skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library
Title: Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library
ABSTRACT Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) ofSalmonella enterica. Despite detailed knowledge of the PrgI needle’s assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle’s mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS’s assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system fromSalmonella entericacan impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.  more » « less
Award ID(s):
2043973
PAR ID:
10545665
Author(s) / Creator(s):
; ; ; ; ;
Editor(s):
Ellermeier, Craig D
Publisher / Repository:
American Society for Microbiology
Date Published:
Journal Name:
mSphere
Volume:
9
Issue:
8
ISSN:
2379-5042
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Microbial Cell Factories)
    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. 
    more » « less
  2. ; ; (, Microbial Cell Factories)
    Abstract BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in theBacillusgenus. Using an industrially relevantB. megateriumhost, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for anE. colihost producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in theBacillushost system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor. 
    more » « less
  3. ; ; (, PLOS Pathogens)
    Navarre, William (Ed.)
    The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such asSalmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to theS.entericaflagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certainSalmonellaserotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of theSalmonellaflagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility inS. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host generaSalmonella,Escherichia, orSerratia. 
    more » « less
  4. ; ; ; ; ; (, mSystems)
    Dyson, Zoe A (Ed.)
    ABSTRACT Predators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains ofEscherichia coliandSalmonella entericaby competing a generalist T5-like phage able to infect both prey against P22vir, anS. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that thein vitrocost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest. IMPORTANCEThere is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and anin vitrosystem consisting ofEscherichia coli,Salmonella enterica, a T5-like generalist phage, and the specialist phage P22virto highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Microbiology Spectrum)
    Wang, Luxin (Ed.)
    ABSTRACT Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens likeSalmonella entericaandListeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detectionof S. enterica and L. monocytogenesby qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n= 1,990) was analyzed by multiplex qPCR specific forS. entericaandL. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection ofS. entericaandL. monocytogeneswere 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter forS. enterica; forL. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for bothS. entericaandL. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCEDetecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence ofSalmonella entericaandListeria monocytogenesin irrigation water samples examined in this study. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email