Molecular Mechanisms Used by Salmonella to Evade the Immune System

Human and animal pathogens are able to circumvent, at least temporarily, the sophisticated immune defenses of their hosts. Several serovars of the Gram-negative bacterium Salmonella enterica have been used as models for the study of pathogen-host interactions. In this review we discuss the strategies used by Salmonella to evade or manipulate three levels of host immune defenses: physical barriers, innate immunity and adaptive immunity. During its passage through the digestive system, Salmonella has to face the acidic pH of the stomach, bile and antimicrobial peptides in the intestine, as well as the competition with resident microbiota. After host cell invasion, Salmonella manipulates inflammatory pathways and the autophagy process. Finally, Salmonella evades the adaptive immune system by interacting with dendritic cells, and T and B lymphocytes. Mechanisms allowing the establishment of persistent infections are also discussed.European Regional Development Fund SAF2013-46229-R, SAF2016-75365-

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targetsSpanish Ministerio de Economía, Industria y Competitividad , Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016‐75365‐REuropean Union’s Horizon 2020 e Marie Skłodowska‐Curie grant agreement No 84262

Patterns of expression and translocation of the ubiquitin ligase SlrP in Salmonella enterica serovar typhimurium.

SlrP is an E3 ubiquitin ligase that can be translocated into eukaryotic host cells by the two type III secretion systems that are expressed by Salmonella enterica serovar Typhimurium and are encoded in Salmonella pathogenicity islands 1 (SPI1) and 2 (SPI2). Expression of slrP and translocation of its product were examined using lac, 3×FLAG, and cyaA′ translational fusions. Although slrP was expressed in different media, optimal expression was found under conditions that imitate the intravacuolar environment and promote synthesis of the SPI2-encoded type III secretion system. Translocation into mammalian cells took place through the SPI1- or the SPI2-encoded type III secretion system, depending on specific host cell type and timing. A search for genetic factors involved in controlling the expression of slrP unveiled LeuO, Lon, and the two-component system PhoQ/PhoP as novel regulators of slrP. Our experiments suggest that LeuO and Lon act through HilD under SPI1-inducing conditions, whereas PhoP directly interacts with the slrP promoter to activate transcription under SPI2 inducing conditions

DsbA and MgrB regulate steA expression through the two-component system PhoQ/PhoP in Salmonella enterica.

SteA is a protein that can be translocated into host cells through the two virulence-related type III secretion systems that are present in Salmonella enterica. We used the T-POP system to carry out general screens for loci that exhibited activation or repression of a steA::lacZ fusion. These screens identified the histidine kinase PhoQ and the response regulator PhoP as positive regulators of steA. Transcription of this gene is σ70 dependent, and the promoter of steA contains a PhoP-binding site that mediates direct regulation by PhoP. Our screens also detected MgrB (also known as YobG) as a negative regulator of the expression of steA. Disruption of the gene encoding the periplasmic disulfide oxidoreductase DsbA or addition of the reducing agent dithiothreitol increases transcription of steA. The effects of MgrB and DsbA on steA are mediated by PhoP. These results suggest that the cellular redox status is a factor contributing to regulation of steA and, probably, other virulence genes regulated by the PhoQ/PhoP two-component syste

Salmonella type III secretion effector SlrP is an E3 ubiquitin ligase for mammalian thioredoxin

Salmonella enterica encodes two virulence-related type III secretion systems in Salmonella pathogenicity islands 1 and 2, respectively. These systems mediate the translocation of protein effectors into the eukaryotic host cell, where they alter cell signaling and manipulate host cell functions. However, the precise role of most effectors remains unknown. Using a genetic screen, we identified the small, reduction/ oxidation-regulatory protein thioredoxin as a mammalian binding partner of the Salmonella effector SlrP. The interaction was confirmed by affinity chromatography and coimmunoprecipitation. In vitro, SlrP was able to mediate ubiquitination of ubiquitin and thioredoxin. A Cys residue conserved in other effectors of the same family that also possess E3 ubiquitin ligase activity was essential for this catalytic function. Stable expression of SlrP in HeLa cells resulted in a significant decrease of thioredoxin activity and in an increase of cell death. The physiological significance of these results was strengthened by the finding that Salmonella was able to trigger cell death and inhibit thioredoxin activity in HeLa cells several hours post-infection. This study assigns a functional role to the Salmonella effector SlrP as a binding partner and an E3 ubiquitin ligase for mammalian thioredoxin.Ministerio de Ciencia e Innovación SAF2007-60738Junta de Andalucía P08-CVI-0348

Global impact of Salmonella type III secretion effector SteA on host cells

Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell-cell adhesion and migration.Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, Spain (grant number P08-CVI-03487).Spanish Ministry of Science and Innovation and the European Regional Development Fund (grant number SAF2010-15015

SrfJ, a salmonella type III secretion system effector regulated by PhoP, RcsB, and IolR.

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of srfJ, a Salmonella enterica serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by Salmonella pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of srfJ was positively regulated by the PhoP/PhoQ system in an SsrBdependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an srfJ-lacZ transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in myo-inositol utilization, as an srfJ repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in myo-inositol-rich environments

Host cell type-dependent translocation and PhoP-mediated positive regulation of the effector SseK1 of Salmonella enterica.

Salmonella enterica expresses two virulence-related type III secretion systems (T3SSs) encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. SseK1 is a poorly characterized substrate of the SPI2-encoded T3SS. Here, we show that this effector is essential to get full virulence both in oral and intraperitoneal mice infections, in spite of not having a role in invasion or intracellular proliferation in cultured mammalian cells. In vitro, expression of sseK1 was higher in media mimicking intracellular conditions, when SPI2 was induced, but it was also significant under SPI1 inducing conditions. A detailed analysis of translocation of SseK1 into host cells unveiled that it was a substrate of both, T3SS1 and T3SS2, although with different patterns and kinetics depending on the specific host cell type (epithelial, macrophages, or fibroblasts). The regulation of the expression of sseK1 was examined using lacZ and bioluminescent lux fusions. The two-component system PhoQ/PhoP is a positive regulator of this gene. A combination of sequence analysis, directed mutagenesis and electrophoretic mobility shift assays showed that phosphorylated PhoP binds directly to the promoter region of sseK1 and revealed a PhoP binding site located upstream of the predicted -35 hexamer of this promoter

The salmonella type III secretion effector, Salmonella Leucine-rich Repeat Protein (SlrP), targets the human chaperone ERdj3

Effectors of the type III secretion systems (T3SS) are key elements in the interaction between many Gram-negative pathogens and their hosts. SlrP is an effector that is translocated into the eukaryotic host cell through the two virulence-associated T3SS of Salmonella enterica. We found previously that this effector is an E3 ubiquitin ligase for mammalian thioredoxin. Here, we identified ERdj3, an endoplasmic reticulum lumenal chaperone of the Hsp40/DnaJ family, as a new target for SlrP. Experiments with truncated forms of ERdj3 showed that domain II was essential for the interaction with SlrP. Confocal microscopy and subcellular fractionation demonstrated that, in transfected HeLa cells, SlrP was partially located in the endoplasmic reticulum. The presence of SlrP interfered with the binding of ERdj3 to a denatured substrate. Taken together, these data suggest that the role of SlrP in the interaction between Salmonella and the host cell is exerted through the modulation of the function of two independent targets: thioredoxin in the cytosol, and ERdj3 in the endoplasmic reticulum.Ministerio de Ciencia e Innovación SAF2007-60738Junta de Andalucía P08-CVI-0348

Implantación en Mallorca de un programa de Tratamiento multimodal de la Carcinomatosis Peritoneal mediante Quimioterapia Sistémica, Cirugía Citorreductora y Quimioterapia Intraperitoneal Hipertérmica (HIPEC)

Introducción: Las investigaciones de Sugarbaker et al demostraron desde principios de los 90 que muchos tumores de origen colorrectal y apendicular con extensión peritoneal pueden permanecer limitados al abdomen durante períodos relativamente largos antes de desarrollar metástasis a distancia. Con ello, la carcinomatosis peritoneal pasó de considerarse desde una perspectiva sistémica y prácticamente terminal a concebirse como una enfermedad loco-regional, susceptible de rescate quirúrgico y oncológico, con un aumento de la supervivencia. Los estudios experimentales de la cirugía citorreductora radical asociada a la quimioterapia intraperitoneal hipertérmica ofrecen mejores resultados en términos de supervivencia a largo plazo y de calidad de vida que los tratamientos convencionales. El tratamiento actual de la carcinomatosis peritoneal es multimodal, con tres líneas de acción: 1. Quimioterapia intravenosa preoperatoria de inducción; 2. Cirugía citorreductora y peritonectomía, y 3. Quimioterapia intraperitoneal hipertérmica. Su base científica se fundamenta en el sinergismo entre la hipertermia y varios quimioterápicos (Mitomicina-C, Cisplatino, Oxaliplatino, etc), en la citotoxicidad directa del calor sobre las células tumorales y en las ventajas farmacocinéticas de la quimioterapia administrada por vía intraperitoneal. Metodología: Objetivos del Programa: 1. Desarrollo del Protocolo de tratamiento multimodal oncológico y quirúrgico. 2. Formación continuada del grupo de trabajo multidisciplinario. 3. Formación continuada en el manejo de la técnica de perfusión de quimioterapia intraperitoneal hipertérmica. 4. Aplicación de un protocolo de seguridad laboral. 5. Definición de indicadores de resultados. 6. Diseño de base de datos informatizada para explotación de resultados. 7. Seguimiento y registro de morbilidad y mortalidad. 8. Seguimiento y análisis de supervivencia a medio y largo plazo. Evaluación clínica preoperatoria y estudio de extensión tumoral. 1. Indicaciones clínicas. Tipos de tumores. 2. Selección de pacientes. Criterios de inclusión y de exclusión. 3. Evaluación oncológica preoperatoria. 4. Evaluación radiológica preoperatoria. 5. Algoritmo de actuación. 6. Seguimiento de pacientes. 7. Seguridad laboral. Resultados: Hace cinco años se puso en marcha en el Hospital Son Llàtzer el programa completo de cirugía citorreductora con quimioterapia intraperitoneal hipertérmica para el tratamiento de la carcinomatosis peritoneal. Se han intervenido 46 pacientes (40 mujeres y 6 hombres), con una edad media de 58.3 años (47-73 años). De los 46 pacientes, 30 lo eran con cáncer de ovario, y en ellos se realizó cirugía citorreductora seguida de quimioterapia sistémica sin aplicación de quimioterapia intraperitoneal. El programa de cirugía citorreductora y quimioterapia intraperitoneal se compone de 17 pacientes en los que se realizaron un total de 20 procedimientos. El origen del tumor fue colorrectal en 11 casos, apendicular en 4 casos, gástrico en un caso y un mesotelioma peritoneal multiquísitico difuso. En 9 casos se realizó quimioterapia intraperitoneal normotérmica y en 11 casos quimioterapia intraperitoneal hipertérmica (HIPEC). La mortaliad perioperatoria fue de un 4.3% (tres pacientes). El índice de resecabildad tumoral fue de un 90.9%. En 17 casos (85%), se pudo conseguir una resección completa (CC-0). La morbilidad global fue de un 40%, siendo la complicación más frecuente la infección de catéter central. Fueron reintervenidos tres pacientes (hemorragia, fístula duodenal e isquemia mesentérica segmentaria). La duración media de la intervención fue de 6.5 horas y la estancia media de 18.2 días. La supervivencia media de los pacientes con diseminación peritoneal de origen colorrectal y apendicular ha sido de 27 y 31 meses respectivamente. La supervivencia global de los pacientes con carcinomatosis peritoneal de origen colorrectal al año, dos y tres años fue del 71%, 60% y 50% respectivamente. Conclusiones: a) La aplicación de un tratamiento multidisciplinar y multimodal puede mejorar de forma significativa la supervivencia en un grupo específico de pacientes con carcinomatosis peritoneal; b) Este tratamiento es costoso y complejo y supone una importante carga de trabajo para los profesionales y las instituciones, por lo que es esencial aplicar criterios rigurosos de selección de pacientes, fundamentados en la experiencia y en la evaluación por equipos multidisciplinares. Es esencial el diagnóstico precoz, el tratamiento de las complicaciones, el seguimiento de los pacientes y la evaluación continua de los resultados de morbilidad y supervivencia. c) los estudios randomizados en curso aportarán previsiblementeIntroduction: Research promoted since the early nineties by Sugarbaker et al in Washington, have demonstrated that many tumors of colorectal and appendiceal origen with peritoneal extension, may remain limited to the abdomen during relatively long periods before developing distant metastases. These findings have radically altered the consideration of peritoneal carcinomatosis from a systemic and almost terminal state, to a locoregional disease, with possibility of surgical and oncological rescue, and thus, increasing the survival. The results of experimental studies of radical cytoreductive surgery associated with hyperthermic intraperitoneal chemotherapy, have demonstrated better results in terms of long-term survival and quality of life than conventional treatments. The rationale for the current treatment of peritoneal carcinomatosis is organized around a multimodal treatment with three courses of action: 1. Preoperative intravenous induction chemotherapy, 2. Cytoreductive surgery and peritonectomy, and 3. Hyperthermic intraperitoneal chemotherapy (HIPEC). The scientific basis of this multimodal treatment is based on the synergism between the hyperthermia and several cytostatic agents (mitomycin-C, cisplatin, oxaliplatin, etc.), the direct heat cytotoxicity on tumor cells, and the pharmacokinetic advantages of chemotherapy administered intraperitoneally. Methodology: Program Objectives were the following: 1. Development of Multimodal surgical and oncologic treatment protocol; 2. Continuing education of the multidisciplinary group; 3. Ongoing training in the hyperthermic intraperitoneal perfusion chemotherapy; 4. Application of a Work Safety Protocol; 5. Definition of indicators; 6. Design of database for exploitation of results; 7. Monitoring of morbidity and mortality, and 8. Monitoring and analysis of survival. Clinical evaluation and preoperative tumor extension study. 1. Clinical Indications. Types of tumors; 2. Patient selection. Inclusion and exclusion criteria; 3. Preoperative Evaluation Oncology; 4. Preoperative radiological evaluation; 5. Clinical pathway algorithm; 6. Patients follow-up, and 7. Laboral safety. Results: Five years ago, we started cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for treatment of peritoneal carcinomatosis in the Hospital Son Llàtzer. 46 patients were operated (40 women and 6 men) with a mean age of 58.3 years (47-73 years). Of these 46 patients, 30 were patients with ovarian cancer, and underwent cytoreductive surgery followed by chemotherapy without applying systemic intraperitoneal chemotherapy. The program consisted of 17 patients who underwent a total of 20 procedures. The origin of the tumor was colorectal in 11 cases, appendiceal in 4 cases, gastric in 1 case, and one case with a diffuse multicystic peritoneal mesothelioma. The intraperitoneally chemotherapy administered was normothermic in 9 patients, and hyperthermic (HIPEC) in 11 cases. The perioperative mortality was 4.3% (three patients). The resectability index was 90.9%. In 17 cases (85%) it was possible to get a complete resection (CC-0). Overall morbidity was 40%. The most frequent complication was central catheter infection. Three patients required reoperation (postoperative bleeding, duodenal fistula, and segmental mesenteric ischemia). The mean operative duration was 6.5 hours. The average stay was 18.2 days. The median survival of patients with peritoneal dissemination of appendiceal and colorectal origin was 27 and 31 months respectively. Overall survival of patients with peritoneal carcinomatosis of colorectal origin was at one, two, and three years 71%, 60% and 50% respectively. Conclusions: a) The application of a multidisciplinary and multimodal treatment can significantly improve survival in a specific group of patients with peritoneal carcinomatosis. b) This treatment is expensive, complex and represents a significant workload for professionals and institutions, so it is essential to apply strict criteria for patient selection, based on experience and the evaluation by multidisciplinary teams. Similarly, it is essential to achieve an early diagnosis, treatment of complications, patient monitoring, and continuous evaluation of morbidity outcomes and survival. c) Randomized studies currently underway will provide more evidence on the expected benefit of this treatment, and establish lines to improve results