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

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

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

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

Tubulin Folding Cofactor TBCB is a Target of the Salmonella Effector Protein SseK1

Salmonella enterica serovar Typhimurium is a human and animal pathogen that uses type III secretion system effectors to manipulate the host cell and fulfill infection. SseK1 is a Salmonella effector with glycosyltransferase activity. We carried out a yeast two-hybrid screen and have identified tubulin-binding cofactor B (TBCB) as a new binding partner for this effector. SseK1 catalyzed the addition of N-acetylglucosamine to arginine on TBCB, and its expression promoted the stabilization of the microtubule cytoskeleton of HEK293T cells. The conserved Asp-x-Asp (DxD) motif that is essential for the activity of SseK1 was required for the binding and modification of TBCB and for the effect on the cytoskeleton. Our study has identified a novel target for SseK1 and suggests that this effector may have a role in the manipulation of the host cell microtubule network to provide a safe niche for this pathogen.España Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016-75365-R;European Union’s Horizon 2020 grant agreement No 842629

The Structure of the SlrP-hTrx1 Complex Sheds Light on the Autoinhibition Mechanism of the Type-III Secretion System Effectors of the NEL Family

Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type-III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP/Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain. Grant P08-CVI-03487Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund. SAF2010-15015 and SAF2013-46229-RSpanish Ministry of Science and Innovation. Grant FR2009-0103Programme Picasso-2010 from the Partenariat Hubert Curie

SNRPD2 Is a Novel Substrate for the Ubiquitin Ligase Activity of the Salmonella Type III Secretion Effector SlrP

SlrP is a protein with E3 ubiquitin ligase activity that is translocated by Salmonella enterica serovar Typhimurium into eukaryotic host cells through a type III secretion system. A yeast two-hybrid screen was performed to find new human partners for this protein. Among the interacting proteins identified by this screen was SNRPD2, a core component of the spliceosome. In vitro ubiquitination assays demonstrated that SNRPD2 is a substrate for the catalytic activity of SlrP, but not for other members of the NEL family of E3 ubiquitin ligases, SspH1 and SspH2. The lysine residues modified by this activity were identified by mass spectrometry. The identification of a new ubiquitination target for SlrP is a relevant contribution to the understanding of the role of this Salmonella effector.Ministerio de Ciencia e Innovación PID2019-106132RB-I00/AEI/10.13039/501100011033Junta de Andalucía P20_00576, US-138080

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2

Análisis de la función de S1rP, un efector de los sistemas de secreción de tipo III de Salmonella enterica, en la interacción con la célula hospedadora

Salmonella enterica en una especie de bacterias patógenas que pueden producir gastroenteritis o enfermedades sistémicas. Salmonella posee dos sistemas de secreción de tipo III (SST3) relacionados con la virulencia, que son elementos claves en la interacción con la célula hospedadora. Estos sistemas median la translocación de proteínas efectoras al citosol de la célula hospedadora donde pueden alte rar las rutas de señalización y manipular las funciones celulares. Sin embargo, se desconoce el papel específico de muchos de estos efectores. SlrP es un efector de Salmonella enterica que puede ser translocado por los dos SST3 de esta bacteria y del que no se conocía su función en el momento de iniciar este estudio. Por tanto, nos planteamos como objetivo realizar un análisis funcional de SlrP en la interacción con la célula hospedadora.A través de un escrutinio genético se identificaron dos proteínas humanas que interaccionaban con SlrP: la tiorredoxina citosólica (Trx) y ERdj3. La primera es una proteína que protege a la célula del daño oxidativo, mientras que la segunda es una chaperona localizada en el retículo endoplásmico. Ambas interacciones se confirmaron por diversos métodos independientes. Experimentos in vitro, demostraron que SlrP tenía actividad ligasa de ubiquitina y era capaz de usar Trx como sustrato. En cultivos celulares que expresaban SlrP de forma constitutiva se observó un descenso en la actividad reductora de Trx y un aumento en la muerte celular. Ambos efectos se observaron también en cultivos de células epiteliales humanas infectadas con Salmonella.Por otro lado, se demostró que el dominio II de ERdj3 era imprescindible para la interacción con SlrP y que la presencia de SlrP impedía la unión de la chaperona a un sustrato desnaturalizado. Además, mediante microscopía confocal y fraccionamiento celular, se demostró que SlrP se localiza parcialmente en el retículo endoplásmico de células HeLa transfectadas.Todos estos datos sugieren que SlrP podría favorecer la muerte de la célula hospedadora a través de la modulación de la función de dos proteínas dianas, de forma independiente: Trx en el citosol y ERdj3 en el retículo endoplásmico. La última parte de la Tesis consistió en un análisis estructural, mediante cristalografía de rayos X de SlrP en complejo con Trx. Se obtuvieron cristales del complejo que difractaron a 3,5 Å de resolución con objeto de resolver la estructura del complejo. El análisis se basó, parcialmente, en las estructuras conocidas de efectores de la misma familia de SlrP y de la Trx humana