Échappement de Leptospira interrogans aux récepteurs NOD1 et NOD2 et stratégies basées sur les agonistes NODs pour restaurer et renforcer les fonctions des macrophages

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis. Chronically infected rodents excrete bacteria in their urine contributing to the spread of the disease. Currently, there is no cross-protective vaccine for more than 400 leptospiral serovars. Innate immune cells such as macrophages quickly respond to microbes through pattern-recognition receptors (PRRs). PRRs recognize conserved microbial-associated molecular patterns (MAMPs) and trigger innate immune responses. For instance, NOD1 and NOD2 receptors sense peptidoglycan (PG) and activate anti-microbial responses, among them autophagy. Furthermore, PRR engagement can trigger the so-called innate immune memory or trained immunity (TI). It has been shown that leptospires escape some innate immune sensing and the role of macrophages during leptospirosis has not yet been clearly established. Previous results from our laboratory established that the LipL21 lipoprotein binds to the leptospiral PG, impairing NOD sensing. My contribution was to confirm the specific role of LipL21 on NOD escape using the heterologous host E. coli that also harbors Gram-negative meso-Dap type PG (Ratet, et al PLoS Pathog. 2017). Next we studied the consequences of NOD escape in macrophages. First, in vitro infections suggested that leptospires were internalized independently of actin polymerization and survive in murine and human macrophages for 48h. Also, leptospires did not trigger nor block autophagy in murine macrophages. In addition, infection selectively induced the accumulation of the autophagy adaptor p62. Moreover, the pharmacological triggering of autophagy on infected cells had a marked effect decreasing the viability of leptospires. Co-infection of leptospires with NOD agonists induced autophagy and p62 degradation. In addition, preliminary data suggest that leptospires do not induce autophagy in the mouse model and that it can be restored with NOD agonists. Finally, using CL429, a NOD2/TLR2 agonist, we demonstrated that TI protects from leptospiral infection (Santecchia et al., PLoS Pathog. 2019). This phenotype was independent of B- and T-cells and characterized by the enhanced pro-inflammatory response of peritoneal macrophages (PMs) and splenic NK-cells towards different pathogenic serovars of L. interrogans. In addition, CL429 influences bone marrow cells, enhancing the antibacterial response of bone marrow-derived macrophages (BMMs). Both PMs and BMMs showed enhanced bactericidal activity alongside more nitric oxide production that has not been previously associated with TI. Finally, ex vivo training with CL429 renders human monocytes more responsive to pathogenic leptospires. In conclusion, we showed that leptospires escape autophagy and that it can be partially restored using NOD agonists. Moreover, training of macrophages with NOD agonists enhance their response toward different pathogenic serovars. Both NOD-based strategies could be exploited for prophylaxis or vaccination.Les bactéries Leptospira interrogans sont des spirochètes pathogènes responsables de la leptospirose. Les rongeurs chroniquement infectés excrètent les bactéries dans leur urine, contribuant ainsi à la propagation de la maladie. Il n'existe actuellement aucun vaccin qui puissent conférer une protection croisée pour les 400 sérovars identifiés de leptospires. Les cellules immunitaires innées telles que les macrophages répondent rapidement aux microbes par le biais de récepteurs (PRR). Les PRR reconnaissent les motifs moléculaires conservés associés aux microbes (MAMP) et déclenchent des réponses immunitaires innées. Par exemple, les récepteurs NOD1 et NOD2 détectent le peptidoglycane (PG) et activent les réponses antimicrobiennes, parmi lesquelles l'autophagie. De plus, l'engagement des PRR peut déclencher une réponse mémoire immunitaire innée ou immunité entraînée (IE). Il a été démontré que les leptospires échappent à certaines réponses immunitaires innées. Le rôle des macrophages au cours de la leptospirose n'a pas encore été clairement établi. Les résultats antérieurs de notre laboratoire ont établi que la lipoprotéine LipL21 se lie au PG des leptospires, altérant la détection par les protéines NOD. Ma contribution a consisté à confirmer le rôle spécifique de la LipL21 sur l'évasion des NOD, en utilisant un système hétérologue d'expression chez E. coli, une bactérie à Gram négatif qui possède également un PG de type méso-Dap (Ratet, Santecchia et al. PLoS Pathog. 2017). Nous avons ensuite étudié les conséquences de l'échappement aux NOD dans les macrophages. Premièrement, des infections in vitro ont suggéré que les leptospires étaient internalisés indépendamment de la polymérisation de l'actine et survivaient dans les macrophages murins et humains pendant 48h. De plus, les leptospires ne déclenchent ni ne bloquent l'autophagie chez les macrophages murins. En outre, l'infection a induit sélectivement l'accumulation de l'adaptateur d'autophagie p62. De plus, le déclenchement pharmacologique de l'autophagie dans les cellules infectées a montré un effet marqué diminuant la viabilité des leptospires. La co-infection de leptospires avec des agonistes des NOD a induit l'autophagie et une dégradation de p62. De plus, des données préliminaires suggèrent que les leptospires ne provoquent pas d'autophagie dans le modèle murin et que l'autophagie au cours de l'infection par les leptospires peut être restaurée avec des agonistes des NOD. Enfin, en utilisant CL429, un agoniste de NOD2 / TLR2, nous avons démontré que l'IE protège de l'infection par les leptospires. Ce phénotype est indépendant des cellules B et T et est caractérisé par une réponse pro-inflammatoire accrue des macrophages péritonéaux et des cellules NK spléniques vis-à-vis de différents sérovars pathogènes de L. interrogans. En outre, le CL429 influence les cellules de la moelle osseuse, renforçant la réponse antibactérienne des macrophages dérivés de la moelle osseuse (BMM). Les PM et les BMM ont montré une activité bactéricide accrue parallèlement à une production accrue d'oxyde nitrique qui n'a jamais été associée auparavant à l'IE. Enfin, un entraînement ex vivo avec CL429 a également rendu les monocytes humains plus sensibles aux leptospires pathogènes (Santecchia et al., PLoS Pathog. 2019, in press). En conclusion, nous avons montré que les leptospires échappent à l'autophagie, qui peut être partiellement restaurée à l'aide d'agonistes des NOD. De plus, la stimulation de macrophages avec des agonistes des NOD améliore leur réponse à différents sérovars pathogènes. Ces deux stratégies basées sur l'utilisation des agonistes des NOD pourraient être exploitées pour la prophylaxie ou la vaccination contre L. interrogans

Échappement de Leptospira interrogans aux récepteurs NOD1 et NOD2 et stratégies basées sur les agonistes NODs pour restaurer et renforcer les fonctions des macrophages

Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis. Chronically infected rodents excrete bacteria in their urine contributing to the spread of the disease. Currently, there is no cross-protective vaccine for more than 400 leptospiral serovars. Innate immune cells such as macrophages quickly respond to microbes through pattern-recognition receptors (PRRs). PRRs recognize conserved microbial-associated molecular patterns (MAMPs) and trigger innate immune responses. For instance, NOD1 and NOD2 receptors sense peptidoglycan (PG) and activate anti-microbial responses, among them autophagy. Furthermore, PRR engagement can trigger the so-called innate immune memory or trained immunity (TI). It has been shown that leptospires escape some innate immune sensing and the role of macrophages during leptospirosis has not yet been clearly established. Previous results from our laboratory established that the LipL21 lipoprotein binds to the leptospiral PG, impairing NOD sensing. My contribution was to confirm the specific role of LipL21 on NOD escape using the heterologous host E. coli that also harbors Gram-negative meso-Dap type PG (Ratet, et al PLoS Pathog. 2017). Next we studied the consequences of NOD escape in macrophages. First, in vitro infections suggested that leptospires were internalized independently of actin polymerization and survive in murine and human macrophages for 48h. Also, leptospires did not trigger nor block autophagy in murine macrophages. In addition, infection selectively induced the accumulation of the autophagy adaptor p62. Moreover, the pharmacological triggering of autophagy on infected cells had a marked effect decreasing the viability of leptospires. Co-infection of leptospires with NOD agonists induced autophagy and p62 degradation. In addition, preliminary data suggest that leptospires do not induce autophagy in the mouse model and that it can be restored with NOD agonists. Finally, using CL429, a NOD2/TLR2 agonist, we demonstrated that TI protects from leptospiral infection (Santecchia et al., PLoS Pathog. 2019). This phenotype was independent of B- and T-cells and characterized by the enhanced pro-inflammatory response of peritoneal macrophages (PMs) and splenic NK-cells towards different pathogenic serovars of L. interrogans. In addition, CL429 influences bone marrow cells, enhancing the antibacterial response of bone marrow-derived macrophages (BMMs). Both PMs and BMMs showed enhanced bactericidal activity alongside more nitric oxide production that has not been previously associated with TI. Finally, ex vivo training with CL429 renders human monocytes more responsive to pathogenic leptospires. In conclusion, we showed that leptospires escape autophagy and that it can be partially restored using NOD agonists. Moreover, training of macrophages with NOD agonists enhance their response toward different pathogenic serovars. Both NOD-based strategies could be exploited for prophylaxis or vaccination.Les bactéries Leptospira interrogans sont des spirochètes pathogènes responsables de la leptospirose. Les rongeurs chroniquement infectés excrètent les bactéries dans leur urine, contribuant ainsi à la propagation de la maladie. Il n'existe actuellement aucun vaccin qui puissent conférer une protection croisée pour les 400 sérovars identifiés de leptospires. Les cellules immunitaires innées telles que les macrophages répondent rapidement aux microbes par le biais de récepteurs (PRR). Les PRR reconnaissent les motifs moléculaires conservés associés aux microbes (MAMP) et déclenchent des réponses immunitaires innées. Par exemple, les récepteurs NOD1 et NOD2 détectent le peptidoglycane (PG) et activent les réponses antimicrobiennes, parmi lesquelles l'autophagie. De plus, l'engagement des PRR peut déclencher une réponse mémoire immunitaire innée ou immunité entraînée (IE). Il a été démontré que les leptospires échappent à certaines réponses immunitaires innées. Le rôle des macrophages au cours de la leptospirose n'a pas encore été clairement établi. Les résultats antérieurs de notre laboratoire ont établi que la lipoprotéine LipL21 se lie au PG des leptospires, altérant la détection par les protéines NOD. Ma contribution a consisté à confirmer le rôle spécifique de la LipL21 sur l'évasion des NOD, en utilisant un système hétérologue d'expression chez E. coli, une bactérie à Gram négatif qui possède également un PG de type méso-Dap (Ratet, Santecchia et al. PLoS Pathog. 2017). Nous avons ensuite étudié les conséquences de l'échappement aux NOD dans les macrophages. Premièrement, des infections in vitro ont suggéré que les leptospires étaient internalisés indépendamment de la polymérisation de l'actine et survivaient dans les macrophages murins et humains pendant 48h. De plus, les leptospires ne déclenchent ni ne bloquent l'autophagie chez les macrophages murins. En outre, l'infection a induit sélectivement l'accumulation de l'adaptateur d'autophagie p62. De plus, le déclenchement pharmacologique de l'autophagie dans les cellules infectées a montré un effet marqué diminuant la viabilité des leptospires. La co-infection de leptospires avec des agonistes des NOD a induit l'autophagie et une dégradation de p62. De plus, des données préliminaires suggèrent que les leptospires ne provoquent pas d'autophagie dans le modèle murin et que l'autophagie au cours de l'infection par les leptospires peut être restaurée avec des agonistes des NOD. Enfin, en utilisant CL429, un agoniste de NOD2 / TLR2, nous avons démontré que l'IE protège de l'infection par les leptospires. Ce phénotype est indépendant des cellules B et T et est caractérisé par une réponse pro-inflammatoire accrue des macrophages péritonéaux et des cellules NK spléniques vis-à-vis de différents sérovars pathogènes de L. interrogans. En outre, le CL429 influence les cellules de la moelle osseuse, renforçant la réponse antibactérienne des macrophages dérivés de la moelle osseuse (BMM). Les PM et les BMM ont montré une activité bactéricide accrue parallèlement à une production accrue d'oxyde nitrique qui n'a jamais été associée auparavant à l'IE. Enfin, un entraînement ex vivo avec CL429 a également rendu les monocytes humains plus sensibles aux leptospires pathogènes (Santecchia et al., PLoS Pathog. 2019, in press). En conclusion, nous avons montré que les leptospires échappent à l'autophagie, qui peut être partiellement restaurée à l'aide d'agonistes des NOD. De plus, la stimulation de macrophages avec des agonistes des NOD améliore leur réponse à différents sérovars pathogènes. Ces deux stratégies basées sur l'utilisation des agonistes des NOD pourraient être exploitées pour la prophylaxie ou la vaccination contre L. interrogans

Leptospira interrogans' escape from NOD1 and NOD2 receptors and NOD-based strategies to restore and boost macrophage functions

Les bactéries Leptospira interrogans sont des spirochètes pathogènes responsables de la leptospirose. Les rongeurs chroniquement infectés excrètent les bactéries dans leur urine, contribuant ainsi à la propagation de la maladie. Il n'existe actuellement aucun vaccin qui puissent conférer une protection croisée pour les 400 sérovars identifiés de leptospires. Les cellules immunitaires innées telles que les macrophages répondent rapidement aux microbes par le biais de récepteurs (PRR). Les PRR reconnaissent les motifs moléculaires conservés associés aux microbes (MAMP) et déclenchent des réponses immunitaires innées. Par exemple, les récepteurs NOD1 et NOD2 détectent le peptidoglycane (PG) et activent les réponses antimicrobiennes, parmi lesquelles l'autophagie. De plus, l'engagement des PRR peut déclencher une réponse mémoire immunitaire innée ou immunité entraînée (IE). Il a été démontré que les leptospires échappent à certaines réponses immunitaires innées. Le rôle des macrophages au cours de la leptospirose n'a pas encore été clairement établi. Les résultats antérieurs de notre laboratoire ont établi que la lipoprotéine LipL21 se lie au PG des leptospires, altérant la détection par les protéines NOD. Ma contribution a consisté à confirmer le rôle spécifique de la LipL21 sur l'évasion des NOD, en utilisant un système hétérologue d'expression chez E. coli, une bactérie à Gram négatif qui possède également un PG de type méso-Dap (Ratet, Santecchia et al. PLoS Pathog. 2017). Nous avons ensuite étudié les conséquences de l'échappement aux NOD dans les macrophages. Premièrement, des infections in vitro ont suggéré que les leptospires étaient internalisés indépendamment de la polymérisation de l'actine et survivaient dans les macrophages murins et humains pendant 48h. De plus, les leptospires ne déclenchent ni ne bloquent l'autophagie chez les macrophages murins. En outre, l'infection a induit sélectivement l'accumulation de l'adaptateur d'autophagie p62. De plus, le déclenchement pharmacologique de l'autophagie dans les cellules infectées a montré un effet marqué diminuant la viabilité des leptospires. La co-infection de leptospires avec des agonistes des NOD a induit l'autophagie et une dégradation de p62. De plus, des données préliminaires suggèrent que les leptospires ne provoquent pas d'autophagie dans le modèle murin et que l'autophagie au cours de l'infection par les leptospires peut être restaurée avec des agonistes des NOD. Enfin, en utilisant CL429, un agoniste de NOD2 / TLR2, nous avons démontré que l'IE protège de l'infection par les leptospires. Ce phénotype est indépendant des cellules B et T et est caractérisé par une réponse pro-inflammatoire accrue des macrophages péritonéaux et des cellules NK spléniques vis-à-vis de différents sérovars pathogènes de L. interrogans. En outre, le CL429 influence les cellules de la moelle osseuse, renforçant la réponse antibactérienne des macrophages dérivés de la moelle osseuse (BMM). Les PM et les BMM ont montré une activité bactéricide accrue parallèlement à une production accrue d'oxyde nitrique qui n'a jamais été associée auparavant à l'IE. Enfin, un entraînement ex vivo avec CL429 a également rendu les monocytes humains plus sensibles aux leptospires pathogènes (Santecchia et al., PLoS Pathog. 2019, in press). En conclusion, nous avons montré que les leptospires échappent à l'autophagie, qui peut être partiellement restaurée à l'aide d'agonistes des NOD. De plus, la stimulation de macrophages avec des agonistes des NOD améliore leur réponse à différents sérovars pathogènes. Ces deux stratégies basées sur l'utilisation des agonistes des NOD pourraient être exploitées pour la prophylaxie ou la vaccination contre L. interrogans.Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis. Chronically infected rodents excrete bacteria in their urine contributing to the spread of the disease. Currently, there is no cross-protective vaccine for more than 400 leptospiral serovars. Innate immune cells such as macrophages quickly respond to microbes through pattern-recognition receptors (PRRs). PRRs recognize conserved microbial-associated molecular patterns (MAMPs) and trigger innate immune responses. For instance, NOD1 and NOD2 receptors sense peptidoglycan (PG) and activate anti-microbial responses, among them autophagy. Furthermore, PRR engagement can trigger the so-called innate immune memory or trained immunity (TI). It has been shown that leptospires escape some innate immune sensing and the role of macrophages during leptospirosis has not yet been clearly established. Previous results from our laboratory established that the LipL21 lipoprotein binds to the leptospiral PG, impairing NOD sensing. My contribution was to confirm the specific role of LipL21 on NOD escape using the heterologous host E. coli that also harbors Gram-negative meso-Dap type PG (Ratet, et al PLoS Pathog. 2017). Next we studied the consequences of NOD escape in macrophages. First, in vitro infections suggested that leptospires were internalized independently of actin polymerization and survive in murine and human macrophages for 48h. Also, leptospires did not trigger nor block autophagy in murine macrophages. In addition, infection selectively induced the accumulation of the autophagy adaptor p62. Moreover, the pharmacological triggering of autophagy on infected cells had a marked effect decreasing the viability of leptospires. Co-infection of leptospires with NOD agonists induced autophagy and p62 degradation. In addition, preliminary data suggest that leptospires do not induce autophagy in the mouse model and that it can be restored with NOD agonists. Finally, using CL429, a NOD2/TLR2 agonist, we demonstrated that TI protects from leptospiral infection (Santecchia et al., PLoS Pathog. 2019). This phenotype was independent of B- and T-cells and characterized by the enhanced pro-inflammatory response of peritoneal macrophages (PMs) and splenic NK-cells towards different pathogenic serovars of L. interrogans. In addition, CL429 influences bone marrow cells, enhancing the antibacterial response of bone marrow-derived macrophages (BMMs). Both PMs and BMMs showed enhanced bactericidal activity alongside more nitric oxide production that has not been previously associated with TI. Finally, ex vivo training with CL429 renders human monocytes more responsive to pathogenic leptospires. In conclusion, we showed that leptospires escape autophagy and that it can be partially restored using NOD agonists. Moreover, training of macrophages with NOD agonists enhance their response toward different pathogenic serovars. Both NOD-based strategies could be exploited for prophylaxis or vaccination

Animal Models of Leptospirosis: Of Mice and Hamsters

International audiencePathogenic Leptospira sp. are spirochetal bacteria responsible for leptospirosis, an emerging worldwide zoonosis. These spirochetes are very successful pathogens that infect a wide range of hosts such as fish, reptiles, birds, marsupials, and mammals. Transmission occurs when chronically infected animals excrete live bacteria in their urine, contaminating the environment. Leptospira sp. enter their hosts through damaged skin and mucosa. Chronically infected rats and mice are asymptomatic and are considered as important reservoirs of the disease. Infected humans may develop either a flu-like, usually mild illness with or without chronic asymptotic renal colonization, or a severe acute disease with kidney, liver, and heart failure, potentially leading to death. Leptospirosis is an economic burden on society due to health-care costs related to elevated morbidity of humans and loss of animals of agricultural interest. There are no effective vaccines against leptospirosis. Leptospira sp. are difficult to genetically manipulate which delays the pace of research progress. In this review, we discuss in an historical perspective how animal models have contributed to further our knowledge of leptospirosis. Hamsters, guinea pigs, and gerbils have been instrumental to study the pathophysiology of acute lethal leptospirosis and the Leptospira sp. genes involved in virulence. Chronic renal colonization has been mostly studied using experimentally infected rats. A special emphasis will be placed on mouse models, long thought to be irrelevant since they survive lethal infection. However, mice have recently been shown to be good models of sublethal infection leading to chronic colonization. Furthermore, congenic and transgenic mice have proven essential to study how innate immune cells interact with the pathogen and to understand the role of the toll-like receptor 4, which is important to control Leptospira sp. load and disease. The use of inbred and transgenic mouse models opens up the field to the comprehensive study of immune responses to Leptospira sp. infection and subsequent pathophysiology of inflammation. It also allows for testing of drugs and vaccines in a biological system that can avail of a wealth of molecular tools that enable understanding of the mechanisms of action of protective vaccines

The extreme C terminus of the Pseudomonas aeruginosa effector ExoY is crucial for binding to its eukaryotic activator, F-actin

International audienceBacterial nucleotidyl cyclase toxins are potent virulence factors that upon entry into eukaryotic cells are stimulated by endogenous cofactors to catalyze the production of large amounts of 35-cyclic nucleoside monophosphates. The activity of the effector ExoY from Pseudomonas aeruginosa is stimulated by the filamentous form of actin (F-actin). Utilizing yeast phenotype analysis, site-directed mutagenesis, functional biochemical assays, and confocal microscopy, we demonstrate that the last nine amino acids of the C terminus of ExoY are crucial for the interaction with F-actin and, consequently, for ExoY's enzymatic activity in vitro and toxicity in a yeast model. We observed that isolated C-terminal sequences of P. aeruginosa ExoY that had been fused to a carrier protein bind to F-actin and that synthetic peptides corresponding to the extreme ExoY C terminus inhibit ExoY enzymatic activity in vitro and compete with the full-length enzyme for F-actin binding. Interestingly, we noted that various P. aeruginosa isolates of the PA14 family, including highly virulent strains, harbor ExoY variants with a mutation altering the C terminus of this effector. We found that these naturally occurring ExoY variants display drastically reduced enzymatic activity and toxicity. Our findings shed light on the molecular basis of the ExoY-F-actin interaction, revealing that the extreme C terminus of ExoY is critical for binding to F-actin in target cells and that some P. aeruginosa isolates carry C-terminally mutated, low-activity ExoY variants

Phagocyte Escape of <i>Leptospira</i>: The Role of TLRs and NLRs

The spirochetal bacteria Leptospira spp. are causative agents of leptospirosis, a globally neglected and reemerging zoonotic disease. Infection with these pathogens may lead to an acute and potentially fatal disease but also to chronic asymptomatic renal colonization. Both forms of disease demonstrate the ability of leptospires to evade the immune response of their hosts. In this review, we aim first to recapitulate the knowledge and explore the controversial data about the opsonization, recognition, intracellular survival, and killing of leptospires by scavenger cells, including platelets, neutrophils, macrophages, and dendritic cells. Second, we will summarize the known specificities of the recognition or escape of leptospire components (the so-called microbial-associated molecular patterns; MAMPs) by the pattern recognition receptors (PRRs) of the Toll-like and NOD-like families. These PRRs are expressed by phagocytes, and their stimulation by MAMPs triggers pro-inflammatory cytokine and chemokine production and bactericidal responses, such as antimicrobial peptide secretion and reactive oxygen species production. Finally, we will highlight recent studies suggesting that boosting or restoring phagocytic functions by treatments using agonists of the Toll-like or NOD receptors represents a novel prophylactic strategy and describe other potential therapeutic or vaccine strategies to combat leptospirosis.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Phagocyte Escape of Leptospira: The Role of TLRs and NLRs

International audienceThe spirochetal bacteria Leptospira spp. are causative agents of leptospirosis, a globally neglected and reemerging zoonotic disease. Infection with these pathogens may lead to an acute and potentially fatal disease but also to chronic asymptomatic renal colonization. Both forms of disease demonstrate the ability of leptospires to evade the immune response of their hosts. In this review, we aim first to recapitulate the knowledge and explore the controversial data about the opsonization, recognition, intracellular survival, and killing of leptospires by scavenger cells, including platelets, neutrophils, macrophages, and dendritic cells. Second, we will summarize the known specificities of the recognition or escape of leptospire components (the so-called microbial-associated molecular patterns; MAMPs) by the pattern recognition receptors (PRRs) of the Toll-like and NOD-like families. These PRRs are expressed by phagocytes, and their stimulation by MAMPs triggers pro-inflammatory cytokine and chemokine production and bactericidal responses, such as antimicrobial peptide secretion and reactive oxygen species production. Finally, we will highlight recent studies suggesting that boosting or restoring phagocytic functions by treatments using agonists of the Toll-like or NOD receptors represents a novel prophylactic strategy and describe other potential therapeutic or vaccine strategies to combat leptospirosis

Innate immune memory through TLR2 and NOD2 contributes to the control of Leptospira interrogans infection

International audienceLeptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis

Genome mining of lipolytic exoenzymes from Bacillus safensis S9 and Pseudomonas alcaliphila ED1 isolated from a dairy wastewater lagoon

Dairy production plants produce highly polluted wastewaters rich in organic molecules such as lactose, proteins and fats. Fats generally lead to low overall performance of the treatment system. In this study, a wastewater dairy lagoon was used as microbial source and different screening strategies were conducted to select 58 lipolytic microorganisms. Exoenzymes and RAPD analyses revealed genetic and phenotypic diversity among isolates. Bacillus safensis, Pseudomonas alcaliphila and the potential pathogens, B. cereus, Aeromonas and Acinetobacter were identified by 16S-rRNA, gyrA, oprI and/or oprL sequence analyses. Five out of 10 selected isolates produced lipolytic enzymes and grew in dairy wastewater. Based on these abilities and their safety, B. safensis S9 and P. alcaliphila ED1 were selected and their genome sequences determined. The genome of strain S9 and ED1 consisted of 3,794,315 and 5,239,535 bp and encoded for 3990 and 4844 genes, respectively. Putative extracellular enzymes with lipolytic (12 and 16), proteolytic (20) or hydrolytic (10 and 15) activity were identified for S9 and ED1 strains, respectively. These bacteria also encoded other technological relevant proteins such as amylases, proteases, glucanases, xylanases and pectate lyases.Fil: Ficarra, Florencia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Santecchia, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Lagorio, Sebastián H.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Alarcon, Sergio Hugo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin