scholarly journals Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

2012 ◽  
Vol 80 (7) ◽  
pp. 2454-2463 ◽  
Author(s):  
Stephen J. Forbes ◽  
Daniel Martinelli ◽  
Chyongere Hsieh ◽  
Jeffrey G. Ault ◽  
Michael Marko ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Integrity ◽  
Effector Proteins ◽  
Content Type ◽  
O Antigen ◽  
Serovar Typhimurium ◽  
Type 3

ABSTRACTInvasion of intestinal epithelial cells bySalmonella entericaserovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as theSalmonellapathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry ofS. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface ofS. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of theS. Typhimurium cell envelope and temporarily renders the bacterium avirulent.

scholarly journals Display of Recombinant Proteins on Bacterial Outer Membrane Vesicles by Using Protein Ligation

2018 ◽  
Vol 84 (8) ◽  
pp. e02567-17 ◽  
Author(s):  
H. Bart van den Berg van Saparoea ◽  
Diane Houben ◽  
Marien I. de Jonge ◽  
Wouter S. P. Jong ◽  
Joen Luirink
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Vesicles ◽  
Therapeutic Agents ◽  
Outer Membrane Vesicles ◽  
High Density ◽  
Content Type ◽  
Protein Ligation ◽  
Serovar Typhimurium

ABSTRACT The Escherichia coli virulence factor hemoglobin protease (Hbp) has been engineered into a surface display system that can be expressed to high density on live E. coli and Salmonella enterica serovar Typhimurium cells or derived outer membrane vesicles (OMVs). Multiple antigenic sequences can be genetically fused into the Hbp core structure for optimal exposure to the immune system. Although the Hbp display platform is relatively tolerant, increasing the number, size, and complexity of integrated sequences generally lowers the expression of the fused constructs and limits the density of display. This is due to the intricate mechanism of Hbp secretion across the outer membrane and the efficient quality control of translocation-incompetent chimeric Hbp molecules in the periplasm. To address this shortcoming, we explored the coupling of purified proteins to the Hbp carrier after its translocation across the outer membrane using the recently developed SpyTag/SpyCatcher protein ligation system. As expected, fusion of the small SpyTag to Hbp did not hamper display on OMVs. Subsequent addition of purified proteins fused to the SpyCatcher domain resulted in efficient covalent coupling to Hbp-SpyTag. Using in addition the orthogonal SnoopTag/SnoopCatcher system, multiple antigen modules could be coupled to Hbp in a sequential ligation strategy. Not only antigens proved suitable for Spy-mediated ligation but also nanobodies. Addition of this functionality to the platform might allow the targeting of live bacterial or OMV vaccines to certain tissues or immune cells to tailor immune responses.IMPORTANCE Outer membrane vesicles (OMVs) derived from Gram-negative bacteria attract increasing interest in the development of vaccines and therapeutic agents. We aim to construct a semisynthetic OMV platform for recombinant antigen presentation on OMVs derived from attenuated Salmonella enterica serovar Typhimurium cells displaying an adapted Escherichia coli autotransporter, Hbp, at the surface. Although this autotransporter accepts substantial modifications, its capacity with respect to the number, size, and structural complexity of the antigens genetically fused to the Hbp carrier is restricted. Here we describe the application of SpyCatcher/SpyTag protein ligation technology to enzymatically link antigens to Hbp present at high density in OMVs. Protein ligation was apparently unobstructed by the membrane environment and allowed a high surface density of coupled antigens, a property we have shown to be important for vaccine efficacy. The OMV coupling procedure appears versatile and robust, allowing fast production of experimental vaccines and therapeutic agents through a modular plug-and-display procedure.

scholarly journals aro Mutations in Salmonella enterica Cause Defects in Cell Wall and Outer Membrane Integrity

2008 ◽  
Vol 190 (9) ◽  
pp. 3155-3160 ◽  
Author(s):  
Alena Sebkova ◽  
Daniela Karasova ◽  
Magdalena Crhanova ◽  
Eva Budinska ◽  
Ivan Rychlik
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Membrane Integrity ◽  
Innate Immune ◽  
Multicopy Plasmid ◽  
Oral Vaccines ◽  
Serovar Typhimurium ◽  
Serum Albumen

ABSTRACT In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.

scholarly journals Cytosporone B, an Inhibitor of the Type III Secretion System of Salmonella enterica Serovar Typhimurium

2013 ◽  
Vol 57 (5) ◽  
pp. 2191-2198 ◽  
Author(s):  
Jianfang Li ◽  
Chao Lv ◽  
Weiyang Sun ◽  
Zhenyu Li ◽  
Xiaowei Han ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Bacterial Resistance ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACTBacterial virulence factors have been increasingly regarded as attractive targets for development of novel antibacterial agents. Virulence inhibitors are less likely to generate bacterial resistance, which makes them superior to traditional antibiotics that target bacterial viability.Salmonella entericaserovar Typhimurium, an important food-borne human pathogen, has type III secretion system (T3SS) as its major virulence factor. T3SS secretes effector proteins to facilitate invasion into host cells. In this study, we identified several analogs of cytosporone B (Csn-B) that strongly block the secretion ofSalmonellapathogenicity island 1 (SPI-1)-associated effector proteins, without affecting the secretion of flagellar protein FliCin vitro. Csn-B and two other derivatives exhibited a strong inhibitory effect on SPI-1-mediated invasion to HeLa cells, while no significant toxicity to bacteria was observed. Nucleoid proteins Hha and H-NS bind to the promoters of SPI-1 regulator geneshilD,hilC, andrtsAto repress their expression and consequently regulate the expression of SPI-1 apparatus and effector genes. We found that Csn-B upregulated the transcription ofhhaandhns, implying that Csn-B probably affected the secretion of effectors through the Hha–H-NS regulatory pathway. In summary, this study presented an effective SPI-1 inhibitor, Csn-B, which may have potential in drug development against antibiotic-resistantSalmonella.

scholarly journals Dormant Intracellular Salmonella enterica Serovar Typhimurium Discriminates among Salmonella Pathogenicity Island 2 Effectors To Persist inside Fibroblasts

2013 ◽  
Vol 82 (1) ◽  
pp. 221-232 ◽  
Author(s):  
Cristina Núñez-Hernández ◽  
Ana Alonso ◽  
M. Graciela Pucciarelli ◽  
Josep Casadesús ◽  
Francisco García-del Portillo
Keyword(s):  
Salmonella Enterica ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Intracellular Bacteria ◽  
Secretion Systems ◽  
Cultured Fibroblasts ◽  
Content Type ◽  
Type Iii Secretion Systems ◽  
Serovar Typhimurium

ABSTRACTSalmonella entericauses effector proteins delivered by type III secretion systems (TTSS) to colonize eukaryotic cells. Recentin vivostudies have shown that intracellular bacteria activate the TTSS encoded bySalmonellapathogenicity island-2 (SPI-2) to restrain growth inside phagocytes. Growth attenuation is also observedin vivoin bacteria colonizing nonphagocytic stromal cells of the intestinal lamina propria and in cultured fibroblasts. SPI-2 is required for survival of nongrowing bacteria persisting inside fibroblasts, but its induction mode and the effectors involved remain unknown. Here, we show that nongrowing dormant intracellular bacteria use the two-component system OmpR-EnvZ to induce SPI-2 expression and the PhoP-PhoQ system to regulate the time at which induction takes place, 2 h postentry. Dormant bacteria were shown to discriminate the usage of SPI-2 effectors. Among the effectors tested, SseF, SseG, and SseJ were required for survival, while others, such as SifA and SifB, were not. SifA and SifB dispensability correlated with the inability of intracellular bacteria to secrete these effectors even when overexpressed. Conversely, SseJ overproduction resulted in augmented secretion and exacerbated bacterial growth. Dormant bacteria produced other effectors, such as PipB and PipB2, that, unlike what was reported for epithelial cells, did not to traffic outside the phagosomal compartment. Therefore, permissiveness for secreting only a subset of SPI-2 effectors may be instrumental for dormancy. We propose that theS. entericaserovar Typhimurium nonproliferative intracellular lifestyle is sustained by selection of SPI-2 effectors that are produced in tightly defined amounts and delivered to phagosome-confined locations.

scholarly journals Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Mediates Ubiquitination-Dependent STAT3 Activation upon Salmonella enterica Serovar Typhimurium Infection

2017 ◽  
Vol 85 (8) ◽  
Author(s):  
Hai-Hua Ruan ◽  
Zhen Zhang ◽  
Su-Ying Wang ◽  
Logan M. Nickels ◽  
Li Tian ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Salmonella Enterica ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Effector Proteins ◽  
Host Cells ◽  
Associated Factor ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Necrosis Factor

ABSTRACT Salmonella enterica serovar Typhimurium can inject effector proteins into host cells via type III secretion systems (T3SSs). These effector proteins modulate a variety of host transcriptional responses to facilitate bacterial growth and survival. Here we show that infection of host cells with S. Typhimurium specifically induces the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). This TRAF6 ubiquitination is triggered by the Salmonella pathogenicity island 1 (SPI-1) T3SS effectors SopB and SopE2. We also demonstrate that TRAF6 is involved in the SopB/SopE2-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a signaling event conducive to the intracellular growth of S. Typhimurium. Specifically, TRAF6 mediates lysine-63 ubiquitination within the Src homology 2 (SH2) domain of STAT3, which is an essential step for STAT3 membrane recruitment and subsequent phosphorylation in response to S. Typhimurium infection. TRAF6 ubiquitination participates in STAT3 phosphorylation rather than serving as only a hallmark of E3 ubiquitin ligase activation. Our results reveal a novel strategy in which S. Typhimurium T3SS effectors broaden their functions through the activation of host proteins in a ubiquitination-dependent manner to manipulate host cells into becoming a Salmonella-friendly zone.

scholarly journals Salmonella enterica Serovar Typhimurium SPI-1 and SPI-2 Shape the Global Transcriptional Landscape in a Human Intestinal Organoid Model System

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Anna-Lisa E. Lawrence ◽  
Basel H. Abuaita ◽  
Ryan P. Berger ◽  
David R. Hill ◽  
Sha Huang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Intestinal Epithelium ◽  
Salmonella Enterica ◽  
Secretion System ◽  
Host Responses ◽  
Secretion Systems ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Type 3

ABSTRACT The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica Typhimurium. While the interaction of S. Typhimurium with the mammalian host has been well studied in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestine. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells and a supporting mesenchymal cell layer. HIOs contain luminal space that supports bacterial replication, are more amenable to experimental manipulation than animals and are more reflective of physiological host responses. Here, we use the HIO model to define host transcriptional responses to S. Typhimurium infection, also determining host pathways dependent on Salmonella pathogenicity island-1 (SPI-1)- and -2 (SPI-2)-encoded type 3 secretion systems (T3SS). Consistent with prior findings, we find that S. Typhimurium strongly stimulates proinflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient, and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, S. Typhimurium infection led to significant downregulation of host genes associated with cell cycle and DNA repair, leading to a reduction in cellular proliferation, dependent on SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as mediators of S. Typhimurium-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reinforce host cell proliferation as a process targeted by Salmonella. IMPORTANCE Salmonella enterica serovar Typhimurium (S. Typhimurium) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, physiological human host responses driven by major virulence determinants of Salmonella have been more challenging to evaluate. Here, we use the three-dimensional human intestinal organoid model to define early responses to infection with wild-type S. Typhimurium and mutants defective in the SPI-1 or SPI-2 type-3 secretion systems. While both secretion system mutants show defects in mouse models of oral Salmonella infection, the specific contributions of each secretion system are less well understood. We show that S. Typhimurium upregulates proinflammatory pathways independently of either secretion system, while the downregulation of the host cell cycle pathways relies on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

scholarly journals Structural and functional studies of Salmonella effector proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C584-C584
Author(s):  
Caishuang Xu ◽  
Michal Boniecki ◽  
Maia Cherney ◽  
Rong Shi ◽  
Miroslaw Cygler
Keyword(s):  
Host Cell ◽  
Salmonella Enterica ◽  
Effector Proteins ◽  
Bacterial Survival ◽  
Vesicle Traffic ◽  
Functional Studies ◽  
Serovar Typhimurium ◽  
Membrane Bound ◽  
Type 3 Secretion System ◽  
Type 3

Gram-negative bacteria of the Salmonella enterica species are ubiquitous facultative intracellular pathogens one of the most infective in humans, causing diseases from gastroenteritis to typhoid fever. Salmonella secretes a range of proteins called effectors to gain entry and colonize the host cell. These effectors are secreted by type 3 secretion system. Upon endocytic internalization by the host cell the bacterium resides in a membrane-bound compartment – the Salmonella containing vacuole (SCV). The effector proteins prevent conversion of SCV into lysosomes and promote bacterial survival and replication within this compartment. The function of effectors varies from interfering protein synthesis and host cell signaling pathways, mediating vesicle traffic to rearranging actin cytoskeleton. We have undertaken studies of several effectors from Salmonella enterica serovar Typhimurium, such as SopD2, GtgE and SpvB, to understand their mechanism of action at the molecular level. We have expressed and purified these proteins and undertaken their crystallization. We will present our most recent results.

scholarly journals Yersinia enterocolitica Inhibits Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Cellular Uptake

2013 ◽  
Vol 82 (1) ◽  
pp. 174-183 ◽  
Author(s):  
Fabien Habyarimana ◽  
Matthew C. Swearingen ◽  
Glenn M. Young ◽  
Stephanie Seveau ◽  
Brian M. M. Ahmer
Keyword(s):  
Listeria Monocytogenes ◽  
Yersinia Enterocolitica ◽  
Salmonella Enterica ◽  
Effector Proteins ◽  
Cell Uptake ◽  
Secretion Systems ◽  
Content Type ◽  
T3ss Effector ◽  
Luxr Homolog ◽  
Serovar Typhimurium

ABSTRACTYersinia enterocoliticabiovar 1B employs two type three secretion systems (T3SS), Ysa and Ysc, which inject effector proteins into macrophages to prevent phagocytosis. Conversely,Salmonella entericaserovar Typhimurium uses a T3SS encoded bySalmonellapathogenicity island 1 (SPI1) to actively invade cells that are normally nonphagocytic and a second T3SS encoded by SPI2 to survive within macrophages. Given the distinctly different outcomes that occur with regard to host cell uptake ofS. Typhimurium andY. enterocolitica, we investigated how each pathogen influences the internalization outcome of the other.Y. enterocoliticareducesS. Typhimurium invasion of HeLa and Caco-2 cells to a level similar to that observed using anS. Typhimurium SPI1 mutant alone. However,Y. enterocoliticahad no effect onS. Typhimurium uptake by J774.1 or RAW264.7 macrophage-like cells.Y. enterocoliticawas also able to inhibit the invasion of epithelial and macrophage-like cells byListeria monocytogenes.Y. enterocoliticamutants lacking either the Ysa or Ysc T3SS were partially defective, while double mutants were completely defective, in blockingS. Typhimurium uptake by epithelial cells.S. Typhimurium encodes a LuxR homolog, SdiA, which detectsN-acylhomoserine lactones (AHLs) produced byY. enterocoliticaand upregulates the expression of an invasin (Rck) and a putative T3SS effector (SrgE). Two different methods of constitutively activating theS. Typhimurium SdiA regulon failed to reverse the uptake blockade imposed byY. enterocolitica.

scholarly journals Roles of the Outer Membrane Protein AsmA of Salmonella enterica in the Control of marRAB Expression and Invasion of Epithelial Cells

2009 ◽  
Vol 191 (11) ◽  
pp. 3615-3622 ◽  
Author(s):  
Ana I. Prieto ◽  
Sara B. Hernández ◽  
Ignacio Cota ◽  
M. Graciela Pucciarelli ◽  
Yuri Orlov ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Outer Membrane ◽  
Salmonella Enterica ◽  
Transmembrane Domain ◽  
In Silico Analysis ◽  
Sodium Deoxycholate ◽  
Oral Route ◽  
Serovar Typhimurium ◽  
Bile Resistance

ABSTRACT A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of ∼70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA + and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

scholarly journals Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

2014 ◽  
Vol 58 (11) ◽  
pp. 6528-6535 ◽  
Author(s):  
Nicolás F. Cordeiro ◽  
José A. Chabalgoity ◽  
Lucía Yim ◽  
Rafael Vignoli
Keyword(s):  
Growth Rate ◽  
Antibiotic Resistance ◽  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Virulent Strain ◽  
Constitutive Expression ◽  
Fitness Cost ◽  
Resistant Bacteria ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTAntibiotic resistance, especially due to β-lactamases, has become one of the main obstacles in the correct treatment ofSalmonellainfections; furthermore, antibiotic resistance determines a gain of function that may encompass a biological cost, or fitness reduction, to the resistant bacteria. The aim of this work was to determinein vitroif the production of the class B β-lactamase VIM-2 determined a fitness cost forSalmonella entericaserovar Typhimurium. To that end the geneblaVIM-2was cloned into the virulent strainS. Typhimurium SL1344, using both the tightly regulated pBAD22 vector and the natural plasmid pST12, for inducible and constitutive expression, respectively. Fitness studies were performed by means of motility, growth rate, invasiveness in epithelial cells, and plasmid stability. The expression ofblaVIM-2was accompanied by alterations in micro- and macroscopic morphology and reduced growth rate and motility, as well as diminished invasiveness in epithelial cells. These results suggest that VIM-2 production entails a substantial fitness cost forS. Typhimurium, which in turn may account for the extremely low number of reports of metallo-β-lactamase-producingSalmonellaspp.

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