Thomas J. Lamkin

Rapid Targeted Gene Disruption in Bacillus Anthracis

BackgroundAnthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome.ResultsGroup II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion.ConclusionsThe design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics.

High-Throughput Screening for Small-Molecule Inhibitors of Staphylococcus epidermidis RP62a Biofilms

High-throughput screening (HTS) of 42 865 compounds was performed to identify compounds that inhibit formation of or kill Staphylococcus epidermidis RP62a biofilms. Three biological processes were assayed, including (1) growth of planktonic/biofilm bacteria, (2) assessment of metabolically active biofilm bacteria using a resazurin assay, and (3) assessment of biofilm biomass by crystal violet staining. After completing the three tiers (primary screening, hit confirmation, and dose-response curves), 352 compounds (representing ~0.8%) were selected as confirmed hit compounds from the HTS assay. The compounds were divided into groups based on their effectiveness on S. epidermidis biofilm properties. The majority of these affected both inhibition and killing of bacterial biofilm cultures. Only 16 of the confirmed hit compounds that have either an AC50 lower than 10 µM and/or Sconst ≥70 from those processed were selected for further study by confocal laser scanning microscopy (CLSM). The CLSM was used to evaluate the confirmed hit compounds on (1) inhibition of biofilm formation and (2) killing of preexisting S. epidermidis biofilms. Taken together, with further testing (e.g., disease-related conditions), such compounds may have applications as broad antimicrobial/antibiofilm use for prophylactic or therapeutic intervention to combat infections in surgical and intensive care clinics and battlefield settings.

Manganese binding to antioxidant peptides involved in extreme radiation resistance in Deinococcus radiodurans

A decapeptide, DEHGTAVMLK (DP1), and its random scrambled version, THMVLAKGED (DP2), have been studied for their interactions with manganese. The amino acid composition of the peptides was selected to include the majority of the most prevalent amino acids present in a Deinococcus radiodurans bacterium cell-free extract that contains components capable of conferring extreme resistance to ionizing radiation. The extract appears to be rich in Mn(II) complexes which seem to be responsible for protecting proteins from Reactive Oxygen Species damage. We focused our attention on the interaction of the decapeptides with Mn(II) ion with the aim of obtaining information on the possible complexes formed, by using NMR, EPR, and ESI-MS techniques.

Automated Analysis and Classification of Infected Macrophages Using Bright-Field Amplitude Contrast Data

This article presents a methodology for acquisition and analysis of bright-field amplitude contrast image data in high-throughput screening (HTS) for the measurement of cell density, cell viability, and classification of individual cells into phenotypic classes. We present a robust image analysis pipeline, where the original data are subjected to image standardization, image enhancement, and segmentation by region growing. This work develops new imaging and analysis techniques for cell analysis in HTS and successfully addresses a particular need for direct measurement of cell density and other features without using dyes.

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253‐, P2cysZK‐, and P1‐driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT‐mini‐Tn7T‐Km, pmini‐Tn7‐gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP‐ and RFP‐tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild‐type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high‐throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS)

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.

Elucidation of Small RNAs that Activate Transcription in Bacteria

Small non-coding RNA (sRNA) control of gene expression has been shown to play a prominent role in genetic regulation. While the majority of identified bacterial sRNAs exert their control at the translational level, a few examples of bacterial sRNAs that inhibit transcription have also been identified. Using an engineered combinatorial RNA library, we have elucidated bacterial sRNAs that activate transcription of a target gene in E. coli to varying degrees. Mutation of the strongest activator modified its activation potential. Our results suggest that transcriptional activation of our target gene results from recruitment of the bacterial RNA polymerase complex to the promoter region. These data, coupled with the malleability of RNA, provide a context to define synthetic control of genes in bacteria at the transcriptional level.

A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene's 5′ Untranslated Region in the Radiation and Desiccation Response

ABSTRACT Tight regulation of gene expression is important for the survival of Deinococcus radiodurans, a model bacterium of extreme stress resistance. Few studies have examined the use of regulatory RNAs as a possible contributing mechanism to ionizing radiation (IR) resistance, despite their proffered efficient and dynamic gene expression regulation under IR stress. This work presents a transcriptome-based approach for the identification of stress-responsive regulatory 5′ untranslated region (5′-UTR) elements in D. radiodurans R1 that can be broadly applied to other bacteria. Using this platform and an in vivo fluorescence screen, we uncovered the presence of a radiation-responsive regulatory motif in the 5′ UTR of the DNA gyrase subunit A gene. Additional screens under H2O2-induced oxidative stress revealed the specificity of the response of this element to IR stress. Further examination of the sequence revealed a regulatory motif of the radiation and desiccation response (RDR) in the 5′ UTR that is necessary for the recovery of D. radiodurans from high doses of IR. Furthermore, we suggest that it is the preservation of predicted RNA structure, in addition to DNA sequence consensus of the motif, that permits this important regulatory ability. IMPORTANCE Deinococcus radiodurans is an extremely stress-resistant bacterium capable of tolerating up to 3,000 times more ionizing radiation than human cells. As an integral part of the stress response mechanism of this organism, we suspect that it maintains stringent control of gene expression. However, understanding of its regulatory pathways remains incomplete to date. Untranslated RNA elements have been demonstrated to play crucial roles in gene regulation throughout bacteria. In this work, we focus on searching for and characterizing responsive RNA elements under radiation stress and propose that multiple levels of gene regulation work simultaneously to enable this organism to efficiently recover from exposure to ionizing radiation. The model we propose serves as a generic template to investigate similar mechanisms of gene regulation under stress that have likely evolved in other bacterial species.

Mapping Infected Cell Phenotype

Quantitative modeling of the phenotypic changes in the host cell during the bacterial infection makes it possible to explore an empirical relation between the infection stages and the quantifiable host-cell phenotype. A statistically reliable model of this relation can facilitate therapeutic defense against threats due to natural and genetically engineered bacterium. In the preliminary experiment, we have collected several thousand cell images over a period of 72 h of infection with a 2-h sampling frequency that covers various stages of infection by Francisella tularenesis (Ft). Segmentation of macrophages in images was accomplished using a fully automatic, parallel region growing technique. Over two thousand feature descriptors for the host cell were calculated. Multidimensional scaling, followed by hierarchical clustering, was used to group the cells. Preliminary results show that the host-cell phenotype, as defined by the set of measureable features, groups into different classes that can be mapped to the stages of infection.

Cell analytics in compound hit selection of bacterial inhibitors

Identifying novel drugs that inhibit bacterial infection has gained a considerable amount of attention in recent years. This is in part due to the increased number of highly resistant bacteria and the serious health threat it poses around the world. In order to combat this threat, a significant hurdle to overcome is the relatively low success rate of identifying novel chemical compounds that are effective at inhibiting bacterial infection. Despite increasing the vast amount of data that is currently generated during drug discovery endeavors, traditional analysis methods have not increased the overall success rate. In this paper, we investigate whether multivariate Image-based high content screening (HCS) platforms can identify chemical compounds using significantly reduced data while retaining its competitiveness. Image-based HCS is still predominantly used in biological compound activity assessments (bioassays) with univariate methods, not utilizing the data to its full potential. We propose a novel method that uses a small number of cells in high dimensional space to analyze interactions between cells, bacteria, and chemical compounds. Our results further indicate that our method can identify compounds that inhibit bacterial infection with a fraction of the control data generated.