Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Physiological and transcriptional immune responses of a non-model arthropod to infection with different entomopathogenic groups.

Insect immune responses to multiple pathogen groups including viruses, bacteria, fungi, and entomopathogenic nematodes have traditionally been documented in model insects such as Drosophila melanogaster, or medically important insects such as Aedes aegypti. Despite their potential importance in understanding the efficacy of pathogens as biological control agents, these responses are infrequently studied in agriculturally important pests. Additionally, studies that investigate responses of a host species to different pathogen groups are uncommon, and typically focus on only a single time point during infection. As such, a robust understanding of immune system responses over the time of infection is often lacking in many pest species. This study was conducted to understand how 3rd instar larvae of the major insect pest Helicoverpa zea responded through the course of an infection by four different pathogenic groups: viruses, bacteria, fungi, and entomopathogenic nematodes; by sampling at three different times post-inoculation. Physiological immune responses were assessed at 4-, 24-, and 48-hours post-infection by measuring hemolymph phenoloxidase concentrations, hemolymph prophenoloxidase concentrations, hemocyte counts, and encapsulation ability. Transcriptional immune responses were measured at 24-, 48-, and 72-hours post-infection by quantifying the expression of PPO2, Argonaute-2, JNK, Dorsal, and Relish. This gene set covers the major known immune pathways: phenoloxidase cascade, siRNA, JNK pathway, Toll pathway, and IMD pathway. Our results indicate H. zea has an extreme immune response to Bacillus thuringiensis bacteria, a mild response to Helicoverpa armigera nucleopolyhedrovirus, and little-to-no detectable response to either the fungus Beauveria bassiana or Steinernema carpocapsae nematodes.

A Short-Type Peptidoglycan Recognition Protein 1 (PGRP1) Is Involved in the Immune Response in Asian Corn Borer, Ostrinia furnacalis (Guenée).

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.

Glyphosate inhibits melanization and increases susceptibility to infection in insects.

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Analysis of the Glyphodes pyloalis larvae immune transcriptome in response to parasitization by its endoparasitoid, Aulacococentrum confusum.

The interaction between a host and its parasitoid is one of the most fascinating relationships of insects. Immune-related genes play crucial roles in this association. Nevertheless, until now, identification of these genes on a large scale has not received much attention. To gain insight into the parasitic effects of the endoparasitoid Aulacocentrum confusum (Hymenoptera: Braconidae) on Glyphodes pyloalis (Lepidoptera: Pyralidae) larva, which is a destructive pest of mulberry (Morus alba L.) trees in China, we presented a transcriptome dataset for uncovering immune-related genes in parasitized G. pyloalis larvae. In total, 91,118,138 and 92,778,814 clean reads were obtained from parasitized and healthy host larvae, respectively, and de novo assembly generated 57,122 unigenes. The transcriptional profile of G. pyloalis larvae was remarkably influenced by parasitism. A total of 3259 differentially expressed genes (DEGs) were identified in parasitized and nonparasitized G. pyloalis larvae and 55 genes related to immune response were screened from these DEGs. Among the 55 DEGs, 37 genes were significantly upregulated, and 18 genes were downregulated. qRT-PCR validated the sequencing results and revealed that the expression levels of selected immune-related genes depended on the parasitization and duration after parasitization. Knocking down the C type lectin gene (CTL) changed the expression of serine proteinase, serine protease inhibitor, antimicrobial peptide, prophenoloxidase activating enzymes and peroxiredoxin in G. pyloalis larvae, suggesting CTL can modulate the immune response after parasitization by A. confusum females. The present study provides a foundation for revealing the molecular mechanisms of immune response in G. pyloalis larvae when they are parasitized by A. confusum and promotes the development of novel biological control practices for G. pyloalis.

Transcriptome sequencing reveals Cnaphalocrocis medinalis against baculovirus infection by oxidative stress.

Cnaphalocrocis medinalis granulovirus (CnmeGV) is a potential microbial agent against the rice leaffolder. Innate immunity is essential for insects to survive pathogenic infection. Therefore, to clarify the immune response of Cnaphalocrocis medinalis to the viral colonization, the gene expression profile of C. medinalis infected with CnmeGV was constructed by RNA-seq. A total of 8,503 differentially expressed genes (DEGs) were found including 5,304 up-regulated and 3,199 down-regulated unigenes. Gene enrichment analysis indicated that these DEGs were mainly linked to protein synthesis and metabolic process as well as ribosome and virus-infection pathways. Specifically, a significantly up-regulated PiggyBac-like transposon gene was identified suggested that the enhancement of transposon activity is related to host immunity. Further, the DEGs encoding oxidative stress related genes were identified and validated by RT-qPCR. Overall, 9 antioxidant enzyme genes and 4 antioxidant protein genes were up-regulated, and the extensive glutathione S-transferase genes were down-regulated. Our results provide a basis for understanding the molecular mechanisms of baculovirus action and oxidative stress response in C. medinalis and other insects.

Immunological regulation by a ß-adrenergic-like octopamine receptor gene in crowded larvae of the oriental Armyworm, Mythmina separata.

Recent reports demonstrate that octopamine plays an important immunological role in crowded larvae of the Oriental Armyworm, Mythmina separata. We identified an octopamine receptor, the ß-adrenergic-like gene (designated MsOctß2R), with a 1191 bp open reading frame that encodes 396 amino acids and contains seven conserved hydrophobic transmembrane domains. Multiple sequence alignments and a phylogenetic analysis indicated that MsOctß2R was orthologous to Octß2R that is present in other lepidopterans. MsOctß2R was expressed throughout all developmental stages with higher relative expression during the fourth instar and adult stages. MsOctß2R was highly expressed in the ventral nerve cord and the fat body relative to other examined tissues. Elevated MsOctß2R expression was observed in larvae that were under higher-density conditions (7 and 10 larvae per jar). Silencing MsOctß2R expression via dsRNA injections in larvae from higher-density conditions significantly decreased phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival rates against Beauveria bassiana infections (54.06%, 9.91%, 36.22%, and 23.53%, respectively) when compared with control larvae. These results suggest that high-density conditions might alter prophylactic immunity in larvae by regulating the MsOctß2R gene in M. separara and provide new insights into density-dependent prophylaxis in insects.

CD109 antigen-like gene is induced by ecdysone signaling and involved in the cellular immunity of Helicoverpa armigera.

Cellular immunity is evolutionarily conserved in invertebrates and vertebrates. In insects, cellular immune response is provided by the hemocytes, and its molecular mechanisms are currently not fully understood. Here, we identified a CD109 antigen-like gene (HaCD109) from Helicoverpa armigera which is highly expressed in the hemocytes of larvae. Stimulation by Escherichia coli and chromatography beads significantly upregulated HaCD109 expression. In vivo HaCD109 silencing significantly increased bacterial load in larval hemolymphs and reduced the hemocyte spread. 20-Hydroxyecdysone (20E) can induce HaCD109 expression through its receptors, EcR and USP. In vivo HaCD109 silencing nearly abolished 20E-induced bacterial clearance and hemocyte spread. These results suggested that HaCD109 plays an important role in cellular immunity, and the 20E-induced cellular immune response in H. armigera requires HaCD109 involvement. Our study contributes to the understanding of regulatory mechanisms for innate immune response and provides new insights into the interaction between innate immunity and steroid hormone signaling.

Heat shock proteins (HSP 90, 70, 60, and 27) in Galleria mellonella (Lepidoptera) hemolymph are affected by infection with Conidiobolus coronatus (Entomophthorales).

Invertebrates are becoming more popular models for research on the immune system. The innate immunity possessed by insects shows both structural and functional similarity to the resistance displayed by mammals, and many processes occurring in insect hemocytes are similar to those that occur in mammals. The humoral immune response in insects acts by melanization, clotting and the production of reactive oxygen species and antimicrobial peptides, while the cellular immunity system is based on nodulation, encapsulation and phagocytosis. An increasingly popular insect model in biological research is Galleria mellonella, whose larvae are sensitive to infection by the entomopathogenic fungus Conidiobolus coronatus, which can also be dangerous to humans. One group of factors that modulate the response of the immune system during infection in mammals are heat shock proteins (HSPs). The aim of this study was to investigate whether infection by C. coronatus in G. mellonella hemolymph is accompanied by an increase of HSP90, HSP70, HSP60 and HSP27. Larvae (five-day-old last instar) were exposed for 24 hours to fully-grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24) or 24 hours later (F48). The concentration of the HSPs in hemolymph was determined using ELISA. Immunolocalization in hemocytes was performed using fluorescence microscopy and flow cytometry. HSP90, HSP70, HSP60 and HSP27 were found to be present in the G. mellonella hemocytes. HSP60 and HSP90 predominated in healthy insects, with HSP70 and HSP27 being found in trace amounts; HSP60 and HSP27 were elevated in F24 and F48, and HSP90 was elevated in F48. The fungal infection had no effect on HSP70 levels. These findings shed light on the mechanisms underlying the interaction between the innate insect immune response and entomopathogen infection. The results of this innovative study may have a considerable impact on research concerning innate immunology and insect physiology.

Effect of thermal stress on the immune responses of Chilo suppressalis walker (Lepidoptera: Crambidae) to Beauveria bassiana.

Temperature is one of the important environmental elements affecting ecological fitness of insects through alterations in physiological systems. In the current study, a comparison was made on the cellular and humoral immune responses of the Chilo suppressalis larvae exposed to thermal stress (34 °C) and optimal rearing temperature (24 °C). Although total hemocyte count increased in the injected larvae by Beauveria bassiana, elevation of hemocyte numbers was significantly different in the larvae exposed to 34 °C for a short-time period compared to long-term exposure and control. A similar trend was observed in plasmatocyte and granulocyte counts as well as phenoloxidase activity. Gene expression of some antimicrobial peptides, including attacin1, attacin2, cecropin1, cecropin2, defensin, gallerimycin, lysozyme and prophenoloxidase-activating proteinase-3, was compared in the larvae exposed to thermal regimes and injection challenges. In all cases, expression of the target genes was relatively higher in the larvae injected by B. bassiana and short-term exposure at 34 °C. The present results confirmed that C. suppressalis could modulate the immune system in response to different thermal stress conditions mainly over a short period.

Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans.

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.

A powerful in vivo alternative model in scientific research: Galleria mellonella.

Murine models are suggested as the gold standard for scientific research, but they have many limitations of ethical and logistical concern. Then, the alternative host models have been developed to use in many aspects especially in invertebrate animals. These models are selected for many areas of research including genetics, physiology, biochemistry, evolution, disease, neurobiology, and behavior. During the past decade, Galleria mellonella has been used for several medical and scientific researches focusing on human pathogens. This model commonly used their larvae stage due to their easy to use, non-essential special tools or special technique, inexpensive, short life span, and no specific ethical requirement. Moreover, their innate immune response close similarly to mammals, which correlate with murine immunity. In this review, not only the current knowledge of characteristics and immune response of G. mellonella, and the practical use of these larvae in medical mycology research have been presented, but also the better understanding of their limitations has been provided.

Parasitoid envenomation alters the Galleria mellonella midgut microbiota and immunity, thereby promoting fungal infection.

Gut bacteria influence the development of different pathologies caused by bacteria, fungi and parasitoids in insects. Wax moth larvae became more susceptible to fungal infections after envenomation by the ectoparasitoid Habrobracon hebetor. In addition, spontaneous bacterioses occurred more often in envenomated larvae. We analyzed alterations in the midgut microbiota and immunity of the wax moth in response to H. hebetor envenomation and topical fungal infection (Beauveria bassiana) alone or in combination using 16S rRNA sequencing, an analysis of cultivable bacteria and a qPCR analysis of immunity- and stress-related genes. Envenomation led to a predominance shift from enterococci to enterobacteria, an increase in CFUs and the upregulation of AMPs in wax moth midguts. Furthermore, mycosis nonsignificantly increased the abundance of enterobacteria and the expression of AMPs in the midgut. Combined treatment led to a significant increase in the abundance of Serratia and a greater upregulation of gloverin. The oral administration of predominant bacteria (Enterococcus faecalis, Enterobacter sp. and Serratia marcescens) to wax moth larvae synergistically increased fungal susceptibility. Thus, the activation of midgut immunity might prevent the bacterial decomposition of envenomated larvae, thus permitting the development of fungal infections. Moreover, changes in the midgut bacterial community may promote fungal killing.

Susceptibility to entomopathogens and modulation of basal immunity in two insect models at different temperatures.

In this work, we analysed the efficacy of different commercial bio-insecticides (Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora and Bacillus thuringiensis) by valuating the mortality induced on two insect models, Galleria mellonella (Lepidoptera) and Sarcophaga africa (Diptera) after exposure to different temperatures (10, 20 and 30 °C). Moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (PO) and lysozyme activity. Our results show that G. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °C with S. carpocapsae and at 30 °C with S. feltiae and B. thuringiensis. S. africa is more susceptible at 30 °C to all bioinsecticides; whereas, when infected at 10 and 20 °C, H. bacteriophora is the most efficient. Temperature modulates PO activity of both G. mellonella and S. africa, otherwise variations in lysozyme activity is observed only in G. mellonella. Except for a possible correlation between the increased lysozyme activity and the delayed Bt efficacy recorded on G. mellonella at 30 °C, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens.

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila.

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF-κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC-Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.

Characterization of PGRP-S1 from the oriental armyworm, Mythimna separata.

Peptidoglycan is the key component forming the backbone of bacterial cell wall. It can be recognized by a group of pattern recognition receptors, known as peptidoglycan recognition proteins (PGRPs) in insects and higher animals. PGRPs may serve as immune receptors or N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). Here, we report the characterization of a short PGRP, PGRP-S1, from the oriental armyworm, Mythimna separata. MsePGRP-S1 cDNA encodes a protein of 197 amino acids (aa) with a PGRP domain of about 150 aa. MsePGRP-S1 was expressed in several tissues of naïve larvae, including hemocytes, midgut, fat body and epidermis. Bacterial challenges caused variable changes in different tissues at the mRNA level. The recombinant protein bound strongly to Staphylococcus aureus and purified peptidoglycans from Staphylococcus aureus and Bacillus subtilis. It can inhibit the growth of gram-negative and gram-positive bacteria by disrupting bacterial surface. It can degrade peptidoglycans from Escherichia coli and Staphylococcus aureus. Taken together, these data demonstrate that M. separata PGRP-S1 is involved in defending against bacteria.

Virulence of Candida haemulonii complex in Galleria mellonella and efficacy of classical antifungal drugs: a comparative study with other clinically relevant non-albicans Candida species.

Candida haemulonii complex has emerged as notorious yeasts causing invasive infections with high rates of treatment failures. Since there is a particular interest in the development of non-mammalian host models to study microbial virulence, with the aim to evade the ethical impact of animal tests, herein we compared the virulence of C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera with non-albicans Candida species (C. tropicalis, C. krusei and C. lusitaniae) on Galleria mellonella and the efficacy of antifungal drugs. All these fungi induced a dose-dependent effect on larvae killing, a decrease in hemocyte density and fungi were phagocytozed by hemocytes in equal proportions. Fungal inoculation caused early larvae melanization after some minutes of injection, followed by an augmented pigmentation after 24 h. Differences among species virulence can be explained, in part, by differences in growth rate and production of hydrolytic enzymes. First-line antifungals were tested with equivalent therapeutic doses and MIC profile in vitro was correlated with in vivo antifungal efficacy. Additionally, fungal burden increased in infected larvae along time and only caspofungin reduced the number of CFUs of C. haemulonii species complex. So, G. mellonella offers a simple and feasible model to study C. haemulonii complex virulence and drug efficacy.

Galleria mellonella as a model invertebrate host for the study of muriform cells of dematiaceous fungi.

AIM: To study the pathogenesis of chromoblastomycosis using the alternative model host Galleria mellonella. METHODOLOGY: We analyzed the virulence of different dematiaceous fungal strains and the host immune responses (hemocytes density and morphological changes) to Fonsecaea monophora by the alternative infection model. Then detected the development of the pathogenic muriform cells within larvae under microscope. RESULTS: Increasing inocula resulted in greater larval mortality and Cladophialophora carrionii was the most virulent. Low inocula activated the humoral immune response significantly. Moreover, the conidia underwent morphological transition to muriform cells within larvae. CONCLUSION: We developed an invertebrate host model that can be used to evaluate the virulence of dematiaceous fungi, which may provide further insights into overcoming current limitations in studying chromoblastomycosis in vivo.

CapC, a Novel Autotransporter and Virulence Factor of Campylobacter jejuni.

Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria, and notably, many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterize the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of C. jejuni The predicted CapC protein has a number of features that are consistent with autotransporters, including the N-terminal signal sequence and the C-terminal ß-barrel domain and was determined to localize to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of nonpolarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced interleukin 8 (IL-8) secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae, indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.IMPORTANCECampylobacter jejuni is a major causative agent of human gastroenteritis, making this zoonotic pathogen of significant importance to human and veterinary public health worldwide. The mechanisms by which C. jejuni interacts with intestinal epithelial cells and causes disease are still poorly understood due, in part, to the heterogeneity of C. jejuni infection biology. Given the importance of C. jejuni to public health, the need to characterize novel and existing virulence mechanisms is apparent. The significance of our research is in demonstrating the role of CapC, a novel virulence factor in C. jejuni that contributes to adhesion and invasion of the intestinal epithelium, thereby in part, addressing the dearth of knowledge concerning the factors involved in Campylobacter pathogenesis and the variation observed in the severity of human infection.