Tao Weitao

Vesiculation from Pseudomonas aeruginosa under SOS

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity.

Association of Acinetobacter baumannii EF-Tu with cell surface, outer membrane vesicles, and fibronectin

A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.

SOS involvement in stress-inducible biofilm formation

Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger the SOS response, in which SOS sensor RecA stimulates auto-cleavage of repressor LexA. These observations lead to a hypothesis of a connection between stress-inducible biofilm formation and the RecA-LexA interplay. To test this hypothesis, three biofilm assays were conducted, viz. the standard 96-well assay, confocal laser scanning microscopy, and the newly developed biofilm-on-paper assay. It was found that biofilm stimulation by the DNA replication inhibitor hydroxyurea was dependent on RecA and appeared repressed by the non-cleavable LexA of Pseudomonas aeruginosa. Surprisingly, deletion of lexA led to reduction of both normal and stress-inducible biofilm formation, suggesting that the wild-type LexA contributes to biofilm formation. The decreases was not the result of poor growth of the mutants. These results suggest SOS involvement in hydroxyurea-inducible biofilm formation. In addition, with the paper biofilm assay, it was found that degradation of the biofilm matrix DNA by DNase I appeared to render the biofilms susceptible to the replication inhibitor. The puzzling questions concerning the roles of LexA in DNA release in the biofilm context are discussed.

Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation.

Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic dsDNA helix could be advantageous for cell replication such as bacterial binary fission and cell mitosis without the need for topoisomerase or helicase to consume additional energy.

Multicellularity of a unicellular organism in response to DNA replication stress.

The bacterium Pseudomonas aeruginosa forms multicellular communities, or biofilms, in response to hydroxyurea, an antiproliferative drug for tumor treatment. This drug inhibits growth of proliferating planktonic bacterial cells but stimulates biofilm formation; analogously, it is used to treat hematopoietic diseases and suppress metastatic populations rather than advanced solid tumors. From computer simulations of biofilm and tumor development, analogies can be drawn between planktonic bacteria and metastatic cells and between bacterial biofilm and solid tumor development. These thoughts lead to a futuristic hypothesis of an evolutionary relationship between bacteria and cancer. Bacteria were traditionally thought of as unicellular organisms and grow strictly as free-living (planktonic) cells. However, under certain conditions bacteria behave like multicellular organisms, forming multicellular communitiesdbiofilms, which involve active communication and cooperation among cells. Such behavior may underscore their evolutionary relationship to multicellular organisms; this thought has led to a futuristic hypothesis of whether biofilm development from planktonic cells is analogous to carcinogenesis from metastatic cells. This analogy may derive from computer simulations of biofilms. Two growth strategies have been suggested in silico for bacteria [3]ehigh growth rate but low yield (HRLY) and low growth rate but high yield (LRHY)ewhere the growth rate is defined as biomass formation per unit of time, and the yield as biomass generation per amount of resources consumed. Planktonic cells tend to grow at maximum growth rates during exponential phase whenever nutrients are available, characteristic of HRLY cells that consume resources egoistically to gain a short-term advantage. In contrast, LRHY subpopulations would utilize resources ecologically for a long-term advantage. Based on such reasoning, it can be predicted that cells in biofilms may grow more slowly than cells in a planktonic subpopulation; in fact, P. aeruginosa grows planktonically at a higher rate than stagnantly in biofilms. Because of the long-term advantage, LRHY may render biofilms predominant in nature [3]. While comprehensive empirical evidence for this model is lacking, we hypothesized that selectively impairing HRLY favors LRHY biofilm formation.

Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation

DNA-damaging antibiotics such as ciprofloxacin induce biofilm formation and the SOS response through autocleavage of SOS-repressor LexA in Pseudomonas aeruginosa. However, the biofilm-SOS connection remains poorly understood. It was investigated with 96-well and lipid biofilm assays. The effects of ciprofloxacin were examined on biofilm stimulation of the SOS mutant and wild-type strains. The stimulation observed in the wild-type in which SOS was induced was reduced in the mutant in which LexA was made non-cleavable (LexAN) and thus SOS non-inducible. Therefore, the stimulation appeared to involve SOS. The possible mechanisms of inducible biofilm formation were explored by subproteomic analysis of outer membrane fractions extracted from biofilms. The data predicted an inhibitory role of LexA in flagellum function. This premise was tested first by functional and morphological analyses of flagellum-based motility. The flagellum swimming motility decreased in the LexAN strain treated with ciprofloxacin. Second, the motility-biofilm assay was performed, which tested cell migration and biofilm formation. The results showed that wild-type biofilm increased significantly over the LexAN. These results suggest that LexA repression of motility, which is the initial event in biofilm development, contributes to repression of SOS-inducible biofilm formation.

Detection of anthrax lef with DNA-based photonic crystal sensors

Bacillus anthracis has posed a threat of becoming biological weapons of mass destruction due to its virulence factors encoded by the plasmid-borne genes, such as lef for lethal factor. We report the development of a fast and sensitive anthrax DNA biosensor based on a photonic crystal structure used in a total-internal-reflection configuration. For the detection of the lef gene, a single-stranded DNA lef probe was biotinylated and immobilized onto the sensor via biotin-streptavidin interactions. A positive control, lef-com, was the complementary strand of the probe, while a negative control was an unrelated single-stranded DNA fragment from the 16S rRNA gene of Acinetobacter baumannii. After addition of the biotinylated lef probe onto the sensor, significant changes in the resonance wavelength of the sensor were observed, resulting from binding of the probe to streptavidin on the sensor. The addition of lef-com led to another significant increase as a result of hybridization between the two DNA strands. The detection sensitivity for the target DNA reached as low as 0.1 nM. In contrast, adding the unrelated DNAs did not cause an obvious shift in the resonant wavelength. These results demonstrate that detection of the anthrax lef by the photonic crystal structure in a total-internal-reflection sensor is highly specific and sensitive.

Bacteria under SOS evolve anticancer phenotypes

BackgroundThe anticancer drugs, such as DNA replication inhibitors, stimulate bacterial adhesion and induce the bacterial SOS response. As a variety of bacterial mutants can be generated during SOS, novel phenotypes are likely to be selected under the drug pressure.Presentation of the hypothesisBacteria growing with cancer cells in the presence of the replication inhibitors undergo the SOS response and evolve advantageous phenotypes for the bacteria to invade the cancer cells in order to evade the drug attack. This hypothesis predicts that bacteria produce the proteins that mediate bacterial capture and invasion of cancer cells--the advantageous phenotypes. Generation of the phenotypes may be facilitated during the SOS response induced by anticancer drugs.Testing the hypothesisExperimental design: 1) Examine attachment and invasion of bacterium Pseudomonas aeruginosa and the SOS mutant control to cancer cells in the presence of the anticancer drugs that inhibit DNA replication enzymes and trigger the SOS response. 2) Reveal the bacterial proteins that exhibit changes in expression. 3) Identify the genes encoding cancer adhesion and invasion. 4) Construct the mutants for the genes, clone and express these genes. 5) Examine the bacterial capture and invasion of cancer cells in contrast to non-cancer control.Expected results: 1) The bacterial proteins will be differentially induced during bacteria-cancer interaction under the SOS response to the anticancer drugs. 2) Knocking out the bacterial cancer-adhesion-invasion genes will disrupt the adhesion-invasion phenotypes of the bacteria. 3) Expressing these genes will direct the bacterial capture and invasion of cancer cells.Implications of the hypothesisBacteria can evolve anticancer phenotypes targeting metastatic cells. If this hypothesis is true, the outcomes will contribute to development of a novel bacterial anti-metastasis regimen.

Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions

DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

Break Breast Cancer Addiction by CRISPR/Cas9 Genome Editing

Breast cancer is the leading diagnosed cancer for women globally. Evolution of breast cancer in tumorigenesis, metastasis and treatment resistance appears to be driven by the aberrant gene expression and protein degradation encoded by the cancer genomes. The uncontrolled cancer growth relies on these cellular events, thus constituting the cancerous programs and rendering the addiction towards them. These programs are likely the potential anticancer biomarkers for Personalized Medicine of breast cancer. This review intends to delineate the impact of the CRSPR/Cas-mediated genome editing in identification and validation of these anticancer biomarkers. It reviews the progress in three aspects of CRISPR/Cas9-mediated editing of the breast cancer genomes: Somatic genome editing, transcription and protein degradation addictions.