The bacterium Bacillus anthracis, the causative agent for the disease anthrax, possesses two plasmids that contribute significantly to virulence. Besides plasmids, certain chromosomal genes also contribute. In previous studies, our lab discovered that the chromosomally encoded ClpX gene is essential for virulence in B. anthracis. ClpX is an ATPase that is part of the ClpXP proteasome found in many bacteria. Loss of ClpX in B. anthracis Sterne results in increased susceptibility to cell wall targeting antibiotics like penicillin and daptomycin. However, the mechanism behind ClpX’s role in antibiotic resistance is not understood as it is likely that multiple pathways are affected by the loss of this global protease. We recently conducted a microarray to find which genes are up or down regulated in ClpX compared to wild-type (WT) B. anthracis. 119 genes had disrupted regulation and several of these had been connected to cell-wall active antibiotics like penicillin. In this study, we focused on three of these genes: MsrA, GlpF, and SigM. We confirmed the microarray results and showed that MsrA, GlpF, and SigM gene expression in our ClpX strains significantly differs from the wild-type B. anthracis Sterne via QPCR. Insertional knockout mutants were made for GlpF and SigM to test whether these genes were necessary for antibiotic resistance. We are currently testing these mutants in penicillin and daptomycin to assess their phenotypes. We found that loss of SigM results in increased susceptibility to penicillin and are currently studying the effect of daptomycin on SigM and GlpF. We will test the virulence of both mutants in our invertebrate animal model G. mellonella. This will hopefully provide better understanding on the mechanism behind ClpX’s antibiotic resistance.

Studies have shown that males and females differ with regard to their ability to survive pathogen infections. The fathead minnow is a newly developed model for immunotoxicity; however, few studies have compared male and female immune responses following pathogen exposure. The purpose of this study was to examine sex-based differences in pathogen resistance and immune responses following exposure to a pathogen in adult fathead minnows (Pimephales promelas). To accomplish this, fish were bacterially infected with Yersinia ruckeri and the immune system’s ability to respond was monitored. Additionally, genes that are known to be expressed during the immune response initiation were measured quantitatively, providing insight into the molecular effect in minnows. At the whole organism level, male fish were less able to survive pathogen infection relative to female fish. At the tissue level, both male and female pathogen-injected fish had decreased hematocrit percentages compared to the fish injected with a saline solution, but did not differ from each other. At the molecular level, increased gene expression of interleukin 1β was seen in pathogen-injected males compared to pathogen-injected females and both sham-injected sexes, indicating that pathogen-injected males mounted a larger inflammatory response at the molecular level. Taken together, this evidence suggests that the increased mortality observed among males earlier in the exposure to the pathogen may be due to the upregulated inflammatory response rather than the effects of the pathogen itself.

Bacillus anthracis is a gram-positive, spore-forming bacterium and the causative agent of the deadly disease anthrax. The B. anthracis genome consists of chromosomal genes and the pXO1 and pXO2 plasmids that strongly contribute to the bacteria’s deadly nature. While the virulence factors associated with the plasmids have been extensively studied, we believe there are still undiscovered chromosomal genes that may also have important virulence factors. To identify novel chromosomal genes associated with B. anthracis virulence, we screened a transposon mutant library of B. anthracis Sterne strain for increased sensitivity to reactive oxygen species. Reactive oxygen species, such as hydrogen peroxide, have many functions in mammalian immune defenses and wild type B. anthracis is able to subvert this host defense. Sensitivity to reactive oxygen species was tested through in vitro hydrogen peroxide assays and after several rounds of screening, eight mutants were confirmed as susceptible. We next tested whether any of these mutants were attenuated in vivo using our invertebrate animal model, Galleria mellonella and found several mutants with decreased virulence. We are currently working on determining the location of the transposon insertion to find which chromosomal gene is disrupted. This could lead to the discovery of novel B. anthracis virulence genes and eventually possible treatment targets for future anthrax outbreaks and attacks.

The swim performance assay is a behavioral assessment used to measure cardiovascular function in fish. Previously, the laminar flow assay (LFA) has been the standard method of assessing swim performance in adult fish to measure their cardiac output. The spinning task assay (STA) is a novel, accessible method of assessing swim performance; however, previous studies have not compared the two methods. Additionally, there is little documentation of swim performance in larval fish, a more sensitive study subject for toxicological research. Therefore, the aim of this research is to compare the swim performance of fish in the LFA to those in the STA to determine which method is better for assessing swim performance in larval fathead minnows (Pimephales promelas). In this study, the percent of fish that fail to swim in the LFA is inversely proportional to the age of the fish, but in the STA, there is no correlation between percent failure and fish age. Results show that as fish increase in size, swim performance in the LFA improves, making it a more representative, predictable assay. Results from the STA indicate that swim performance in fish does not improve with size and performance in the STA is not correlated with performance in the LFA. Ucrit values from the LFA have less variation than those from the STA. The results of this study show that the LFA is a more suitable modality for assessing swim performance in larval fathead minnows.

Porous silicon nanoparticles exhibit great potential as drug delivery vectors due to their high surface-area-to-volume ratio allowing for increased efficacy of surface functionalization and therapeutic loading capabilities. This data set demonstrates the fabrication of a class of plant-derived materials which are sub-micron in size and capable of functionalization with primary amine groups through the addition of APTES.
The production of porous silicon particles (pSi) is achieved through magnesiothermic reduction of silica containing Tabasheer powder isolated from the nodal joints of the Bambuseae plant. Efficacy of this reduction is evaluated using techniques including X-ray diffraction and Energy-dispersive X-ray spectroscopy which show successful reduction of silica starting material to porous silicon.
High energy ball milling followed by reduction is used to produce pSi particles of sub-micrometer size while also allowing for a significantly higher yield (~90%) of material than previous methods. Particle size is confirmed via electron microscopy and dynamic light scattering (DLS).
Following reduction, surface functionalization of silicon nanoparticles with primary amine groups was carried out using a 4% (v/v) solution of APTES in acetone. The evaluation of this functionalization was conducted using techniques including zeta potential and infrared spectroscopy (IR). Zeta potential values are found to be approximately -10 mV. This data demonstrates successful amino silanization.
The results achieved through these methods suggest successful fabrication of pSi nanoparticles and subsequent functionalization for future use as a drug delivery vector.