Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

Role of pathogen-derived cell wall carbohydrates and prostaglandin E2 in immune response and suppression of fish immunity by the oomycete Saprolegnia parasitica.

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1ß1 [IL-1ß1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin [corrected] E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis.

BACKGROUND: Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. Candida albicans is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution. RESULTS: Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-α, IFN-γ and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mice. CONCLUSIONS: Gomesin proved to be effective against experimental Candida albicans infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin's mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.

Cellular Immune Responses in Rainbow Trout (Onchorhynchus mykiss) Following Vaccination and Challenge Against Salmonid Alphavirus (SAV).

Viral disease outbreaks remain a significant limiting factor for aquaculture. The majority of licensed vaccines used in the industry are administered as oil-adjuvanted formulations carrying inactivated whole pathogens. Cell-mediated immune responses, in particular those based on virus-specific cytotoxic T-cells (CTLs) to conventional inactivated oil-based vaccines, are largely unexplored. As vaccines cannot be optimized against viral pathogens if knowledge of host cellular immune mechanisms remains unknown, in this study we examined fundamental cell-mediated immune responses after vaccination of rainbow trout with an oil-adjuvanted inactivated vaccine against salmonid alphavirus (SAV) and after infection with SAV. A unique in vitro model system was developed to examine MHC class I restricted CTL responses in a clonal line of rainbow trout. The levels of cell-mediated cytotoxicity were compared to pathology, virus load, specific antibody response, changes in immune cell populations, and mRNA expression. Our results hint that different protective mechanisms are being triggered by infection compared to vaccination. While vaccination itself did not cause a strong cytotoxic or humoral response, subsequent challenge of vaccinated fish resulted in significantly stronger and faster specific cytotoxicity, alongside reduced viral titers and pathology. Hence, testing a vaccine on the capacity to induce cell-mediated cytotoxicity will still require a challenge test. Examination of cellular markers additionally indicates that the initial innate response induced by the vaccine could play an important role in steering adaptive mechanisms.

Arrest of oogenesis in the bug Rhodnius prolixus challenged with the fungus Aspergillus niger is mediated by immune response-derived PGE2.

In this work we characterized the immune response of the insect Rhodnius prolixus to a direct injection into the hemocoel of the non-entomopathogenic fungus Aspergillus niger, and evaluated its consequences on host oogenesis. These animals were able to respond by mounting effective cellular and humoral responses to this fungus; these responses were shown, however, to have reproductive fitness costs, as the number of eggs laid per female was significantly reduced. The disturbance of egg formation during infectious process correlated with an elevation in the titer of hemolymph prostaglandin E2 48 h post-challenge. Administration of Zymosan A as an immunogenic non-infectious challenge produced similar effects on phenoloxidase and prophenoloxidase activities, oocyte development and prostaglandin E2 titer, precluding the hypothesis of an effect mediated by fungal metabolites in animals challenged with fungus. Ovaries at 48 h post-challenge showed absence of vitellogenic ovarian follicles, and the in vivo administration of prostaglandin E2 or its receptor agonist misoprostol, partially reproduced this phenotype. Together these data led us to hypothesize that immune-derived prostaglandin E2 raised from the insect response to the fungal challenge is involved in disturbing follicle development, contributing to a reduction in host reproductive output and acting as a host-derived adaptive effector to infection.

Seguridad de la cirugía de luxofracturas vertebrales toracolumbares según la oportunidad quirúrgica / Safety of surgical treatment for thoracolumbar fracture-dislocations according to surgical timing

Introducción: Las luxofracturas vertebrales toracolumbares se producen por traumatismos de alta energía, representan el 10% de las lesiones traumáticas de la columna vertebral y se asocian frecuentemente con otras lesiones. El objetivo de este estudio fue comparar las complicaciones tempranas en pacientes con una luxofractura toracolumbar según la oportunidad quirúrgica, antes o después de las 24 h del trauma. Materiales y métodos: Estudio multicéntrico, analítico, observacional y retrospectivo de una cohorte de pacientes operados por una luxofractura toracolumbar, desde el 1 de enero de 2014 hasta el 1 de enero de 2023. Se inclu-yó a pacientes de ambos sexos, >18 años, operados por una luxofractura vertebral de alta energía. Se los agrupó según si habían sido operados de columna antes o después de las 24 h del trauma. Se registraron las complicaciones totales y agrupadas. Resultados: Se evaluó a 72 pacientes, 64 hombres (88,9%) y 8 mujeres (11,1%), con una edad promedio de 35.94 años. Predominaron las instituciones laborales (n = 60; 83,3%). El mecanismo de lesión más frecuente fueron los accidentes de tránsito (n = 42; 58,3%), seguidos de las caídas de altura (n = 26; 36,1%). El 86% sufrió una o más lesiones asociadas. Se registraron 283 complicaciones en 67 (93,1%) pacientes y 45 complicaciones quirúrgicas en 26 pacientes (36,1%). La mediana de complicaciones fue mayor en pacientes operados tardíamente (p = 0,004). Conclusiones:Los pacientes con luxofractura toracolumbar operados después de las primeras 24 h presentaron una mediana de complicaciones totales significativamente mayor que los operados precozmente. Nnivel de Evidencia: IV

Introduction: Thoracolumbar fracture-dislocations account for 10% of traumatic spinal injuries and typically occur in the context of high-energy trauma. Our objective is to compare early complications in patients with thoracolumbar fracture-dislocation based on surgical timing, either before or after 24 hours from the trauma.materials and methods: This is a multicenter, retrospective cohort study of patients surgically treated for thoracolumbar dislocations, from January 1, 2014 to January 1, 2023. We included adult patients (>18 years old) of any gender, surgically treated for high-energy thoracolumbar fracture-dislocations. Patients were grouped based on when they underwent spinal surgery: before or after 24 hours following trauma. Total and grouped complications were recorded. Results: Our sample comprised 72 patients, with 64 men (88.9%) and 8 women (11.1%) at an average age of 35.94 years. Occupational health care centers were predominant (n=60; 83.3%). Road traffic accidents (n=42; 58.3%) were the most frequent cause of injury, followed by falls from height (n=26; 36.1%). Furthermore, 86% of patients had one or more associated injuries. In total, 283 complications were recorded, with 67 patients (93.1%) suffering at least one complication, and 26 patients (36.1%) experiencing surgical complications. The median number of complications was significantly higher in late-operated patients (p=0.004). Conclusions: Patients with thoracolumbar dislocations who underwent surgery after the first 24 hours following trauma had a significantly higher median rate of complications than those who underwent early surgery. Level of Evidence: IV

Tratamiento de las fracturas cervicales aisladas de tipo "masa lateral flotante": serie de casos / Treatment of isolated "floating lateral mass" fractures of the cervical spine: A case series

Objetivo: Presentar una serie de casos de pacientes con fracturas cervicales de tipo "masa lateral flotante" con énfasis en describir el rol de la lesión discal en la potencial inestabilidad segmentaria. Materiales y métodos: Se realizó un estudio descriptivo y retrospectivo de una serie de pacientes con fracturas facetarias de tipo "masa lateral flotante", aisladas, diagnosticadas entre el 1 de enero de 2016 y el 1 de enero de 2022. Se incluyeron pacientes con lesiones de tipo "masa lateral flotante" según la clasificación AO, diagnosticadas por tomografía computarizada y, al menos, 6 meses de seguimiento. Se excluyó a aquellos con fracturas patológicas, lesiones por fragilidad ósea y registros incompletos. Resultados: Se analizó a 16 pacientes (media de la edad 42.86; DE 12,396), con predominio del sexo masculino (81,25%). El 68,75% tenía una lesión del disco intervertebral en el segmento fracturado y el 18,75%, anterolistesis. A 11 pacientes se les propuso un tratamiento conservador durante 12 semanas. La fractura consolidó en el 45,4% y 6 (54,6%) evolucionaron con traslación. El fracaso del tratamiento conservador se asoció con lesión del disco intervertebral. Once pacientes fueron operados, en su mayoría, con artrodesis cervical anterior monosegmentaria. Conclusiones: En esta serie de casos, la presencia de una lesión asociada del disco intervertebral fue más frecuente cuando el tratamiento conservador fracasó y cuando se decidió una artrodesis como tratamiento inicial. La mayoría de las cirugías se realizaron por vía anterior con discectomía y artrodesis cervical anterior en un único nivel, y se lograron buenos resultados. Nivel de Evidencia: IV

Introduction: We report a series of patients with "floating lateral mass" cervical fractures, focusing on the role of disc injury in potential segmental instability. materials and methods: We conducted a descriptive and retrospective study on a case series of isolated floating lateral mass facet fractures diagnosed between January 1, 2016 and January 1, 2022. Patients with floating lateral mass lesions according to the AO classification, diagnosed by computed tomography, and at least 6 months of follow-up were included. Patients with pathological fractures, bone fragility injuries and incomplete records were excluded. Results: We included 16 cases, the average age was 42.86 (SD 12.396), and the majority were male (n=13; 81.25%). 68.75% (n=11) had intervertebral disc injury in the fractured segment, while 18.75% (n=3) had anterolisthesis. Conservative treatment was proposed for 12 weeks in 11 patients (68.75%), of whom 5 (45.4%) achieved fracture healing and 6 (54.6%) progressed to translation. Cases where conservative treatment failed were associated with intervertebral disc injury. Eleven patients were treated surgically, mostly with monosegmental anterior cervical arthrodesis. Conclusions: We report a series of cases in which the existence of an associated intervertebral disc injury was more frequent in patients with failed conservative treatment and in those initially treated with arthrodesis. Most of the surgical cases were treated using an anterior approach with discectomy and anterior cervical arthrodesis at a single level, with favorable outcomes. Level of Evidence: IV

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

Análisis radiográfico de los parámetros espinopélvicos obtenidos con el dispositivo de TLIF anterior. Estudio multicéntrico / Radiographic analysis of the spinopelvic parameters obtained with an anterior TLIF device. Multicenter study

Objetivo: Comunicar los resultados obtenidos según la posición del dispositivo de TLIF anterior. Materiales y Métodos: Estudio multicéntrico, observacional, analítico, transversal, de recuperación retrospectiva. Se evaluaron los parámetros espinopélvicos pre- y posoperatorios de espinogramas de 20 pacientes que fueron operados entre septiembre de 2019 y agosto de 2021. Se incluyó a pacientes sometidos a artrodesis lumbar con implante de tipo TLIF anterior. Se excluyó a pacientes sin espinograma pre- o posquirúrgico y más de un dispositivo. Resultados: La media de la lordosis monosegmentaria fue de 13,33° antes de la cirugía y de 18,81° después (p <0,001). La media de la lordosis monosegmentaria fue de 7,32°; 2,95° y 6,24° para las posiciones I, II y III, respectivamente. La media de la altura discal fue de 6,22 mm en el preoperatorio y 11,06 mm en el posoperatorio (p >0,001). Conclusiones: Los resultados de la colocación de este tipo de dispositivos y su relación con la lordosis segmentaria fueron alentadores, se comprendió la importancia de la disposición de estos en el extremo anterior del espacio discal. Nivel de Evidencia: IV

Objective: To report the results obtained according to the position of an anterior TLIF device. Materials and Methods: Multicenter, observational, analytical, cross-sectional, retrospective recovery study. We evaluated the pre and post-operative spinopelvic pa-rameters of the spinograms of 20 patients who underwent surgery between September 2019 and August 2021. Patients who had undergone lumbar arthrodesis with an anterior TLIF implant were included, whereas patients without a pre or post-surgical spinogram and more than one device were excluded. Results: The mean monosegmental lordosis was 13.33° preoperatively and 18.81° postoperatively (p <0.001). The mean monosegmental lordosis was 7.32°, 2.95°, and 6.24° for positions I, II, and III, respectively. The mean disc height was 6.22 mm for the preoperative period and 11.06 mm for the postoperative period (p >0.001). Conclusion: We found encouraging results on the placement of this type of device and its relationship with segmental lordosis, understanding the importance of its placement at the anterior end of the disc space. Level of Evidence: IV

Integrative Model of Oxidative Stress Adaptation in the Fungal Pathogen Candida albicans.

The major fungal pathogen of humans, Candida albicans, mounts robust responses to oxidative stress that are critical for its virulence. These responses counteract the reactive oxygen species (ROS) that are generated by host immune cells in an attempt to kill the invading fungus. Knowledge of the dynamical processes that instigate C. albicans oxidative stress responses is required for a proper understanding of fungus-host interactions. Therefore, we have adopted an interdisciplinary approach to explore the dynamical responses of C. albicans to hydrogen peroxide (H2O2). Our deterministic mathematical model integrates two major oxidative stress signalling pathways (Cap1 and Hog1 pathways) with the three major antioxidant systems (catalase, glutathione and thioredoxin systems) and the pentose phosphate pathway, which provides reducing equivalents required for oxidative stress adaptation. The model encapsulates existing knowledge of these systems with new genomic, proteomic, transcriptomic, molecular and cellular datasets. Our integrative approach predicts the existence of alternative states for the key regulators Cap1 and Hog1, thereby suggesting novel regulatory behaviours during oxidative stress. The model reproduces both existing and new experimental observations under a variety of scenarios. Time- and dose-dependent predictions of the oxidative stress responses for both wild type and mutant cells have highlighted the different temporal contributions of the various antioxidant systems during oxidative stress adaptation, indicating that catalase plays a critical role immediately following stress imposition. This is the first model to encapsulate the dynamics of the transcriptional response alongside the redox kinetics of the major antioxidant systems during H2O2 stress in C. albicans.

An investigation into the protein composition of the teneral Glossina morsitans morsitans peritrophic matrix.

BACKGROUND: Tsetse flies serve as biological vectors for several species of African trypanosomes. In order to survive, proliferate and establish a midgut infection, trypanosomes must cross the tsetse fly peritrophic matrix (PM), which is an acellular gut lining surrounding the blood meal. Crossing of this multi-layered structure occurs at least twice during parasite migration and development, but the mechanism of how trypanosomes do so is not understood. In order to better comprehend the molecular events surrounding trypanosome penetration of the tsetse PM, a mass spectrometry-based approach was applied to investigate the PM protein composition using Glossina morsitans morsitans as a model organism. METHODS: PMs from male teneral (young, unfed) flies were dissected, solubilised in urea/SDS buffer and the proteins precipitated with cold acetone/TCA. The PM proteins were either subjected to an in-solution tryptic digestion or fractionated on 1D SDS-PAGE, and the resulting bands digested using trypsin. The tryptic fragments from both preparations were purified and analysed by LC-MS/MS. RESULTS: Overall, nearly 300 proteins were identified from both analyses, several of those containing signature Chitin Binding Domains (CBD), including novel peritrophins and peritrophin-like glycoproteins, which are essential in maintaining PM architecture and may act as trypanosome adhesins. Furthermore, 27 proteins from the tsetse secondary endosymbiont, Sodalis glossinidius, were also identified, suggesting this bacterium is probably in close association with the tsetse PM. CONCLUSION: To our knowledge this is the first report on the protein composition of teneral G. m. morsitans, an important vector of African trypanosomes. Further functional analyses of these proteins will lead to a better understanding of the tsetse physiology and may help identify potential molecular targets to block trypanosome development within the tsetse.

Purification and characterization of Hb 98-114: a novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus.

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50µM of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized.

Secretion, delivery and function of oomycete effector proteins.

Oomycetes are responsible for multi-billion dollar damages in aquaculture, agriculture and forestry. One common strategy they share with most cellular disease agents is the secretion of effector proteins. Effectors are molecules that change host physiology by initiating and allowing an infection to develop. Oomycetes secrete both extracellular and intracellular effectors. Studying secretion, delivery and function of effectors will hopefully lead to alternative control measures, which is much needed as several chemicals to control plant and animal pathogenic oomycetes cannot be used anymore; due to resistance in the host, or because the control measures have been prohibited as a result of toxicity to the environment and/or consumers. Here the latest findings on oomycete effector secretion, delivery and function are discussed.

Immune gene expression in trout cell lines infected with the fish pathogenic oomycete Saprolegnia parasitica.

The oomycete Saprolegnia parasitica causes significant losses in the aquaculture industry, mainly affecting salmon, trout and catfish. Since the ban of malachite green, effective control measures are currently not available prompting a re-evaluation of the potential for immunological intervention. In this study, the immune response of salmonid cells is investigated at the transcript level, by analysis of a large set of immune response genes in four different rainbow trout cell lines (RTG-2, RTGill, RTL and RTS11) upon infection with S. parasitica. Proinflammatory cytokine transcripts were induced in all four cell lines, including IL-1ß1, IL-8, IL-11, TNF-α2, as well as other components of the innate defences, including COX-2, the acute phase protein serum amyloid A and C-type lectin CD209a and CD209b. However, differences between the four cell lines were found. For example, the fold change of induction was much higher in the epithelial RTL and macrophage-like RTS11 cell lines compared to the fibroblast cell lines RTG-2 and RTGill. Several antimicrobial peptides (AMPs) were also up-regulated in response to Saprolegnia infection, including hepcidin and cathelicidin 1 (rtCATH1) and 2 (rtCATH2). An rtCATH2 peptide was synthesised and tested for activity and whilst it showed no killing activity for zoospores, it was able to delay sporulation of S. parasitica. These results demonstrate that particular immune genes are up-regulated in response to S. parasitica infection and that AMPs may play a crucial role in the first line of defence against oomycetes in fish.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

BACKGROUND: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. RESULTS: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. CONCLUSIONS: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone.

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.