Comparative analysis of surface sanitization protocols on the bacterial community structures in the hospital environment.

OBJECTIVES: In hospital hygiene, it remains unclear to what extent surface contamination might represent a potential reservoir for nosocomial pathogens. This study investigates the effects of different sanitization strategies on the microbial structures and the ecological balance of the environmental microbiome in the clinical setting. METHODS: Three cleaning regimes (disinfectants, detergents, and probiotics) were applied subsequently in nine independent patient rooms at a neurological ward (Charité, Berlin). Weekly sampling procedures included three different environmental sites: floor, door handle, and sink. Characterization of the environmental microbiota and detection of antibiotic resistance genes (ARGs) were performed by 16S rRNA sequencing and multiplex Taq-Man qPCR assays, respectively. RESULTS: Our results showed a displacement of the intrinsic environmental microbiota after probiotic sanitization, which reached statistical significance in the sink samples (median 16S-rRNA copies = 138.3; IQR: 24.38-379.5) when compared to traditional disinfection measures (median 16S rRNA copies = 1343; IQR: 330.9-9479; p < 0.05). This effect was concomitant with a significant increase in the alpha-diversity metrics in both the floor (p < 0.001) and the sink samples (p < 0.01) during the probiotic strategy. We did not observe a sanitization-dependent change in relative pathogen abundance at any tested site, but there was a significant reduction in the total ARG counts in the sink samples during probiotic cleaning (mean ARGs/sample: 0.095 ± 0.067) when compared to the disinfection strategy (mean ARGs/sample: 0.386 ± 0.116; p < 0.01). DISCUSSION: The data presented in this study suggest that probiotic sanitization is an interesting strategy in hospital hygiene management to be further analyzed and validated in randomized clinical studies.

Inhibition of the NLRP3/IL-1ß axis protects against sepsis-induced cardiomyopathy.

BACKGROUND: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1ß (IL-1ß), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1ß in sepsis-induced cardiomyopathy and cardiac atrophy. METHODS: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1ß effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1ß on cardiomyocytes. RESULTS: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 µL vs. 24.6 ± 8.7 µL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 µL vs. 29.9 ± 9.9 µL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1ß decreased contractility (cell shortening: IL-1ß: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1ß) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1ß: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1ß), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1ß reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1ß activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA. CONCLUSIONS: IL-1ß causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1ß pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Bacterial colonization dynamics and antibiotic resistance gene dissemination in the hospital environment after first patient occupancy: a longitudinal metagenetic study.

BACKGROUND: Humans spend the bulk of their time in indoor environments. This space is shared with an indoor ecosystem of microorganisms, which are in continuous exchange with the human inhabitants. In the particular case of hospitals, the environmental microorganisms may influence patient recovery and outcome. An understanding of the bacterial community structure in the hospital environment is pivotal for the prevention of hospital-acquired infections and the dissemination of antibiotic resistance genes. In this study, we performed a longitudinal metagenetic approach in a newly opened ward at the Charité Hospital (Berlin) to characterize the dynamics of the bacterial colonization process in the hospital environment after first patient occupancy. RESULTS: The sequencing data showed a site-specific taxonomic succession, which led to stable community structures after only a few weeks. This data was further supported by network analysis and beta-diversity metrics. Furthermore, the fast colonization process was characterized by a significant increase of the bacterial biomass and its alpha-diversity. The compositional dynamics could be linked to the exchange with the patient microbiota. Over a time course of 30 weeks, we did not detect a rise of pathogenic bacteria in the hospital environment, but a significant increase of antibiotic resistance determinants on the hospital floor. CONCLUSIONS: The results presented in this study provide new insights into different aspects of the environmental microbiome in the clinical setting, and will help to adopt infection control strategies in hospitals and health care-related buildings. Video Abstract.

Context-dependent regulation of immunoglobulin mutagenesis by p53.

p53 plays a major role in genome maintenance. In addition to multiple p53 functions in the control of DNA repair, a regulation of DNA damage bypass via translesion synthesis has been implied in vitro. Somatic hypermutation of immunoglobulin genes for affinity maturation of antibody responses is based on aberrant translesion polymerase action and must be subject to stringent control to prevent genetic alterations and lymphomagenesis. When studying the role of p53 in somatic hypermutation in vivo, we found altered translesion polymerase-mediated A:T mutagenesis in mice lacking p53 in all organs, but notably not in mice with B cell-specific p53 inactivation, implying that p53 functions in non-B cells may alter mutagenesis in B cells. During class switch recombination, when p53 prevents formation of chromosomal translocations, we in addition detected a B cell-intrinsic role for p53 in altering G:C and A:T mutagenesis. Thus, p53 regulates translesion polymerase activity and shows differential activity during somatic hypermutation versus class switch recombination in vivo. Finally, p53 inhibition leads to increased somatic hypermutation in human B lymphoma cells. We conclude that loss of p53 function may promote genetic instability via multiple routes during antibody diversification in vivo.

Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities.

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/TH17 polarization. In murine NEC, pro-inflammatory type 3 NKp46-RORγt+Tbet+ innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46+RORγt+ ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.

A simple sequence-based filtering method for the removal of contaminants in low-biomass 16S rRNA amplicon sequencing approaches.

Controlling for contaminant sequences in microbiome experiments involving low-biomass samples is a highly challenging task which still lacks of standardized protocols. Here we propose a simple sequence-based filtering method for 16S rRNA gene microbial profiling approaches, and validate its efficiency using mock community dilution series and environmental samples collected in a clinical setting.

Early Bacterial Colonization and Antibiotic Resistance Gene Acquisition in Newborns.

Several studies have recently identified the main factors contributing to the bacterial colonization of newborns and the dynamics of the infant microbiome development. However, most of these studies address large time periods of weeks or months after birth, thereby missing on important aspects of the early microbiome maturation, such as the acquisition of antibiotic resistance determinants during postpartum hospitalization. The pioneer bacterial colonization and the extent of its associated antibiotic resistance gene (ARG) dissemination during this early phase of life are largely unknown. Studies addressing resistant bacteria or ARGs in neonates often focus only on the presence of particular bacteria or genes from a specific group of antibiotics. In the present study, we investigated the gut-, the oral-, and the skin-microbiota of neonates within the first 72 h after birth using 16S rDNA sequencing approaches. In addition, we screened the neonates and their mothers for the presence of 20 different ARGs by directed TaqMan qPCR assays. The taxonomic analysis of the newborn samples revealed an important shift of the microbiota during the first 72 h after birth, showing a clear site-specific colonization pattern in this very early time frame. Moreover, we report a substantial acquisition of ARGs during postpartum hospitalization, with a very high incidence of macrolide resistance determinants and mecA detection across different body sites of the newborns. This study highlights the importance of antibiotic resistance determinant dissemination in neonates during hospitalization, and the need to investigate the implication of the mothers and the hospital environment as potential sources of ARGs.

Unaltered Fungal Burden and Lethality in Human CEACAM1-Transgenic Mice During Candida albicans Dissemination and Systemic Infection.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is a receptor for Candida albicans. It is crucial for the immune response of intestinal epithelial cells to this opportunistic pathogen. Moreover, CEACAM1 is of importance for the mucosal colonization by different bacterial pathogens. We therefore studied the influence of the human CEACAM1 receptor in human CEACAM1-transgenic mice on the C. albicans colonization and infection utilizing a colonization/dissemination and a systemic infection mouse model. Our results showed no alterations in the host response between the transgenic mice and the wild-type littermates to the C. albicans infections. Both mouse strains showed comparable C. albicans colonization and mycobiota, similar fungal burdens in various organs, and a similar survival in the systemic infection model. Interestingly, some of the mice treated with anti-bacterial antibiotics (to prepare them for C. albicans colonization via oral infection) also showed a strong reduction in endogenous fungi instead of the normally observed increase in fungal numbers. This was independent of the expression of human CEACAM1. In the systemic infection model, the human CEACAM1 expression was differentially regulated in the kidneys and livers of Candida-infected transgenic mice. Notably, in the kidneys, a total loss of the largest human CEACAM1 isoform was observed. However, the overwhelming immune response induced in the systemic infection model likely covered any CEACAM1-specific effects in the transgenic animals. In vitro studies using bone marrow-derived neutrophils from both mouse strains also revealed no differences in their reaction to C. albicans. In conclusion, in contrast to bacterial pathogens interacting with CEACAM1 on different mucosal surfaces, the human CEACAM1-transgenic mice did not reveal a role of human CEACAM1 in the in vivo candidiasis models used here. Further studies and different approaches will be needed to reveal a putative role of CEACAM1 in the host response to C. albicans.

AmpliSeq Screening of Genes Encoding the C-Type Lectin Receptors and Their Signaling Components Reveals a Common Variant in MASP1 Associated with Pulmonary Tuberculosis in an Indian Population.

Tuberculosis (TB) is a multifactorial disease governed by bacterial, host and environmental factors. On the host side, growing evidence shows the crucial role that genetic variants play in the susceptibility to Mycobacterium tuberculosis (Mtb) infection. Such polymorphisms have been described in genes encoding for different cytokines and pattern recognition receptors (PRR), including numerous Toll-like receptors (TLRs). In recent years, several members of the C-type lectin receptors (CTLRs) have been identified as key PRRs in TB pathogenesis. Nevertheless, studies to date have only addressed particular genetic polymorphisms in these receptors or their related pathways in relation with TB. In the present study, we screened the main CTLR gene clusters as well as CTLR pathway-related genes for genetic variation associated with pulmonary tuberculosis (PTB). This case-control study comprised 144 newly diagnosed pulmonary TB patients and 181 healthy controls recruited at the Bhagwan Mahavir Medical Research Center (BMMRC), Hyderabad, India. A two-stage study was employed in which an explorative AmpliSeq-based screening was followed by a validation phase using iPLEX MassARRAY. Our results revealed one SNP (rs3774275) in MASP1 significantly associated with PTB in our population (joint analysis p = 0.0028). Furthermore, serum levels of MASP1 were significantly elevated in TB patients when compared to healthy controls. Moreover, in the present study we could observe an impact of increased MASP1 levels on the lectin pathway complement activity in vitro. In conclusion, our results demonstrate a significant association of MASP1 polymorphism rs3774275 and MASP1 serum levels with the development of pulmonary TB. The present work contributes to our understanding of host-Mtb interaction and reinforces the critical significance of mannose-binding lectin and the lectin-complement pathway in Mtb pathogenesis. Moreover, it proposes a MASP1 polymorphism as a potential genetic marker for TB resistance.

Differential regulation of the transcriptomic and secretomic landscape of sensor and effector functions of human airway epithelial cells.

Protein secretion upon TLR, TNFR1, and IFNGR ligation in the human airways is considered to be central for the orchestration of pulmonary inflammatory and immune responses. In this study, we compared the gene expression and protein secretion profiles in response to specific stimulation of all expressed TLRs and in further comparison to TNFR1 and IFNGR in primary human airway epithelial cells. In addition to 22 cytokines, we observed the receptor-induced regulation of 571 genes and 1,012 secreted proteins. Further analysis revealed high similarities between the transcriptional TLR sensor and TNFR1 effector responses. However, secretome to transcriptome comparisons showed a broad receptor stimulation-dependent release of proteins that were not transcriptionally regulated. Many of these proteins are annotated to exosomes with associations to, for example, antigen presentation and wound-healing, or were identified as secretable proteins related to immune responses. Thus, we show a hitherto unrecognized scope of receptor-induced responses in airway epithelium, involving several additional functions for the immune response, exosomal communication and tissue homeostasis.

IL-37 Causes Excessive Inflammation and Tissue Damage in Murine Pneumococcal Pneumonia.

Streptococcus pneumoniae infections can lead to severe complications with excessive immune activation and tissue damage. Interleukin-37 (IL-37) has gained importance as a suppressor of innate and acquired immunity, and its effects have been therapeutic as they prevent tissue damage in autoimmune and inflammatory diseases. By using RAW macrophages, stably transfected with human IL-37, we showed a 70% decrease in the cytokine levels of IL-6, TNF-α, and IL-1ß, and a 2.2-fold reduction of the intracellular killing capacity of internalized pneumococci in response to pneumococcal infection. In a murine model of infection with S. pneumoniae, using mice transgenic for human IL-37b (IL-37tg), we observed an initial decrease in cytokine expression of IL-6, TNF-α, and IL-1ß in the lungs, followed by a late-phase enhancement of pneumococcal burden and subsequent increase of proinflammatory cytokine levels. Additionally, a marked increase in recruitment of alveolar macrophages and neutrophils was noted, while TRAIL mRNA was reduced 3-fold in lungs of IL-37tg mice, resulting in necrotizing pneumonia with augmented death of infiltrating neutrophils, enhanced bacteremic spread, and increased mortality. In conclusion, we have identified that IL-37 modulates several core components of a successful inflammatory response to pneumococcal pneumonia, which lead to increased inflammation, tissue damage, and mortality.

Binding of Candida albicans to Human CEACAM1 and CEACAM6 Modulates the Inflammatory Response of Intestinal Epithelial Cells.

Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans-induced CXCL8 secretion.IMPORTANCE The present study demonstrates for the first time that fungal pathogens can be recognized by at least four members of the immunomodulatory CEACAM receptor family: CEACAM1, -3, -5, and -6. Three of the four receptors (i.e., CEACAM1, -5, and -6) are expressed in mucosal cells of the intestinal tract, where they are implicated in immunomodulation and control of tissue homeostasis. Importantly, the interaction of the major fungal pathogen in humans Candida albicans with CEACAM1 and CEACAM6 resulted in an altered epithelial immune response. With respect to the broad impact of CEACAM receptors on various aspects of the innate and the adaptive immune responses, in particular epithelial, neutrophil, and T cell behavior, understanding the role of CEACAMs in the host response to fungal pathogens might help to improve management of superficial and systemic fungal infections.

Differential Effects of Vitamins A and D on the Transcriptional Landscape of Human Monocytes during Infection.

Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic effects in humans. Beyond their well-documented roles in cellular differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, both vitamins have attracted considerable attention due to their association with-immunological traits. Nevertheless, our knowledge of their immunomodulatory potential during infection is restricted to single gene-centric studies, which do not reflect the complexity of immune processes. In the present study, we performed a comprehensive RNA-seq-based approach to define the whole immunomodulatory role of vitamins A and D during infection. Using human monocytes as host cells, we characterized the differential role of both vitamins upon infection with three different pathogens: Aspergillus fumigatus, Candida albicans and Escherichia coli. Both vitamins showed an unexpected ability to counteract the pathogen-induced transcriptional responses. Upon infection, we identified 346 and 176 immune-relevant genes that were regulated by atRA and vitD, respectively. This immunomodulatory activity was dependent on the inflammatory stimulus, allowing us to distinguish regulatory patterns which were specific for each stimulatory setting. Moreover, we explored possible direct and indirect mechanisms of vitamin-mediated regulation of the immune response. Our findings highlight the importance of vitamin-monitoring in critically ill patients. Moreover, our results underpin the potential of atRA and vitD as therapeutic options for anti-inflammatory treatment.

Massive Effect on LncRNAs in Human Monocytes During Fungal and Bacterial Infections and in Response to Vitamins A and D.

Mycoses induced by C.albicans or A.fumigatus can cause important host damage either by deficient or exaggerated immune response. Regulation of chemokine and cytokine signaling plays a crucial role for an adequate inflammation, which can be modulated by vitamins A and D. Non-coding RNAs (ncRNAs) as transcription factors or cis-acting antisense RNAs are known to be involved in gene regulation. However, the processes during fungal infections and treatment with vitamins in terms of therapeutic impact are unknown. We show that in monocytes both vitamins regulate ncRNAs involved in amino acid metabolism and immune system processes using comprehensive RNA-Seq analyses. Compared to protein-coding genes, fungi and bacteria induced an expression change in relatively few ncRNAs, but with massive fold changes of up to 4000. We defined the landscape of long-ncRNAs (lncRNAs) in response to pathogens and observed variation in the isoforms composition for several lncRNA following infection and vitamin treatment. Most of the involved antisense RNAs are regulated and positively correlated with their sense protein-coding genes. We investigated lncRNAs with stimulus specific immunomodulatory activity as potential marker genes: LINC00595, SBF2-AS1 (A.fumigatus) and RP11-588G21.2, RP11-394l13.1 (C.albicans) might be detectable in the early phase of infection and serve as therapeutic targets in the future.

A common theme in extracellular fluids of beetles: extracellular superoxide dismutases crucial for balancing ROS in response to microbial challenge.

Extracellular Cu/Zn superoxide dismutases (SODs) are critical for balancing the level of reactive oxygen species in the extracellular matrix of eukaryotes. In the present study we have detected constitutive SOD activity in the haemolymph and defensive secretions of different leaf beetle species. Exemplarily, we have chosen the mustard leaf beetle, Phaedon cochleariae, as representative model organism to investigate the role of extracellular SODs in antimicrobial defence. Qualitative and quantitative proteome analyses resulted in the identification of two extracellular Cu/Zn SODs in the haemolymph and one in the defensive secretions of juvenile P. cochleariae. Furthermore, quantitative expression studies indicated fat body tissue and defensive glands as the main synthesis sites of these SODs. Silencing of the two SODs revealed one of them, PcSOD3.1, as the only relevant enzyme facilitating SOD activity in haemolymph and defensive secretions in vivo. Upon challenge with the entomopathogenic fungus, Metarhizium anisopliae, PcSOD3.1-deficient larvae exhibited a significantly higher mortality compared to other SOD-silenced groups. Hence, our results serve as a basis for further research on SOD regulated host-pathogen interactions. In defensive secretions PcSOD3.1-silencing affected neither deterrent production nor activity against fungal growth. Instead, we propose another antifungal mechanism based on MRJP/yellow proteins in the defensive exudates.

Tissue-specific transcript profiling for ABC transporters in the sequestering larvae of the phytophagous leaf beetle Chrysomela populi.

BACKGROUND: Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC) carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi. RESULTS: In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp). RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration. CONCLUSION: We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a homeostasis of plant-derived compounds and offer a basis for future analyses of their physiological function in sequestration processes.

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve.

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant-insect interactions.

Putative sugar transporters of the mustard leaf beetle Phaedon cochleariae: their phylogeny and role for nutrient supply in larval defensive glands.

BACKGROUND: Phytophagous insects have emerged successfully on the planet also because of the development of diverse and often astonishing defensive strategies against their enemies. The larvae of the mustard leaf beetle Phaedon cochleariae, for example, secrete deterrents from specialized defensive glands on their back. The secretion process involves ATP-binding cassette transporters. Therefore, sugar as one of the major energy sources to fuel the ATP synthesis for the cellular metabolism and transport processes, has to be present in the defensive glands. However, the role of sugar transporters for the production of defensive secretions was not addressed until now. RESULTS: To identify sugar transporters in P. cochleariae, a transcript catalogue was created by Illumina sequencing of cDNA libraries. A total of 68,667 transcripts were identified and 68 proteins were annotated as either members of the solute carrier 2 (SLC2) family or trehalose transporters. Phylogenetic analyses revealed an extension of the mammalian GLUT6/8 class in insects as well as one group of transporters exhibiting distinctive conserved motifs only present in the insect order Coleoptera. RNA-seq data of samples derived from the defensive glands revealed six transcripts encoding sugar transporters with more than 3,000 counts. Two of them are exclusively expressed in the glandular tissue. Reduction in secretions production was accomplished by silencing two of four selected transporters. RNA-seq experiments of transporter-silenced larvae showed the down-regulation of the silenced transporter but concurrently the up-regulation of other SLC2 transporters suggesting an adaptive system to maintain sugar homeostasis in the defensive glands. CONCLUSION: We provide the first comprehensive phylogenetic study of the SLC2 family in a phytophagous beetle species. RNAi and RNA-seq experiments underline the importance of SLC2 transporters in defensive glands to achieve a chemical defense for successful competitive interaction in natural ecosystems.

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles.

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi, the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae. The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.