scholarly journals Signal Pathway in Salt-Activated Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System in Salmonella enterica Serovar Typhimurium

2008 ◽  
Vol 190 (13) ◽  
pp. 4624-4631 ◽  
Author(s):  
Hideaki Mizusaki ◽  
Akiko Takaya ◽  
Tomoko Yamamoto ◽  
Shin-Ichi Aizawa
Keyword(s):  
Electron Microscopy ◽  
Real Time ◽  
Protein Secretion ◽  
Real Time Pcr ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Sds Page ◽  
Two Component ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host, S. enterica induces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hilA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activated hilD and hilA transcription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.

scholarly journals Clinical Evaluation of a Type III Secretion System Real-Time PCR Assay for Diagnosing Melioidosis

2006 ◽  
Vol 44 (8) ◽  
pp. 3028-3030 ◽  
Author(s):  
E. M. Meumann ◽  
R. T. Novak ◽  
D. Gal ◽  
M. E. Kaestli ◽  
M. Mayo ◽  
...  
Keyword(s):  
Real Time ◽  
Clinical Evaluation ◽  
Real Time Pcr ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Pcr Assay ◽  
Type Iii

scholarly journals An optimized growth medium for increased recombinant protein secretion titer via the type III secretion system

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lisa Ann Burdette ◽  
Han Teng Wong ◽  
Danielle Tullman-Ercek
Keyword(s):  
Protein Secretion ◽  
Salt Concentration ◽  
Type Iii Secretion ◽  
Heterologous Protein ◽  
Carbon Sources ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
Heterologous Protein Secretion ◽  
Extracellular Environment

Abstract Background Protein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an “outside-in” approach to elucidate the effect of growth medium components on secretion titer. Results We dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements. Conclusions We leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression—one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

scholarly journals Accelerated Type III Secretion System 2-Dependent Enteropathogenesis by a Salmonella enterica Serovar Enteritidis PT4/6 Strain

2009 ◽  
Vol 77 (9) ◽  
pp. 3569-3577 ◽  
Author(s):  
Mrutyunjay Suar ◽  
Balamurugan Periaswamy ◽  
Pascal Songhet ◽  
Benjamin Misselwitz ◽  
Andreas Müller ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Alternative Pathway ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Mucosal Inflammation ◽  
Classical Pathway ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica subsp. I serovars Typhimurium and Enteritidis are major causes of enteric disease. The pathomechanism of enteric infection by serovar Typhimurium has been studied in detail. Serovar Typhimurium employs two pathways in parallel for triggering disease, i.e., the “classical” pathway, triggered by type III secretion system 1 (TTSS-1), and the “alternative” pathway, mediated by TTSS-2. It had remained unclear whether these two pathways would also explain the enteropathogenesis of strains from other serovars. We chose the isolate P125109 of the epidemic serovar Enteritidis PT4/6, generated isogenic mutants, and studied their virulence. Using in vitro and in vivo infection experiments, a dendritic cell depletion strategy, and MyD88−/− knockout mice, we found that P125109 employs both the “classical” and “alternative” pathways for triggering mucosal inflammation. The “classical” pathway was phenotypically similar in serovar Typhimurium strain SL1344 and in P125109. However, the kinetics of the “alternative” pathway differed significantly. Via TTSS-2, P125109 colonized the gut tissue more efficiently and triggered mucosal inflammation approximately 1 day faster than SL1344 did. In conclusion, our data demonstrate that different Salmonella spp. can differ in their capacity to trigger mucosal inflammation via the “alternative” pathway in vivo.

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