Publications by authors named "Miranda J Ridder"

6 Publications

  • Page 1 of 1

Bone Marrow-Derived Macrophage Infection Assay.

Methods Mol Biol 2021 ;2341:133-140

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS, USA.

The use of cultured mammalian cells, whether immortalized cell lines or primary cells, is a well-known technique used as a substitute or prescreen for in vivo virulence potential of bacterial pathogens. This technique is also a way to examine host-pathogen interactions in a less complex environment compared to that found in whole animals. To this end, macrophage infection assays have become a key technique for studying the molecular mechanisms by which bacteria interact with the host. Herein, this chapter describes both how to produce macrophages from mouse bone marrow and the subsequent infection assays.
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http://dx.doi.org/10.1007/978-1-0716-1550-8_16DOI Listing
August 2021

Quantitative Hemolysis Assays.

Methods Mol Biol 2021 ;2341:25-30

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS, USA.

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.
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http://dx.doi.org/10.1007/978-1-0716-1550-8_4DOI Listing
August 2021

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Infect Immun 2020 07 21;88(8). Epub 2020 Jul 21.

Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA

fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin , and that the Δ mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both and Δ mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the Δ mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the Δ mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the Δ mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in Δ mutant-infected mice. The paradoxical findings of decreased Hla but increased necrosis led us to investigate the role of the proteases regulated by FakA. Utilizing Δ and Δ mutants in both the WT and mutant backgrounds, we found that the absence of these proteases in a mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the Δ mutant during skin infection.
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http://dx.doi.org/10.1128/IAI.00163-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375762PMC
July 2020

Contribution of YjbIH to Virulence Factor Expression and Host Colonization in .

Infect Immun 2019 06 21;87(6). Epub 2019 May 21.

University of Kansas Medical Center, Department of Microbiology, Molecular Genetics and Immunology, Kansas City, Kansas, USA

To persist within the host and cause disease, relies on its ability to precisely fine-tune virulence factor expression in response to rapidly changing environments. During an unbiased transposon mutant screen, we observed that disruption of a two-gene operon, , resulted in decreased levels of pigmentation and aureolysin (Aur) activity relative to the wild-type strain. Further analyses revealed that YjbH, a predicted thioredoxin-like oxidoreductase, is predominantly responsible for the observed mutant phenotypes, though a minor role exists for the putative truncated hemoglobin YjbI. These differences were due to significantly decreased expression of and Previous studies found that YjbH targets the disulfide- and oxidative stress-responsive regulator Spx for degradation by ClpXP. The absence of or resulted in altered sensitivities to nitrosative and oxidative stress and iron deprivation. Additionally, aconitase activity was altered in the and mutant strains. Decreased levels of pigmentation and aureolysin (Aur) activity in the mutant were found to be Spx dependent. Lastly, we used a murine sepsis model to determine the effect of the deletion on pathogenesis and found that the mutant was better able to colonize the kidneys and spleens during an acute infection than the wild-type strain. These studies identified changes in pigmentation and protease activity in response to YjbIH and are the first to have shown a role for these proteins during infection.
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http://dx.doi.org/10.1128/IAI.00155-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529663PMC
June 2019

Inactivation of the exogenous fatty acid utilization pathway leads to increased resistance to unsaturated fatty acids in Staphylococcus aureus.

Microbiology (Reading) 2019 02 19;165(2):197-207. Epub 2018 Dec 19.

Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160, USA.

The human pathogen Staphylococcus aureus produces saturated fatty acids, but can incorporate both exogenous saturated and unsaturated fatty acids into its lipid membrane. S. aureus encounters unsaturated fatty acids in the host skin where they serve as an innate immune defence due to their toxicity. Previously, we identified a fatty acid kinase in S. aureus that is necessary for the utilization of exogenous fatty acids. The goal of this study was to determine the effects of fatty acids on mutants deficient in the exogenous fatty acid utilization machinery. We have demonstrated that mutants lacking a functional fatty acid kinase (fakA) or both fatty acid carrier proteins (fakB1 fakB2) are more resistant to unsaturated fatty acids. Previous studies suggested a role for ammonia-producing enzymes in resistance to unsaturated fatty acids, but these enzymes do not contribute to the resistance of the fakA mutant, despite increased urease transcription and protein activity in the mutant. Additionally, while pigment is altered in mutants unable to use exogenous fatty acids, staphyloxanthin does not contribute to fatty acid resistance of an fakA mutant. Because exposure to unsaturated fatty acids probably initiates a stress response, we investigated the role of the alternative sigma factor σ and determined if it is necessary for the fatty acid resistance observed in the fakA mutant. Collectively, this study demonstrates that the inability to incorporate unsaturated fatty acids leads to increased resistance to those fatty acids, and that resistance requires a σ stress response.
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http://dx.doi.org/10.1099/mic.0.000757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600405PMC
February 2019

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Metabolism and Virulence.

Infect Immun 2019 02 24;87(2). Epub 2019 Jan 24.

Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville, Florida, USA

nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The , , and mutants showed comparable defects in endothelial intracellular survival, whereas the and mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant .
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http://dx.doi.org/10.1128/IAI.00570-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346124PMC
February 2019
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