genes. The genomic location of the GH16 β-porphyranase gene (bp1689) and the β-agarase gene (bp1670) is within carbohydrate-related genes that have their closest orthologues in marine bacteria. This observation leads to the hypothesis that the gut symbiont B. plebeius received an unusual set of genes most probably by horizontal gene transfer (HGT) from marine bacteria. This hypothesis is strongly supported by the identification of genes coding for mobilisation proteins at the downstream region of bp1670, which indicate an ancestral HGT transfer of this porphyran utilisation locus from a marine bacterium related to Z. galactanivorans or Microscilla sp. PRE1. Oceanic sample analyses revealed the absence of bacteria containing these genes in open water samples, thus restricting them to coastal samples where red algae are present. The gut metagenome of 13 Japanese volunteers was analysed revealing the presence of seven potential porphyranase homologues in four individuals with 31%-42% identity to PorB, and 83%-100% identity to Bp1689. The presence of these homologues in one mother and her unweaned baby also indicates that the Bacteroides-containing microbiota harbouring these genes may be passed from mother to offspring. The authors also examined the microbiome of 18 North American individuals finding no trace of porphyranase or agarase genes. Altogether, these findings suggest that the HGT leading to the acquisition of porphyranase and agarase genes in Japanese populations took place due to consumption of raw nori. Nori was traditionally served raw in sushi and the constant ingestion of this food source lead to this HGT event in Japanese microbiota. This study by Hehemann and coworkers is the first report of an HGT event occurring as a consequence of dietary habits that could provide us with the ability to exploit additional food sources.
Pyoverdines (PVDs) are the major siderophores produced by a subgroup of Pseudomonas species that due to the production of these molecules has been termed fluorescent pseudomonads. This group includes amongst others: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae and Azotobacter vinelandii. The interest on the study of pyoverdines is due to their role in the virulence, survival and cell-to-cell communication in these species, with a keen eye on the opportunistic pathogen P. aeruginosa and the plant biocontrol species P. fluorescens. Although pyoverdines can bind to other metals with different affinities, iron is considered to be the main target of these siderophores given the iron scarcity and the high affinity of PVD towards this metal. Pyoverdines have a characteristic chromophore, which is responsible for their fluorescence and is an active part of the iron-binding site. The core of these molecules is composed of a peptide of variable composition that is synthesised through the PVD non-ribosomal peptide synthases (NRPS). The identity of these different peptides forming PVD has been used for taxonomical purposes to differentiate amongst different fluorescent species. Currently, the structure of >50 pyoverdines has been elucidated and classification into three different groups has been established using different analytical techniques (isoelectrofocusing analysis, growth stimulation, immunoblot detection of PVD receptor and iron uptake). The first PVD genes to be elucidated in the PVD pathway were those encoding the four NRPS: pvdL, pvdI, pvdJ and pvdD; and subsequently, the genes encoding the PVD receptor fpvA, the PVD regulator pvdS ad its link to the major ferric uptake regulator FUR. Later, the work by the groups of Lamont and Schalk revealed the role of the ATP-binding cassette protein PvdE and the ATP-dependent efflux pump PVDRT-OpmQ involved in the transport and secretion of PVD. Additionally, a group of genes were previously identified as potentially involved in PVD synthesis due to the presence of a PvdS promoter or to homology to genes coding for siderophore and other secondary metabolites’ synthesis (Lamont and Martin, Microbiology 2003). PvdM, PvdN, PvdO, PvdP and PvdQ were the remaining genes whose function needed to be elucidated to complete the full biosynthetic pathway of PVD. In the recent years, PvdQ was demonstrated to be an Ntn-type hydrolase responsible from the removal of the acyl chain of the PVD precursor, ferribactin, during PVD maturation and cellular transport from the cytoplasm to the periplasm. PvdP was shown to be a tyrosinase acting on the tyrosine residue of ferribactin to transform it into the mature chromophore (the iron-binding core) of PVD.
In this manuscript, Ringel, Drager and Bruser elucidate the role of PvdN in transforming the N-terminal glutamic acid to succinamide. This modification was also shown not to be essential for growth under iron-limiting conditions at a broad range of pH. The authors link this modification to the different PVD isoforms that are often found when isolating PVD from a single strain. It is thus plausible that a mixture of PVD exists where PvdN has acted only on deal of them. This work leaves open exciting questions on the role of PvdN: if this enzyme is not necessary for PVD function, why is it highly conserved amongst fluorescent pseudomonads? What is the role of the two isoforms (PvdN- and PvdN+) in these bacteria? And finally, what are the conditions in which each form plays a role? Further studies and elucidation of PvdM function and the recently elucidated PvdO function (Ringel, Drager and Bruser, J. Biol. Chem, 2018) may be essential to understand these questions and complete a pathway that has been a quest for a few decades.
Quorum sensing is a cell-to-cell communication process used by many bacteria to coordinate group behaviour according to population density and species composition. Quorum sensing relies on the production and detection of small signalling molecules termed autoinducers that control different functions in bacteria including bioluminescence, secretion of virulence factors, biofilm formation, and DNA exchange. In the human pathogen Vibrio cholerae, the causative agent of cholera, two quorum-sensing systems have been described. The cholera autoinducer 1, CAI-1 ((S)-3-hydroxytridecan-4-one), which is found in Vibrio species (intra-genus communication) and is produced by the CqsA enzyme and AI-2 (4,5-dihydroxy-2,3-pentanedione) that is synthesized by LuxS, which is present in multiple bacterial species and thus considered as an inter-species signal. CAI-1 and AI-2 are detected by the membrane-bound receptors CqsS and LuxPQ, which in turn activate a phosphorylation cascade that results in the control of multiple phenotypes including biofilm formation through the transcriptional regulator vpsT.
It has been suggested that additional quorum sensing systems exist in V. cholerae. The authors identified the luxR solo VqmA in a recent transcriptome analysis, which at high cell density activates expression of vqmR, which encodes the VqmR sRNA. VqmR activates the expression of several genes including biofilm formation genes and a major V. cholerae toxin. In this manuscript, the authors isolated VqmA together with its ligand, which was shown to be 3,5-dimethylpyrazin-2-ol (DPO), a novel type of autoinducer. DPO bound VqmA represses biofilm formation in V. cholerae. These results are particularly significant for the development of novel antimicrobial agents. In fact, it had previously been shown that the commensal bacterium Ruminococcus obeum limits the severity of V. cholerae infection by impairing colonization. This effect was eliminated when the test animals were infected with a VqmA mutant. The authors propose that the effects observed are a consequence of the high levels of DPO produced in the small intestine.
Quorum sensing is a cell-to-cell communication process used by many bacteria to coordinate group behaviour according to population density. Quorum sensing relies in the production and detection of small signalling molecules termed autoinducers. In Vibrio cholerae, the causative agent of cholera, one of the quorum-sensing circuits consists of the 3,5-dimethylpyrazin-2-ol (DPO) autoinducer and the receptor VqmA. Upon DPO binding, VqmA activates the expression of vqmR a gene coding for the small RNA VqmR, which represses genes required for biofilm formation and virulence factor production.
In bacteria many virulence factors and toxigenic strains have been shown to emerge due to the presence of phages. In V. cholerae, the lysogenic CTXφ temperate phage encodes the major virulence factor (CTX) in this bacterium. In this manuscript, Silpe and Bassler revealed that vibriophage VP882, found in V. parahaemolyticus O3:K6 strain and infecting both V. parahaemolyticus and V. cholerae is able to respond to the DPO quorum-sensing molecule of their host. This vibriophage encodes a VqmA homologue, which the authors term VqmA-phage, which is a homologue of the VqmA QS receptor in V. cholerae. DPO binding to VqmA-phage induces the phage lytic program via induction of the qtip gene (quorum triggered inactivator of cI protein). The Qtip protein (encoded by the gene of the same name) aggregates and binds to the VP882 lysis repressor. While DPO-bound VqmA-phage binds to the vqmR promoter, DPO-bound to the V. cholerae VqmA is unable to bind to vqmR. The authors also report that phages related to VP882 encode DNA-transcription factors and small proteins with identical genomic locations as the vqmA-Phage and qtip present in VP882.
Using this principle the authors designed a VP882 system in Salmonella typhimurium and demonstrated the ability of tis system to target selected pathogens. The results revealed in this manuscript indicate that quorum sensing could be used to program phages to induce their “kill-switch” with environmental, industrial and medical applications.
Antibiotic resistance has often been linked to the spontaneous appearance of mutations that are selected and promoted under selective pressure caused by the use of antibiotics in clinical and farm environments. Despite this, antibiotic resistance is a natural process that occurs often as a result of competitive strategies employed by bacteria to compete amongst other species. The production of antibiotics is a common strategy to eliminate competitors and requires the development of antibiotic resistance towards the antibiotic being produced. In addition, antibiotic resistance also exerts selective pressure on other bacteria in the same ecological niche that may use the same resistance-developing strategy to survive to the antibiotics produced by others. Staphylococcus aureus is a Gram-positive bacterium often associated with life-threatening human infections associated with clinical settings. While S. aureus is a common inhabitant of the human nasopharynx and skin, under certain conditions, some strains may become pathogenic and pose serious risks. These risks become life-threatening when the pathogenic strains are antibiotic-resistant. In this article, Koch and coworkers describe a natural process leading to a spontaneous mutation in S. aureus originating from competition amongst bacterial cells of the same strain. Some of the cells of methicillin-resistant S. aureus (MRSA) evolve into a mutant strain (W strain) that produces high levels of surfactants and a toxic bacteriocin. Subsequently, the wild type cells counteract by producing another mutant (Y strain), which is resistant to this bacteriocin and to an intermediate level of vancomycin. The vancomycin-resistant mutation (VISA for vancomycin-intermediate S. aureus) has been observed regularly in clinical isolates, which are difficult to treat. This study reveals how antibiotic resistance can spontaneously arise in S. aureus as a consequence of competition between cells originating from the same parental strain. Additionally, to demonstrate that the appearance of W and Y strains is not linked exclusively to biofilms formed in agar plates, the authors demonstrate the presence of W and Y strains (in addition to the parental O strain) in mice five days after infection with O-strain S. aureus. The authors aimed as well to understand the phenotypical differences between O, W and Y strain in order to link genotype and phenotype. Their work revealed that in contrast to the O strain, which has a strong orange pigmentation due to the production of the pigment staphyloxanthin, strain W is white and rapidly develops around the orange O strain. Subsequently, the Y strain develops from the orange area at the centre of the colony displaying yellow flares that spread over the white (W) population. Using qRT-PCR, the authors revealed different expression levels of key genes involved in pigment production, biofilm formation, hemolysis and biofilm formation; confirming that the three strains are different from one another. These phenotypes had previously been linked to the agr cell-to-cell communication (quorum sensing) system of S. aureus. Indeed, quorum sensing seems to be strongly repressed in the wild type O strain when compared to the W strain. When analysed genetically, these differences were shown to be due to a loss of function mutation in the sigma factor σB, which represses quorum sensing in this bacterium. As a result of this mutation in the W strain, antibiotics and surfactants are overproduced inhibiting the O strain and rapid expansion of the W strain. That pressure induces the appearance of the Y strain from the O strain, which possesses mutations in the GraRS and WalKR two-component systems that promote resistance to Bsa, the epidermin-like antibiotic produced by the W strain. The mode of action of Bsa is similar to that of vancomycin, targeting lipid II during bacterial cell-wall synthesis. As such, this arms race between O, W and Y strains results in resistance towards vancomycin, a clinically used antibiotic that has been claimed as the last line of defence against multiresistant Gram-positive bacteria.
In this exciting manuscript, Weiss et al. reveal a mechanism that pathogens use to evade the host immune system. Using the tsetse fly (Glossina morsitans) and its facultative mutualist symbiont Sodalis glossinidius as a model system, they explore the genetic factors that allow Sodalis to establish symbiosis with its host. The authors use the common lab strain E. coli K12, which induces fly lethality in tsetse flies, and the fruit fly Drosophila melanogaster, which is resistant to both Sodalis and E. coli infections to analyze the different phenotypes resulting from each bacterial-insect interaction. In a previous study, the authors analyzed the cell-membrane immunogenic components encoded in the genome of S. glossinidius. Their work revealed the presence of a truncated lipopolysaccharide (LPS) that misses the O-antigen, and a modified outer membrane protein (OmpA, a major component of the outer membrane in Enterobacteriaceae). In this study, the authors focused exclusively on OmpA. Initially, they tested E. coli K12 (MG1655; K12OmpA::Tn5Kan-2), an E. coli K12 transposon mutant with a disruption in the ompA gene. They revealed that, contrary to the wild type E. coli K12, this mutant is no longer lethal for tsetse flies. Subsequently, they complemented this mutant with the original E. coli ompA and with the Sodalis ompA gene, restoring lethality only with the E. coli version. Furthermore, they aimed to corroborate whether this effect was also taking place on a Sodalis background. As expected, when expressed in Sodalis, ompA_E. coli induced lethality in tsetse flies whilst ompA_Sodalis maintained Sodalis avirulent. As a control, the authors also injected D. melanogaster with Sodalis expressing OmpA_E. coli, which resulted in the survival of these flies. This excellent control provided two main conclusions: a) it demonstrates that the mortality caused by E. coli is not exclusively due to the faster growth of this bacterium potentially causing septic shock and b) it reveals that the lethality of E. coli OmpA is host-dependent. On the latter point, the authors suggest that the different lifestyles of tsetse flies and fruit flies may have led the second group to evolve a stronger tolerance to pathogens.
The authors then aim to investigate the potential roles that OmpA from these two different bacteria may have in the host. To this aim, they look into the expression of different immune genes in tsetse flies when exposed to avirulent (Sodalis WT, and E. coli-ompA-) and virulent strains (E. coli WT and Sodalis-ompA_E. coli). Using qPCR analysis, they elucidated that the expression levels of antimicrobial peptides (AMPs) that are controlled by Imd and Toll pathways were lower in flies infected with virulent strains. In addition, virulent bacteria also induced expression of the host pgrp-lb, a negative regulator of the Imd pathway. Low Imd levels may have prevented the activation of the innate immune response in flies infected with virulent bacteria. Finally, the authors also demonstrate that avirulent E. coli (ompA- and ompA_Sodalis) end up being cleared from the host whether Sodalis is able to persist. This observation aims to clarify that despite the significance of the findings in this manuscript, the full genetic basis of bacteria-insect symbiosis is much more complex than OmpA, leaving a full niche and lots of interesting discoveries to follow up on this manuscript. In this exciting manuscript, the authors are revealing a crucial mechanism that bacteria may have evolved to “control themselves and behave properly” in order to establish a long-term relationship with their host rather than a short-term pathogenic interaction.
Consumables commonly used in research labs have often elevated prices that may result too costly to weakly funded labs. As a result, these labs may have their research capabilities limited to the number of techniques and experiments that can be performed under a low budget. A direct consequence of this is often the low impact that the outcome of their research may have, hindering their possibilities to obtain competitive funding, recruiting highly qualified personnel and acquiring expensive equipment. This situation perpetuates a system that unfortunately creates big quality gaps between research labs according to funding opportunities that only can be (sometimes) overcome by the high quality and hard work of their members. In this manuscript, Henrici and coworkers developed a pair of plasmids that after enzymatic digestion can be used as DNA ladders in molecular biology labs. As claimed by the authors, 100 ml of E. coli harbouring the plasmids pPSU1 and pPSU2 are able to produce enough DNA ladder for 1000 DNA gels. Also, these plasmids contain a good number of restriction sites allowing the production of different ladders according to different needs. This can be optimal for those interested in distinguishing fragments of similar lengths. The manuscript details the construction and structure of both plasmids, which they have been constructed using the pUC9 backbone vector. This plasmid is a high-copy number plasmid, free of license restrictions and containing an ampicillin resistance cassette for selection and maintenance, which is also one of the cheapest and most widely available antibiotics in the market. The authors also provide a price comparison in table 2 to justify the use of their plasmids based on the low costs. In our experience, these plasmids have now been deposited in Addgene, and must now be purchased from this company at a cost of 65$ each (at the time of purchase). Despite this being a relatively low investment for a material that can be used over and over, these costs should also be taken into consideration when assessing the usefulness of this alternative. It is important as well to remark that the costs of the preparation of the loading dye are not included in these calculations. In our experience, this is a useful tool that can help to decrease the costs in labs that are constantly running DNA gels. It is important to note that after enzymatic digestion, the enzyme should be properly inactivated or the digestion product should be passed through a purification column (additional cost in the later) to avoid degradation of the ladder. Once prepared, the pPSU-derived ladder should be stored in the freezer as any other ladder to keep maintaining its quality. Altogether, a useful approach that can contribute to reducing materials’ costs in some labs in return from some additional invested labour. In addition to this manuscript, other interesting cost-reducing alternatives have been previously reported, as the production of homemade Taq polymerase (Konovalova et al., Data Brief, 2017; Chen et al., Electronic Journal of Biotechnology, 2015), as well as high-fidelity polymerases (available at https://barricklab.org/twiki/ bin/view/Lab/ProtocolsReagentsPfuSso7d); alternatives to restriction enzymes (Okegawa et al., Biochemistry and Biophysics Reports, 2015); and a series of other tools developed with the sole intention to democratise biotechnology labs, a topic that has been massively analysed in various social media.
In this exciting study, Watanabe and colleagues demonstrate how vertically transmitted symbionts are not exclusively transmitted by females. While studying the transmission of a Rickettsia bacterial symbiont in the green rice leafhopper Nephotettix cincticeps, the authors discovered the presence of this bacterium in the spermatozoid of insect males. This infection did not seem to affect the host fitness nor spermatozoid function. The presence of Rickettsia in the spermatozoid of this insect was unexpected since bacterial symbionts are more prone to take advantage of the rich egg's cytoplasm as a growing environment. On the contrary, spermatozoid cells discard their cytoplasm during spermatogenesis and consist of condensed nuclei (in the head) and microtubule bundles (in the tail), altogether a much less rich environment than eggs to support bacterial growth. In addition, the presence of bacteria in the spermatozoid's head is likely to affect the genetic material and the spermatozoid function. In the green rice leafhopper, Rickettsia cells have been found not only in the cytoplasm but also in the nucleus of various cells and tissues. It thus seems obvious the preference that Rickettsia has for cell nuclei. Using Rickettsia infected and uninfected strains of leafhoppers with identical genetic background, the authors proved that although being less efficient (61.8% vs 100%), Rickettsia could efficiently be paternally transmitted. Paternally derived Rickettsia-infections were 100 % established in the subsequent generations. A result that reveals that only a few Rickettsia cells are enough to establish full infection. This work opens many possibilities in the fascinating world of bacterial endosymbionts. If this Rickettsia lineage can be transmitted both by the eggs and spermatozoid, it is plausible that different strains of Rickettsia may enter in contact in the same host. Could this competition affect some of the reproductive abnormalities that some of these bacteria cause in various hosts (cytoplasmic incompatibility, male-killing, parthenogenesis and feminization)? Could competition derive into virulent strains that are no longer able to establish symbiosis and result thus in pathogenesis? What consequences would that have in the insect population as well as potential human infections? Finally, this discovery opens also the door for the potential development of genetic transformation and material delivery through male gametes.
In this article, Kroiss and coworkers provide an excellent example of how observations and keen interest to understand the “tiny” world around us may prove highly valuable for the finding of novel bioactive molecules for their use in human health. This manuscript reveals a previously unknown symbiosis between beewolf digger wasps (Philantus spp.) and actinobacteria from the genus Streptomyces (Candidatus Streptomyces philanthi). The authors were puzzled by how the eggs of these wasps that are laid in warm and humid underground environments would survive potential bacterial and fungal infections. Their observations revealed that female beewolves cover the brood cells (where their eggs are being laid) with a substance originating from their “particularly thick” antennae. Upon analysis, they revealed that female beewolves cultivate Streptomyces in unique antennal glands and that the application of these Streptomyces cultures to the cells significantly increases the survival of their larvae.
Further analysis of the beewolf cocoons led to the discovery of nine antibiotic substances, which were further analyzed for their antimicrobial activity. The antibiotics isolated were streptochlorin and a complex of eight piericidin derivatives (piericidin A1, piericidin B1, glucopiericidin A, piericidin A5, piericidin C1, 9’-demethyl-piericidin A1, piericidin B5, and piericidin IT-143-B). Despite all these antibiotics that had previously been isolated from Streptomyces spp. inhabiting terrestrial and marine environments, this particular antibiotic cocktail had never been described. After testing this cocktail on ten different fungal and bacterial species (fungi: Aspergillus fumigatus, Aspergillus flavus, Penicillium notatum, Penicillium avellaneum, Sporobolomyces salmonicolor, Fusarium oxysporum, Metarhizium anisopliae, Beauveria bassiana; and bacteria: Bacillus subtilis, and Paenibacillus larvae), the results revealed that this cocktail strongly inhibits the growth of all tested microbes. While most microbes were most susceptible to the most abundant antibiotic (piericidin A1), some species were more inhibited by the other antibiotics, proving the effectiveness of the complex antibiotic cocktail.
In their exquisite work, the authors reveal how behavioral observations (a mother wasp grooming its antennae and petting right after her eggs and brood cells) and admiration for the natural world can help us both, to understand the complex interactions taking place between kingdoms, and additionally provide a novel source of bioactive molecules that may be invaluable to the healthcare industry and human wellbeing. Further and similar research may unleash a complete set of novel bioactive molecules or combinations with great potential uses.
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