Publications by authors named "Jeffrey L Bose"

52 Publications

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

Measuring Staphylococcal Promoter Activities Using a Codon-Optimized β-Galactosidase Reporter.

Methods Mol Biol 2021 ;2341:37-44

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

The lacZ gene and corresponding β-galactosidase enzyme has been a mainstay for bacterial reporter systems for decades. We have used this versatile reporter to analyze expression profiles from strains grown both on solid media and from broth culture. The standard broth protocol can also be adapted for a 96-well plate to allow high-throughput screening of promoter reporter constructs under a variety of conditions. Furthermore, codon-optimization of the E. coli lacZ gene has greatly improved activity levels of β-galactosidase in S. aureus, facilitating improved sensitivity for screening assays, detection of low-activity promoters, and use of small sample volumes. In this chapter, details are provided for both standard and high-throughput quantitative assays that we have routinely used for S. aureus transcriptional profiling.
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http://dx.doi.org/10.1007/978-1-0716-1550-8_6DOI 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

Analysis of Murein Hydrolases and Proteases Through Zymography.

Methods Mol Biol 2021 ;2341:9-16

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

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.
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http://dx.doi.org/10.1007/978-1-0716-1550-8_2DOI Listing
August 2021

Inhibition of Pyruvate Kinase From by IMP Is Independent of the Extra-C Domain.

Front Microbiol 2021 17;12:628308. Epub 2021 Feb 17.

Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, KS, United States.

The pyruvate kinase (PYK) isozyme from (TsPYK) has previously been used in metabolic engineering for improved ethanol production. This isozyme belongs to a subclass of PYK isozymes that include an extra C-domain. Like other isozymes that include this extra C-domain, we found that TsPYK is activated by AMP and ribose-5-phosphate (R5P). Our use of sugar-phosphate analogs generated a surprising result in that IMP and GMP are allosteric inhibitors (rather than activators) of TsPYK. We believe this to be the first report of any PYK isozyme being inhibited by IMP and GMP. A truncated protein that lacks the extra C-domain is also inhibited by IMP. A screen of several other bacterial PYK enzymes (include several that have the extra-C domain) indicates that the inhibition by IMP is specific to only a subset of those isozymes.
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http://dx.doi.org/10.3389/fmicb.2021.628308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925390PMC
February 2021

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

mSphere 2020 06 10;5(3). Epub 2020 Jun 10.

Azitra Inc., Farmington, Connecticut, USA

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, , for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of that requires exogenously supplied d-alanine. The NRRL B-4268 Δ Δ Δ strain (SE) contains deletions of three biosynthetic genes: two alanine racemase genes, and (SE1674 and SE1079), and the d-alanine aminotransferase gene, (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SE colonized and increased expression of human β-defensin 2 in cultured human skin models SE showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic These studies support the potential safety and utility of SE as a live biotherapeutic strain whose growth can be controlled by d-alanine. The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans.
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http://dx.doi.org/10.1128/mSphere.00360-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289707PMC
June 2020

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

Exogenous Fatty Acids Remodel Staphylococcus aureus Lipid Composition through Fatty Acid Kinase.

J Bacteriol 2020 06 25;202(14). Epub 2020 Jun 25.

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

can utilize exogenous fatty acids for phospholipid synthesis. The fatty acid kinase FakA is essential for this utilization by phosphorylating exogenous fatty acids for incorporation into lipids. How FakA impacts the lipid membrane composition is unknown. In this study, we used mass spectrometry to determine the membrane lipid composition and properties of in the absence of We found the mutant to have increased abundance of lipids containing longer acyl chains. Since does not synthesize unsaturated fatty acids, we utilized oleic acid (18:1) to track exogenous fatty acid incorporation into lipids. We observed a concentration-dependent incorporation of exogenous fatty acids into the membrane that required FakA. We also tested how FakA and exogenous fatty acids impact membrane-related physiology and identified changes in membrane potential, cellular respiration, and membrane fluidity. To mimic the host environment, we characterized the lipid composition of wild-type and mutant bacteria grown in mouse skin homogenate. We show that wild-type can incorporate exogenous unsaturated fatty acids from host tissue, highlighting the importance of FakA in the presence of host skin tissue. In conclusion, FakA is important for maintaining the composition and properties of the phospholipid membrane in the presence of exogenous fatty acids, impacting overall cell physiology. Environmental fatty acids can be harvested to supplement endogenous fatty acid synthesis to produce membranes and circumvent fatty acid biosynthesis inhibitors. However, how the inability to use these fatty acids impacts lipids is unclear. Our results reveal lipid composition changes in response to fatty acid addition and when is unable to activate fatty acids through FakA. We identify concentration-dependent utilization of oleic acid that, when combined with previous work, provides evidence that fatty acids can serve as a signal to Furthermore, using mouse skin homogenates as a surrogate for conditions, we showed that can incorporate host fatty acids. This study highlights how exogenous fatty acids impact bacterial membrane composition and function.
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http://dx.doi.org/10.1128/JB.00128-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317038PMC
June 2020

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

Infect Immun 2020 04 20;88(5). Epub 2020 Apr 20.

Division of Biomedical Sciences, University of California Riverside School of Medicine, Riverside, California, USA.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.
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http://dx.doi.org/10.1128/IAI.00859-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171244PMC
April 2020

Genetic Manipulations of Staphylococcal Chromosomal DNA.

Methods Mol Biol 2020 ;2069:103-111

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

Performing genetic manipulation is often key to understanding bacterial gene function. In this chapter, we present the method of allelic exchange using temperature-sensitive plasmids to generate mutations in Staphylococcus, including single-nucleotide mutations, insertions, and gene deletions. In addition, this chapter summarizes other key genetic technologies used for the manipulation of S. aureus, including the CRISPR/Cas9 system and complementation.
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http://dx.doi.org/10.1007/978-1-4939-9849-4_8DOI Listing
December 2020

CidR and CcpA Synergistically Regulate Staphylococcus aureus Expression.

J Bacteriol 2019 12 5;201(23). Epub 2019 Nov 5.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA

The death and lysis of a subpopulation of cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized - and -acting elements essential for the induction of the operon. In addition to a CidR-binding site located within the promoter region, sequence analysis revealed the presence of a putative catabolite responsive element ( box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified site for the induction of the operon was demonstrated by examining the expression of P reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the operon and reveal the complexity of molecular interactions controlling its expression. This work focuses on the characterization of - and -acting elements essential for the induction of the operon in The results of this study are the first to demonstrate the synergistic control of expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in .
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http://dx.doi.org/10.1128/JB.00371-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832070PMC
December 2019

Bacterial Community Succession, Transmigration, and Differential Gene Transcription in a Controlled Vertebrate Decomposition Model.

Front Microbiol 2019 18;10:745. Epub 2019 Apr 18.

Department of Biological Sciences, Mississippi State University, Starkville, MS, United States.

Decomposing remains are a nutrient-rich ecosystem undergoing constant change due to cell breakdown and abiotic fluxes, such as pH level and oxygen availability. These environmental fluxes affect bacterial communities who respond in a predictive manner associated with the time since organismal death, or the postmortem interval (PMI). Profiles of microbial taxonomic turnover and transmigration are currently being studied in decomposition ecology, and in the field of forensic microbiology as indicators of the PMI. We monitored bacterial community structural and functional changes taking place during decomposition of the intestines, bone marrow, lungs, and heart in a highly controlled murine model. We found that organs presumed to be sterile during life are colonized by during later decomposition as the fluids from internal organs begin to emulsify within the body cavity. During colonization of previously sterile sites, gene transcripts for multiple metabolism pathways were highly abundant, while transcripts associated with stress response and dormancy increased as decomposition progressed. We found our model strengthens known bacterial taxonomic succession data after host death. This study is one of the first to provide data of expressed bacterial community genes, alongside transmigration and structural changes of microbial species during laboratory controlled vertebrate decomposition. This is an important dataset for studying the effects of the environment on bacterial communities in an effort to determine which bacterial species and which bacterial functional pathways, such as amino acid metabolism, provide key changes during stages of decomposition that relate to the PMI. Finding unique PMI species or functions can be useful for determining time since death in forensic investigations.
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http://dx.doi.org/10.3389/fmicb.2019.00745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482229PMC
April 2019

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

The Small RNA Teg41 Regulates Expression of the Alpha Phenol-Soluble Modulins and Is Required for Virulence in Staphylococcus aureus.

mBio 2019 02 5;10(1). Epub 2019 Feb 5.

Department of Biological Sciences, Ohio University, Athens, Ohio, USA

Small RNAs (sRNAs) remain an understudied class of regulatory molecules in bacteria in general and in Gram-positive bacteria in particular. In the major human pathogen , hundreds of sRNAs have been identified; however, only a few have been characterized in detail. In this study, we investigate the role of the sRNA Teg41 in virulence. We demonstrate that Teg41, an sRNA divergently transcribed from the locus that encodes the cytolytic alpha phenol-soluble modulin (αPSM) peptides, plays a critical role in αPSM production. Overproduction of Teg41 leads to an increase in αPSM levels and a corresponding increase in hemolytic activity from cells and cell-free culture supernatants. To identify regions of Teg41 important for its function, we performed an RNA-RNA interaction analysis which predicted an interaction between the 3' end of Teg41 and the αPSM transcript. Deleting a 24-nucleotide region from the genome, corresponding to the 3' end of Teg41, led to a 10-fold reduction in αPSM-dependent hemolytic activity and attenuation of virulence in a murine abscess model of infection. Restoration of hemolytic activity in the Teg41Δ3' strain was possible by expressing full-length Teg41 in Restoration of hemolytic activity was also possible by expressing the 3' end of Teg41, suggesting that this region of Teg41 is necessary and sufficient for αPSM-dependent hemolysis. Our results show that Teg41 is positively influencing αPSM production, demonstrating for the first time regulation of the αPSM peptides by an sRNA in The alpha phenol-soluble modulins (αPSMs) are among the most potent toxins produced by Their biological role during infection has been studied in detail; however, the way they are produced by the bacterial cell is not well understood. In this work, we identify a small RNA molecule called Teg41 that plays an important role in αPSM production by Teg41 positively influences αPSM production. The importance of Teg41 is highlighted by the fact that a strain containing a deletion in the 3' end of Teg41 produces significantly less αPSMs and is attenuated for virulence in a mouse abscess model of infection. As the search for new therapeutic strategies to combat infection proceeds, Teg41 may represent a novel target.
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http://dx.doi.org/10.1128/mBio.02484-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428751PMC
February 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

Redirection of Metabolism in Response to Fatty Acid Kinase in Staphylococcus aureus.

J Bacteriol 2018 10 10;200(19). Epub 2018 Sep 10.

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

is capable of phosphorylating exogenous fatty acids for incorporation into the bacterium's membrane via the fatty acid kinase, FakA. Additionally, FakA plays a significant role in virulence factor regulation and skin infections. We previously showed that a mutant displays altered growth kinetics , observed during the late-exponential phase of growth. Here, we demonstrate that the absence of FakA leads to key metabolic changes. First, the mutant has an altered acetate metabolism, with acetate being consumed at an increased rate than in the wild-type strain. Moreover, the growth benefit was diminished with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-based approach, we identified altered concentrations of tricarboxylic acid (TCA) cycle intermediates and both intracellular and extracellular amino acids. Together, these data demonstrate a change in carbohydrate carbon utilization and altered amino acid metabolism in the mutant. Energy status analysis revealed the mutant had a similar ADP/ATP ratio to that of the wild type, but a reduced adenylate energy charge. The inactivation of changed the NAD/NADH and NADP/NADPH ratios, indicating a more oxidized cellular environment. Evidence points to the global metabolic regulatory proteins CcpA and CodY being important contributors to the altered growth in a mutant. Indeed, it was found that directing amino acids from the urea cycle into the TCA cycle via glutamate dehydrogenase was an essential component of growth after glucose depletion. Together, these data identify a previously unidentified role of FakA in the global physiology of , linking external fatty acid utilization and central metabolism. The fatty acid kinase, FakA, of plays several important roles in the cell. FakA is important for the activation of the SaeRS two-component system and secreted virulence factors like α-hemolysin. However, the contribution of FakA to cellular metabolism has not been explored. Here, we highlight the metabolic consequence of removal of FakA from the cell. The absence of FakA leads to altered acetate metabolism and altered redox balance, as well as a change in intracellular amino acids. Additionally, the use of environmental amino acid sources is affected by FakA. Together, these results demonstrate for the first time that FakA provides a link between the pathways for exogenous fatty acid use, virulence factor regulation, and other metabolic processes.
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http://dx.doi.org/10.1128/JB.00345-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148474PMC
October 2018

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

J Bacteriol 2017 03 14;199(5). Epub 2017 Feb 14.

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

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of , , and mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I () and class II () target genes were downregulated during the exponential growth phase in the mutant, compared to the wild-type strain. In addition, expression was decreased in the mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, expression was decreased in the mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in The SaeRS two-component system is a key regulator of virulence determinant production in Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase VfrB as a positive regulatory modulator of SaeRS-mediated transcription of virulence factors in In addition to describing a new regulatory aspect of SaeRS, this study establishes a link between fatty acid kinase activity and virulence factor regulation.
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http://dx.doi.org/10.1128/JB.00828-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309915PMC
March 2017

Generation of a Stable Plasmid for and Studies of Staphylococcus Species.

Appl Environ Microbiol 2016 Dec 16;82(23):6859-6869. Epub 2016 Sep 16.

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

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for species. LAC-p01 was genetically manipulated into an - shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded ( and ) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of systemic infection and successfully complemented an mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both and experiments are powerful new tools for studies of Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations and without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of species.
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http://dx.doi.org/10.1128/AEM.02370-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103085PMC
December 2016

Potassium Uptake Modulates Staphylococcus aureus Metabolism.

mSphere 2016 May-Jun;1(3). Epub 2016 Jun 15.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K(+)) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K(+) uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K(+) deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K(+) uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K(+) uptake in S. aureus revealed that the Ktr-mediated K(+) transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K(+) uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K(+) uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K(+) uptake in establishing efficient carbon utilization.
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http://dx.doi.org/10.1128/mSphere.00125-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911797PMC
June 2016

The LysR-type transcriptional regulator, CidR, regulates stationary phase cell death in Staphylococcus aureus.

Mol Microbiol 2016 09 4;101(6):942-53. Epub 2016 Jul 4.

Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, 68198-5900, USA.

The Staphylococcus aureus LysR-type transcriptional regulator, CidR, activates the expression of two operons including cidABC and alsSD that display pro- and anti-death functions, respectively. Although several investigations have focused on the functions of different genes associated with these operons, the collective role of the CidR regulon in staphylococcal physiology is not clearly understood. Here we reveal that the primary role of this regulon is to limit acetate-dependent potentiation of cell death in staphylococcal populations. Although both CidB and CidC promote acetate generation and cell death, the CidR-dependent co-activation of CidA and AlsSD counters the effects of CidBC by redirecting intracellular carbon flux towards acetoin formation. From a mechanistic standpoint, we demonstrate that CidB is necessary for full activation of CidC, whereas CidA limits the abundance of CidC in the cell.
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http://dx.doi.org/10.1111/mmi.13433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014633PMC
September 2016

The major autolysin is redundant for Staphylococcus aureus USA300 LAC JE2 virulence in a murine device-related infection model.

FEMS Microbiol Lett 2016 05 3;363(9). Epub 2016 Apr 3.

Department of Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland

The major Staphylococcus aureus autolysin, Atl, has been implicated in attachment to surfaces and release of extracellular DNA during biofilm formation under laboratory conditions. Consistent with this, polyclonal antibodies to the amidase and glucosaminidase domains of Atl inhibited in vitro biofilm formation. However, in a murine model of device-related infection the community-associated S. aureus strain USA300 LAC JE2 established a successful infection in the absence of atl These data indicate that Atl activity is not required for biofilm production in this infection model and reveal the importance of characterizing the contribution of biofilm phenotypes to virulence under in vivo conditions.
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http://dx.doi.org/10.1093/femsle/fnw087DOI Listing
May 2016

SrrAB Modulates Staphylococcus aureus Cell Death through Regulation of cidABC Transcription.

J Bacteriol 2016 Jan 25;198(7):1114-22. Epub 2016 Jan 25.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA

Unlabelled: The death and lysis of a subpopulation in Staphylococcus aureus biofilm cells are thought to benefit the surviving population by releasing extracellular DNA, a critical component of the biofilm extracellular matrix. Although the means by which S. aureus controls cell death and lysis is not understood, studies implicate the role of the cidABC and lrgAB operons in this process. Recently, disruption of the srrAB regulatory locus was found to cause increased cell death during biofilm development, likely as a result of the sensitivity of this mutant to hypoxic growth. In the current study, we extended these findings by demonstrating that cell death in the ΔsrrAB mutant is dependent on expression of the cidABC operon. The effect of cidABC expression resulted in the generation of increased reactive oxygen species (ROS) accumulation and was independent of acetate production. Interestingly, consistently with previous studies, cidC-encoded pyruvate oxidase was found to be important for the generation of acetic acid, which initiates the cell death process. However, these studies also revealed for the first time an important role of the cidB gene in cell death, as disruption of cidB in the ΔsrrAB mutant background decreased ROS generation and cell death in a cidC-independent manner. The cidB mutation also caused decreased sensitivity to hydrogen peroxide, which suggests a complex role for this system in ROS metabolism. Overall, the results of this study provide further insight into the function of the cidABC operon in cell death and reveal its contribution to the oxidative stress response.

Importance: The manuscript focuses on cell death mechanisms in Staphylococcus aureus and provides important new insights into the genes involved in this ill-defined process. By exploring the cause of increased stationary-phase death in an S. aureus ΔsrrAB regulatory mutant, we found that the decreased viability of this mutant was a consequence of the overexpression of the cidABC operon, previously shown to be a key mediator of cell death. These investigations highlight the role of the cidB gene in the death process and the accumulation of reactive oxygen species. Overall, the results of this study are the first to demonstrate a positive role for CidB in cell death and to provide an important paradigm for understanding this process in all bacteria.
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http://dx.doi.org/10.1128/JB.00954-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4800867PMC
January 2016

Preface.

Authors:
Jeffrey L Bose

Methods Mol Biol 2016 ;1373:v-vi

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http://dx.doi.org/10.1007/978-1-4939-3158-3DOI Listing
August 2016

Understanding Staphylococcal Nomenclature.

Methods Mol Biol 2016 ;1373:1-7

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, MSN 3029, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA.

Bacteria are often grouped by a variety of properties, including biochemical activity, appearance, and more recently, nucleic acid sequence differences. In the case of human pathogens, significant work goes into "typing" strains to understand relatedness. This is especially true when trying to understand the epidemiology of these organisms. In attempts to group Staphylococci, a variety of methods and nomenclatures have been employed, which can often serve as a point of confusion to those entering the field. Therefore, the intent of this chapter is to give a brief overview of some common methods and associated nomenclature used to type Staphylococci, with S. aureus as an example.
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http://dx.doi.org/10.1007/7651_2015_283DOI Listing
August 2016

Rapid Isolation of DNA from Staphylococcus.

Methods Mol Biol 2016 ;1373:59-62

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, MSN 3029, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA.

Many methods exist to extract DNA from bacteria. Indeed, there is no shortage of kits available from manufacturers that allow for isolation of highly purified DNA. However, for many applications samples do not need to be extremely pure (i.e., free of contaminating proteins or RNA). Furthermore, for quick genetic screening, it is often useful to have a rapid and inexpensive option for DNA isolation from small samples. For these occasions, the method found in this chapter provides a cost-efficient, yet rapid, isolation of DNA.
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http://dx.doi.org/10.1007/7651_2014_184DOI Listing
August 2016

Chemical and UV Mutagenesis.

Authors:
Jeffrey L Bose

Methods Mol Biol 2016 ;1373:111-5

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, MSN 3029, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA.

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.
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http://dx.doi.org/10.1007/7651_2014_190DOI Listing
August 2016

Allelic Exchange.

Methods Mol Biol 2016 ;1373:89-96

Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Methods used to understand the function of a gene/protein are one of the hallmarks of modern molecular genetics. The ability to genetically manipulate bacteria has become a fundamental tool in studying these organisms and while basic cloning has become a routine task in molecular biology laboratories, generating directed mutations can be a daunting task. This chapter describes the method of allelic exchange in Staphylococcus aureus using temperature-sensitive plasmids that have successfully produced a variety of chromosomal mutations, including in-frame deletions, insertion of antibiotic-resistance cassettes, and even single-nucleotide point mutations.
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http://dx.doi.org/10.1007/7651_2014_187DOI Listing
August 2016

Bacteriophage Transduction in Staphylococcus aureus: Broth-Based Method.

Methods Mol Biol 2016 ;1373:63-8

Department of Microbiology, Molecular Genetics, and Immunology, The University of Kansas Medical Center, MSN 3029, 3901 Rainbow Boulevard, Kansas City, KS, 66160, USA.

The ability to move DNA between Staphylococcus strains is essential for the genetic manipulation of this bacterium. Often in the Staphylococci, this is accomplished through transduction using generalized transducing phage and can be performed in different ways and therefore the presence of two transduction procedures in this book. The following protocol is a relatively easy-to-perform, broth-based procedure that we have used extensively to move both plasmids and chromosomal fragments between strains of Staphylococcus aureus.
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http://dx.doi.org/10.1007/7651_2014_185DOI Listing
August 2016

Identification of the amino acids essential for LytSR-mediated signal transduction in Staphylococcus aureus and their roles in biofilm-specific gene expression.

Mol Microbiol 2015 Feb 16;95(4):723-37. Epub 2015 Jan 16.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, 68198, USA.

Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically localized within tower structures during biofilm development. To gain a better understanding of the mechanisms underlying this spatial control of lrgAB expression, we carried out a detailed analysis of the LytSR two-component system. Specifically, a conserved aspartic acid (Asp53) of the LytR response regulator was shown to be the target of phosphorylation, which resulted in enhanced binding to the lrgAB promoter and activation of transcription. In addition, we identified His390 of the LytS histidine kinase as the site of autophosphorylation and Asn394 as a critical amino acid involved in phosphatase activity. Interestingly, LytS-independent activation of LytR was observed during planktonic growth, with acetyl phosphate acting as a phosphodonor to LytR. In contrast, mutations disrupting the function of LytS prevented tower-specific lrgAB expression, providing insight into the physiologic environment within these structures. In addition, overactivation of LytR led to increased lrgAB promoter activity during planktonic and biofilm growth and a change in biofilm morphology. Overall, the results of this study are the first to define the LytSR signal transduction pathway, as well as determine the metabolic context within biofilm tower structures that triggers these signaling events.
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http://dx.doi.org/10.1111/mmi.12902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347461PMC
February 2015
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