Dual Expression of the Salmonella Effector SrfJ in Mammalian Cells and Plants

SrfJ is an effector of the Salmonella pathogenicity island 2-encoded type III secretion system. Salmonella enterica serovar Typhimurium expresses srfJ under two disparate sets of conditions: media with low Mg2+ and low pH, imitating intravacuolar conditions, and media with myo-inositol (MI), a carbohydrate that can be used by Salmonella as sole carbon source. We investigated the molecular basis for this dual regulation. Here, we provide evidence for the existence of two distinct promoters that control the expression of srfJ. A proximal promoter, PsrfJ, responds to intravacuolar signals and is positively regulated by SsrB and PhoP and negatively regulated by RcsB. A second distant promoter, PiolE, is negatively regulated by the MI island repressor IolR. We also explored the in vivo activity of these promoters in different hosts. Interestingly, our results indicate that the proximal promoter is specifically active inside mammalian cells whereas the distant one is expressed upon Salmonella colonization of plants. Importantly, we also found that inappropriate expression of srfJ leads to reduced proliferation inside macrophages whereas lack of srfJ expression increases survival and decreases activation of defense responses in plants. These observations suggest that SrfJ is a relevant factor in the interplay between Salmonella and hosts of different kingdoms.España, Ministerio de Econonomía, Industria y Competitividad SAF2013-46229-REspaña, Ministerio de Econonomía, Industria y Competitividad SAF2016-75365-

Proteomic insight into the effects of the Salmonella ubiquitin ligase SlrP on host cells

The virulence of the human and animal pathogen Salmonella enterica serovar Typhimurium is dependent on two type III secretion systems. These systems translocate proteins called effectors into eukaryotic host cells. SlrP is a Salmonella type III secretion effector with ubiquitin ligase activity. Here, we used two complementary proteomic approaches, two-dimensional gel electrophoresis and iTRAQ (isobaric tags for relative and absolute quantification) to study the consequences of the presence of SlrP in human epithelial cells. We identified 37 proteins that were differentially expressed in HeLa cells expressing slrP compared to control cells. Microarray analysis revealed that more than a half of differentially expressed proteins did not show changes in the transcriptome, suggesting post-transcriptional regulation. A gene ontology overrepresentation test carried out on the differentially expressed proteins revealed enrichment of ontology terms related to several types of junctions mediating adhesion in epithelial cells. Consistently, slrP-transfected cells showed defects in migration and adhesion. Our results suggest that the modification of cell–cell interaction ability of the host could be one of the final consequences of the action of SlrP during an infection.Ministerio de Ciencia e Innovación de España y el Fondo Europeo de Desarrollo Regional (FEDER) SAF2010-15015 y SAF2013-46229-RConsejería de Innovación, Ciencia y Empresa, Junta de Andalucía, España. P08-CVI -0348

Proteomic insight into the effects of the Salmonella ubiquitin ligase SlrP on host cells

The virulence of the human and animal pathogen Salmonella enterica serovar Typhimurium is dependent on two type III secretion systems. These systems translocate proteins called effectors into eukaryotic host cells. SlrP is a Salmonella type III secretion effector with ubiquitin ligase activity. Here, we used two complementary proteomic approaches, two-dimensional gel electrophoresis and iTRAQ (isobaric tags for relative and absolute quantification) to study the consequences of the presence of SlrP in human epithelial cells. We identified 37 proteins that were differentially expressed in HeLa cells expressing slrP compared to control cells. Microarray analysis revealed that more than a half of differentially expressed proteins did not show changes in the transcriptome, suggesting post-transcriptional regulation. A gene ontology overrepresentation test carried out on the differentially expressed proteins revealed enrichment of ontology terms related to several types of junctions mediating adhesion in epithelial cells. Consistently, slrP-transfected cells showed defects in migration and adhesion. Our results suggest that the modification of cell–cell interaction ability of the host could be one of the final consequences of the action of SlrP during an infection.Ministerio de Ciencia e Innovación de España y el Fondo Europeo de Desarrollo Regional (FEDER) SAF2010-15015 y SAF2013-46229-RConsejería de Innovación, Ciencia y Empresa, Junta de Andalucía, España. P08-CVI -0348

Genetic and molecular analysis of the salmonella effector SrfJ and use of type III secretion effectors as carriers for heterologous vaccine design.

Salmonella enterica is a species of bacterial pathogens that can produce different diseases from gastroenteritis to typhoid fever. Salmonella possesses two different virulence-related type III secretion systems (T3SSs) that are key elements in the interaction with the host cell. These systems mediate the translocation of effector proteins into the cytosol of the host cell where they interfere with different cellular processes to allow the pathogen entry and its survival inside vacuoles. We studied the S. enterica serovar Typhimurium SrfJ effector expression. We provide evidence for the existence of two distinct promoters that control the expression of srfJ. A proximal promoter, PsrfJ, responds to intravacuolar signals inside mammalian cells and is positively regulated by SsrB and PhoP and negatively regulated by RcsB. A second distal promoter, PiolE, is negatively regulated by the myo-inositol island repressor IolR, whereas it is expressed upon Salmonella colonization of plants. Importantly, we also found that inappropriate expression of srfJ leads to reduced proliferation inside macrophages whereas lack of srfJ expression increases survival and decreases activation of defense responses in plants. These observations suggest that SrfJ is a relevant factor in the interplay between Salmonella and host of different kingdoms. Transcriptomic carried out in human epithelial HeLa cells and murine RAW264.7 macrophages detected 16 genes that are significantly down-regulated and 12 genes that are significantly upregulated in response to the presence of SrfJ. Proteomic analysis revealed that SrfJ is involved in dephosphorylation of WNK1 and prevention of induction of HSP60. The last part of this thesis was focused on the development of a live Salmonella vaccine against Pseudomonas aeruginosa. As T3SS-mediated translocation can be used for efficient delivery of heterologous antigens to the cytosol of antigen-presenting cells, we tested the possibility of using Salmonella effectors SseJ, SrfJ, SlrP, SteA and SseK1, as carriers in the design of this vaccine. We finally developed a vaccine delivering the Pseudomonas antigen PcrV in fusion with SseJ through the T3SS. This vaccine protected mice against lethal infections with P. aeruginosa.Salmonella enterica es una especie de bacterias patógenas que pueden producir diferentes enfermedades desde gastroenteritis a enfermedades sistémicas. Salmonella posee dos sistemas de secreción tipo III (T3SS) relacionados con la virulencia que son elementos clave en la interacción con la célula hospedadora. Estos sistemas median la translocación de proteínas efectoras al citosol de la célula hospedadora donde interfieren con diferentes procesos celulares para permitir la entrada del patógeno y su supervivencia dentro de vacuolas. Se estudió la expresión del efector SrfJ de S. enterica serovar Typhimurium. Se averiguó la existencia de dos promotores distintos que controlan la expresión de srfJ. Un promotor proximal, PsrfJ, que responde a las señales intravacuolares dentro de las células de mamífero y está regulado positivamente por SsrB y PhoP y negativamente por RcsB. Un segundo promotor distal, PiolE, que está regulado negativamente por el represor de la isla de utilización del mio-inositol, IolR, mientras que se expresa tras la colonización de plantas por Salmonella. A su vez, se estableció que la expresión inapropiada de srfJ conduce a una reducción de la proliferación en macrófagos, mientras que la falta de expresión de srfJ aumenta la supervivencia y disminuye la activación de las respuestas de defensa en plantas. Estas observaciones sugieren que SrfJ es un factor relevante en la interacción entre Salmonella y hospedadores de diferentes reinos. A través del análisis transcriptómico llevado a cabo en células epiteliales humanas HeLa y macrófagos de ratón RAW264.7 se detectaron 16 genes con expresión significativamente reducida y 12 genes con expresión significativamente aumentada en respuesta a la presencia de SrfJ. Un análisis proteómico indicó que SrfJ está implicado en la desfosforilación de WNK1 y en la prevención de la inducción de HSP60. La última parte de esta tesis se centró en el desarrollo de una vacuna viva de Salmonella contra Pseudomonas aeruginosa. Dado que la translocación mediada por T3SS puede usarse para la administración eficiente de antígenos heterólogos al citosol de células presentadoras de antígeno, se probó el uso de los efectores de Salmonella SseJ, SrfJ, SlrP, SteA y SseK1, como portadores en el diseño de la vacuna. Finalmente desarrollamos una vacuna en la que el antígeno PcrV de Pseudomonas en fusión con SseJ se secreta a través de un T3SS. Esta vacuna protegió a los ratones contra una infección letal con P. aeruginosa

Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.Fondo Europeo de Desarrollo Regional (FEDER) de la Unión Europea, y Departament de Recerca i Universitats de la Generalitat de Catalunya - IU16-01580