Publications by Year: 2019

Payne SM. Laboratory Cultivation and Storage of Shigella . Curr Protoc Microbiol. [Internet]. 2019;55 (1) :e93. Publisher's VersionAbstract

Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation of routine growth conditions and media, for storage of the bacteria, and for monitoring of the presence of the virulence plasmid.

Basic Protocol 1: Growth of S. flexneri from frozen stocks or agar stabs

Basic Protocol 2: Growth of S. flexneri in rich liquid medium

Alternate Protocol 1: Growth of S. flexneri in rich defined medium

Alternate Protocol 2: Growth of S. flexneri in minimal medium

Basic Protocol 3: Storage of S. flexneri in frozen stocks

Alternate Protocol 3: Storage of S. flexneri in agar stabs

Butz HA, Mey AR, Ciosek A, Payne SM. Vibrio cholerae CsrA directly regulates varA to increase expression of the three nonredundant Csr sRNAs. mBio [Internet]. 2019;10 (3) :e01042-19. Publisher's VersionAbstract
CsrA, an RNA-binding global regulator, is an essential protein in Vibrio cholerae. V. cholerae CsrA is regulated by three small RNAs (sRNAs), namely, CsrB, CsrC, and CsrD, which act to sequester and antagonize the activity of CsrA. Although the sRNAs were considered to be largely redundant, we found that they differ in expression, half-life, and the ability to regulate CsrA. Further, we identified a feedback loop in the Csr system in which CsrA increases the synthesis of these antagonistic sRNAs. Because the Csr sRNAs are positively regulated by VarA, we determined the effects of CsrA on VarA levels. The level of VarA was reduced in a csrA mutant, and we found that CsrA directly bound to varA mRNA in an electrophoretic mobility shift assay in vitro and in an CsrA-RNA immunoprecipitation assay in vivo. Thus, varA mRNA is an in vivo-verified direct target of CsrA in V. cholerae, and this is the first demonstration of CsrA directly binding to a varA/uvrY/gacA homolog. Additionally, we demonstrated that a varA translational fusion was less active in a csrA mutant than in wild-type V. cholerae, suggesting that CsrA enhances varA translation. We propose that this autoregulatory feedback loop, in which CsrA increases the production of the nonredundant Csr sRNAs by regulating the amount of VarA, provides a mechanism for fine-tuning the availability of CsrA and, thus, of its downstream targets.
Veloria J, Shin M, Devkota AK, Payne SM, Cho EJ, Dalby KN. Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB). SLAS Discovery [Internet]. 2019;24 (5) :597-605. Publisher's VersionAbstract
Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
Shin M, Mey AR, Payne SM. Vibrio cholerae FeoB contains a dual nucleotide-specific NTPase domain essential for ferrous iron uptake. Proceedings of the National Academy of Sciences [Internet]. 2019;116 (10) :4599-4604. Publisher's VersionAbstract
The Feo ferrous iron transporter is widely distributed among bacteria and archaea, but its mechanism of transport has not been fully elucidated. In Vibrio cholerae, the transport system requires three proteins: the small cytosolic proteins FeoA and FeoC and a large cytoplasmic-membrane-associated protein FeoB, which has an N-terminal G-protein domain. We show that, in contrast to Escherichia coliFeoB, which is solely a GTPase, the V. cholerae and Helicobacter pylori FeoB proteins have both GTPase and ATPase activity. In V. cholerae, mutation of the G4 motif, responsible for hydrogen bonding with the guanine base, abolished the GTPase activity but not ATPase activity. The ATPase activity of the G4 motif mutants was sufficient for Feo function in the absence of GTPase. We show that the serine and asparagine residues in the G5 motif likely play a role in the ATPase activity, and substitution of these residues with those found in the corresponding positions in E. coli FeoB resulted in similar nucleotide hydrolysis activity in the E. coli protein. These results add significantly to our understanding of the NTPase domain of FeoB and its role in Feo function.
Koestler BJ, Ward CM, Fisher CR, Rajan A, Maresso AW, Payne SM. Human intestinal enteroids as a model system of Shigella pathogenesis. Infection and Immunity [Internet]. 2019;87 (4). Publisher's VersionAbstract
The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade the colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease, thus cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells and grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigellapathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneriinvades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE pro-inflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneriinvasion via the apical surface.