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May 2014 - present
Publications
Publications (117)
Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an int...
Staphylococcus aureus has evolved mechanisms to cope with low iron (Fe) availability in host tissues. S. aureus uses the ferric uptake transcriptional regulator (Fur) to sense titers of cytosolic Fe. Upon Fe depletion, apo-Fur relieves transcriptional repression of genes utilized for Fe uptake. We demonstrate that an S. aureus Δ fur mutant has decr...
Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAK’s structure and enzymatic mechanism remain poorly understood. Here, we used a combina...
In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, man...
Staphylococcus aureus nitric oxide synthase (saNOS) contributes to oxidative stress resistance, antibiotic tolerance, virulence, and modulation of aerobic and nitrate-based cellular respiration. Despite its involvement in these essential processes, the genetic regulation of nos expression has not been well characterized. 5′ rapid amplification of c...
Previously, our group demonstrated a role for the small RNA (sRNA) Teg41 in regulating production of the alpha phenol-soluble modulin toxins (αPSMs) in Staphylococcus aureus. Overexpressing Teg41 increased αPSM production while deleting the 3' end of Teg41 (Teg41Δ3' strain) resulted in a decrease in αPSM production, reduced hemolytic activity of S....
Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). Studies examining the immune response to S. aureus have been conducted, yet our understanding of the kinetic response to S. aureus subcutaneous skin infection remains incomplete. In this study, we used C57BL/6J mice and USA300 S. aureus to examine the host-pathogen...
Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs). While studies have examined how the immune system responds to S. aureus SSTIs, most are limited in scope to a single time point or outcome. To set a foundation for future studies, we conducted a S. aureus skin infection experiment using female C57BL/6J mice and USA...
The Staphylococcus aureus cidABC and lrgAB operons encode members of a well-conserved family of proteins thought to be involved in programmed cell death (PCD). Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. T...
In Staphylococcus aureus, the two‐component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil‐derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts...
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...
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 e...
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. Th...
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 screeni...
Staphylococcus aureus fatty acid kinase, FakA, is necessary for incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro , and that Δ fakA causes larger lesions compared...
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 S. aureus is unable t...
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 confer...
Implanted medical device-associated infections pose significant health risks as they are often the result of bacterial biofilm formation. Staphylococcus aureus 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 fo...
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. Her...
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...
This work focuses on the characterization of cis - and trans -acting elements essential for the induction of the cidABC operon in S. aureus . The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full ind...
To persist within the host and cause disease, Staphylococcus aureus 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 the two-gene operon, yjbIH , resulted in decreased pigmentation and aureolysin activity...
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 mic...
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 Staphylococcus aureus, 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 S. a...
Staphylococcus aureus 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 are largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory...
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...
The fatty acid kinase, FakA, of Staphylococcus aureus 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 remov...
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 Staphylococcus aureus. In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB, saeR, and saeS mutant strains reveale...
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 in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus...
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 supporti...
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 s...
The majorStaphylococcus aureusautolysin, 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 (AM) and glucosaminidase (GL) domains of Atl inhibitedin vitrobiofilm formation. However, in a murine model of d...
Importance:
This manuscript focuses on cell death mechanisms in Staphylococcus aureus and provides important new insight into the genes involved in this ill-defined process. Exploring the cause of increased stationary phase death in a S. aureus ΔsrrAB regulatory mutant, we found that the decreased viability of this mutant was a consequence of the...
Microbially-mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics...
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 atte...
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 hav...
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 pro...
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 th...
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...
Recent studies have demonstrated that expression of the Staphylococcus aureus lrgAB operon is specifically expressed 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. Specifica...
The Gac/Csr regulatory system is conserved throughout the γ-proteobacteria and controls key pathways in central carbon metabolism, quorum sensing, biofilm formation, and virulence in important plant and animal pathogens. Here we show that elevated intracellular citrate levels in a Vibrio fischeri aconitase mutant correlate with activation of the Ga...
Biofilm communities contain distinct microniches that result in metabolic heterogeneity and variability in gene expression. Previously, these niches were visualized within Staphylococcus aureus biofilms by observing differential expression of the cid and lrg operons during tower formation. In the present study, we examined early biofilm development...
Significance
The enzymes required for the incorporation of host fatty acids into the membrane phospholipids of Gram-positive bacterial pathogens are unknown. Fatty acid kinase (Fak) is a new enzyme in lipid metabolism that requires two proteins to form acyl-phosphate: an ATP binding-domain protein (FakA) that interacts with a fatty acid binding pro...
During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the
production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not...
The ability to genetically manipulate bacteria is essential to understanding gene/protein function in these organisms. While basic cloning has become routine in molecular biology, many still view the ability to make directed mutations as a daunting or intimidating task. To aid the staphylococcal research community, the goal of this treatise is to d...
Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As
in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also
modulated in response to environmental stimuli. Previous studies sug...
Potassium (K(+) ) plays a vital role in bacterial physiology, including regulation of cytoplasmic pH, turgor pressure, and transmembrane electrical potential. Here, we examine the Staphylococcus aureus Ktr system uniquely comprised of two ion-conducting proteins (KtrB and KtrD) and only one regulator (KtrA). Growth of Ktr system mutants was severel...
Staphylococcus aureus is an important pathogen that has a major impact on human health. Although it is notorious for causing skin and soft-tissue infections, it has the ability to infect nearly every organ system in the human body, often with fatal consequences. This remarkable adaptability is due in large part to the wide array of virulence factor...
Unlabelled:
To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methic...
Essential gene comparisons.
Identified phenotypes.
Genes lacking Tn insertions.
The bursa aurealis transposon has been used to create transposon insertion libraries of Bacillus anthracis and Staphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that
allows for the replacement of the transposon with useful genetic markers and fluorescent repor...
Unlabelled:
Subminimal inhibitory concentrations of antibiotics have been shown to induce bacterial biofilm formation. Few studies have investigated antibiotic-induced biofilm formation in Staphylococcus aureus, an important human pathogen. Our goal was to measure S. aureus biofilm formation in the presence of low levels of β-lactam antibiotics. F...
The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protei...
Characterization of LAC-13C and Δatl mutant KB4051. A) Growth of LAC-13C (solid symbols) and Δatl mutant KB4051 (empty symbols) in TSB at 1∶10 media to flask volume ratio. B) Triton X-100 induced autolysis for 3 hr cultures of LAC-13C (solid symbols) and Δatl mutant KB4051 (empty symbols) grown in TSB with 1 M glucose at a 1∶10 media to flask volum...
Zymographic and western blot analyses of the atl point mutants. Three µg of total extracellular proteins from the atlAM mutant (KB5002) and the atlGL mutant (KB5001) with wild-type and point mutation allele expressing plasmids were examined in zymography gels containing either S. aureus (upper left panel) or Micrococcus (upper right panel) cells as...
Visualization of mutant clusters. Wild-type UAMS-1, Δatl mutant KB5000, ΔatlGLU mutant KB5001, and ΔatlAM mutant KB5002 all with pJB128 were grown to mid-exponential phase (3 hrs) in TSB with a 1∶10 media to volume ratio at 37°C with shaking (250 rpm). A sample was removed and Syto-9 added to a final concentration 5 µM of to aid in visualization. C...
Plasmid construction and oligonucleotides.
(DOCX)
Select bacterial strains and plasmids used in this study.
(DOCX)
Cluster size of the atl mutants with complementation plasmids. Wild-type UAMS-1 (black) compared to A) Δatl mutant KB5000 with pJB128 (red) and KB5000 with the atl complement plasmid pJB141 (orange), B) ΔatlAM mutant KB5002 carrying pJB128 (green) or the atlAM complement plasmid pJB111 (brown), and C) ΔatlGLU mutant KB5001 with pJB128 (blue) or the...
The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holi...
Vibrio fischeri serves as a valuable model of bacterial bioluminescence, its regulation, and its functional significance. Light output varies
more than 10,000-fold in wild-type isolates from different environments, yet dim and bright strains have similar organization
of the light-producing lux genes, with the activator-encoding luxR divergently tra...