Shining light on T6SS mode of action and function within single cells and bacterial communities

Bacteria are ubiquitously found in the environment and form the basis for all known ecosystems on our planet. Most bacterial cells reside in complex multi-species bacterial communities, which are often associated with a host, such as the human microbiota. These bacterial communities are shaped by cooperative and competitive interactions amongst their members. Like higher animals, bacteria also compete with their conspecifics for nutrients and space. This evolutionary arms race resulted in a diverse set of strategies for microbial competition. In particular, bacteria residing on solid surfaces can compete with their neighbors through the use of specialized nanomachines, called secretion systems, enabling the direct delivery of toxic effector molecules into by-standing target cells. The most commonly used weapon for contact-dependent antagonism is the bacterial Type VI secretion system (T6SS). The T6SS belongs to the family of contractile injection systems (CISs). All CISs are structurally and functionally related to contractile bacteriophages (e.g. phage T4) and translocate proteins into target cells by means of physical force, which is generated by rapid sheath contraction. This results in the ejection of the inner tube associated with a sharp tip and effector proteins at its end. Effector translocation leads ultimately to target cell death. Importantly, the T6SS is capable translocating effectors across broad ranges of biological membranes making it a powerful weapon in microbial warfare as well as potent virulence mechanism towards eukaryotic host cells. Our current understanding of T6SS mode of action is primarily based on the combination of structural biology and fluorescence live-cell microscopy studies. While in particular cryo-electron microscopy (cryo-EM) revealed the detailed architecture of the T6SS in situ and of isolated subassemblies, fluorescence live-cell microscopy uncovered the remarkable dynamics of T6SS biogenesis. However, a complete understanding of T6SS dynamics is hampered in standard fluorescent microscopy due to: (i) the spatial and temporal resolution limit, (ii) the inability to efficiently label secreted components of the machinery, (iii) the weak signals due to low protein abundance and rapid photobleaching, (iv) the difficulty to perform long-term co-incubation experiments as well as (v) the inability to precisely control spatial and chemical environment. My doctoral thesis aimed to overcome these limitations to allow novel insights into dynamics of the T6SSs of Vibrio cholerae, Pseudomonas aeruginosa and Acinetobacter baylyi. Specifically sheath assembly, initiation of sheath contraction, T6SS mediated protein translocation in to sister cells as well as strategies for prey cell inhibition were studied in this thesis. First, I studied sheath assembly in ampicillin induced V. cholerae spheroplasts. These enlarged cells assemble T6SS sheaths which are up to 10x longer as compared to rod shaped cells. This allowed us to photobleach an assembling sheath structure and demonstrate that new sheath subunits are added to the growing sheath polymer at the distal end opposite the baseplate. Importantly, this was the first direct observation made for any contractile machines studied to date. Moreover, I showed that unlike for all other CISs, T6SS sheath length is not regulated and correlates with cell size. In order to monitor protein translocation into target cells, we developed a T6SS dependent interbacterial protein complementation assay, enabling the indirect detection of translocated T6SS components into the cytosol of recipient cells. This allowed us to demonstrate that secreted T6SS components are exchanged among by-standing sister cells within minutes upon initial cell contact. Importantly, these results were the first experimental indication that T6SS is capable of translocating its components into the cytosol of Gram-negative target cells. Furthermore, we showed that the amount and the composition of the secreted tip influences the number of T6SS assemblies per cell, whereas different concentration of the tube protein influenced sheath length. We also provided evidence that precise aiming of T6SS assemblies through posttranslational regulation in P. aeruginosa increases the efficiency of substrate delivery. In addition, together with two Nanoscience master students we have also been implementing microfluidics in the Basler laboratory. This powerful technology enabled us to control the spatial arrangements of aggressor and prey populations and to follow these populations at single-cell level over time scales of several hours. In collaboration with Prof. Kevin Forster, University of Oxford, we demonstrated that the rate of target cell lysis heavily influences the outcome of contact-dependent T6SS killing and thus drives evolution of lytic effectors. Moreover, microfluidics allows for the dynamic change of the chemical microenvironment during imaging experiments. By following the T6SS dynamics in response to hyperosmotic shocks resulting in a rapid cell volume reduction, we found that physical pressure from the collapsing cell envelope could trigger sheath contraction. This led us to propose a model for sheath contraction under steady-state conditions where continued sheath polymerization against membrane contact site leads to a gradual increase in pressure applied to the assembled sheath. We propose that this could be potentially sensed by the baseplate, which in turn would trigger sheath contraction

Localization matters : new insights into spatio-temporal regulation of type VI secretion systems

Subcellular organization is important for bacterial cell physiology. Especially, bacterial secretion systems are tightly regulated in a temporal and spatial manner to efficiently fulfill their function. Among them, the contact-dependent Type VI secretion (T6SS) has an important role in inter-bacterial competitions and pathogenicity of Gram-negative bacteria. T6SS translocates effector proteins into target cells using the contraction of a long cytosolic sheath, which pushes an inner tube together with a sharp tip and associated effectors across target cell membranes. This mode of action allows bacteria to use T6SS against a broad range of prokaryotic and eukaryotic organisms. However, the contact-dependency limits the target range and the efficiency of effector translocation is low because only a small number of effectors can be delivered per one round of T6SS assembly. Recent advances in live-cell fluorescence and super resolution microscopy led to the revelation that T6SS activation patterns and dynamics are surprisingly diverse in different bacteria. These differences in T6SS assembly dynamics likely reflect different strategies to overcome the disadvantages of T6SS mode of action. However, the spatio-temporal regulation behind these different T6SS firing patterns are not well understood. My PhD thesis provides new insights into how different subcellular localizations of T6SS assembly are achieved. The Threonine phosphorylation pathway (TPP) is a unique posttranslational regulation mechanism, which allows Pseudomonas aeruginosa to activate its T6SS apparatus in response to membrane damage inflicted by an attack from neighboring bacteria and to localize it to the site of attack. While the involved components are identified, it is not clear how the periplasmic sensor module integrates spatial and temporal information for precise and fast T6SS assembly initiation. To test if relocation of TPP components from outer to inner membrane (IM) is important for T6SS activation, I changed their subcellular localization by mutating their N-terminal signal sequences. Relocation of one TPP component to IM indeed hyper-activated T6SS assembly, however, the exact mechanism of T6SS localization remains to be elucidated. In collaboration with Prof. Kevin Foster, University of Oxford, we tested the benefit and cost of TPP-dependent localization of T6SS during bacterial competitions. Our results from in silico and imaging experiments suggested that P. aeruginosa uses TPP to kill competing bacteria by localized and repeated T6SS assemblies and thus inflicting more damage than it encounters from attacking competitors. In collaboration with Prof. Petr Broz, University of Lausanne, we characterized the unique Francisella pathogenicity island (FPI), which encodes a non-canonical T6SS essential for phagosomal escape. The FPI lacks a specialized unfoldase required for recycling of contracted sheaths and for dynamics of canonical T6SS. Furthermore, the FPI encodes genes with unknown function. By live-cell fluorescence microscopy, we showed that F. novicida T6SS dynamics is comparable to canonical T6SS dynamics. Moreover, we found that general-purpose unfoldase ClpB recycles contracted sheaths and is essential for phagosomal escape in vivo. By analyzing T6SS dynamics and virulence of single deletion mutants in vitro and in vivo, we could group FPI components with unknown function into structural components, which are required for T6SS function, and putative effectors, which are critical for virulence but not for T6SS assembly. Moreover, we showed that F. novicida T6SS assembles exclusively at bacterial poles. This unique polar localization raised the question of how Francisella T6SS is localized to the poles and whether it is important for T6SS function. I analyzed the dynamics of membrane complex formation, which is the first step of T6SS assembly, by live-cell fluorescence microscopy and structured illumination microscopy. I showed that the membrane complex is stably formed on the poles even in the absence of other FPI components. In addition, the membrane complex formation was insufficient to initiate sheath assembly indicating that additional signals are required to activate T6SS in F. novicida. To investigate the contribution of FPI components and localization of T6SS to Francisella virulence in more detail, I established Galleria mellonella larvae as infection model. Besides, I constructed two expression plasmids for F. novicida, which are mobilized by conjugation and have tetracyline inducible promoters for tunable gene expression. These new tools will be invaluable in the future research of mechanism required for F. novicida pathogenesis

Targeting the bacterial cell envelope by molecular coevolution and high throughput phenotyping

Microbial species exhibit a wide repertoire of phenotypic responses to their surroundings, be it stresses posed by their environment, or signals from their bacterial community. Despite advances in computer vision, reporting such phenotypic responses is often done in a qualitative manner. In the course of my work I developed a user-friendly software tool to address the lack of a standardized, quantitative method to measure microbial phenotypes macroscopically. This freely available software, called Iris, can quantify a wide range of microbial phenotypes at the colony level and in a high-throughput fashion. Iris is already used by several research groups, and I present some of its diverse applications and potential for hypothesis generation. One such application is the quantification of the impact of each gene on the cell envelope permeability in E. coli. The Gram-negative bacterial cell envelope forms a barrier against antimicrobial drugs, drastically limiting the list of treatments effective against these organisms. To expand our knowledge on how this multi-layered is built and perturbed, we developed a rapid screening method to detect mutants with envelope defects. By screening a systematic gene deletion mutant collection in E. coli across 4 conditions, we identified a number of mutants with defects in envelope assembly. Among those were genes known to be involved in envelope biogenesis, as well as 102 genes of unknown function. In the course of my work I built upon and improved this screening approach, to acquire quantitative membrane permeability measurements that can be used for high- throughput chemical genomics approaches. Gram-negative bacterial envelope is both a permeability barrier, and a structural barrier. The structural component mainly consists of the rigid peptidoglycan (PG) sacculus, which gives the cells the ability to withstand both turgor pressure and environmental insults. Although biosynthesis of PG is central to bacteria and a target of β-lactam antibiotics, its regulation remains largely elusive. Recently, a number of regulators of PG biosynthesis have been identified, and shown to have coevolved with domains in PG synthases. With the aim of uncovering potential regulatory connections, I developed a computational approach to explore the coevolution of domains in proteins involved in cell wall biosynthesis and remodeling with other proteins in the cell. The method correctly identified existing regulatory interactions, and is readily applied to species across the bacterial kingdom

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)

Bio-adhesion and cleanliness in bio-processing

Biofilms are a significant problem for many industries including medical devices, the oil and gas industry, consumer product manufacturers and water distribution systems. Biofilms are communities of microbes that attach and grow on almost any surface. Biofilms are able to establish quickly in a matter of hours and can be very difficult to remove. This thesis was focused on increasing the understanding of biofilm interaction with different substratum materials, for both the initial surface colonisation and for mature biofilm lifecycle stages using a Pseudomonas aeruginosa (PA) model. The surface energy and the surface roughness, for a number of substratums were investigated in this thesis. The effects of a number of proprietary and non-proprietary surface coatings which primarily changed surface energies and or topography were investigated. This work has confirmed that surface energy is important in Pseudomonas bio-adhesion, with correlations observed in both the initial attachment of microbes, but also in high shear cleaning experiments for mature biofilms. Biofilm-substratum interfacial adhesion remains an important region even in mature biofilms, suggesting that surfaces that exhibit lower colonisation rates may also be easier to clean. Initial attachment studies confirm 1/3 less microbe attachment to polymer surfaces compared to metal surfaces; roughness was not a significant parameter. PTFE-AF coatings on 316 stainless steel showed 88 percent decrease in initial microbe attachment coupled with enhanced cleanability. Industries currently using stainless steel in microbe applications could from benefit this coating; limiting future biofilm colonisation rates and improved cleanability. It is demonstrated that Pseudomonas naturally colonises at low levels on selected transparent polycarbonate surfaces and given its good chemical compatibility and low cost would be an alternative to stainless steel. A range of commercial surface coatings, which are meant to reduce microbial adhesion, were also tested in this thesis; this study could not affirm such claimed performance.Open Acces

Microbial Biofilms

In the book Microbial Biofilms: Importance and applications, eminent scientists provide an up-to-date review of the present and future trends on biofilm-related research. This book is divided with four subdivisions as biofilm fundamentals, applications, health aspects, and their control. Moreover, this book also provides a comprehensive account on microbial interactions in biofilms, pyocyanin, and extracellular DNA in facilitating Pseudomonas aeruginosa biofilm formation, atomic force microscopic studies of biofilms, and biofilms in beverage industry. The book comprises a total of 21 chapters from valued contributions from world leading experts in Australia, Bulgaria, Canada, China, Serbia, Germany, Italy, Japan, the United Kingdom, the Kingdom of Saudi Arabia, Republic of Korea, Mexico, Poland, Portugal, and Turkey. This book may be used as a text or reference for everyone interested in biofilms and their applications. It is also highly recommended for environmental microbiologists, soil scientists, medical microbiologists, bioremediation experts, and microbiologists working in biocorrosion, biofouling, biodegradation, water microbiology, quorum sensing, and many other related areas. Scientists in academia, research laboratories, and industry will also find it of interest

Program and Proceedings: The Nebraska Academy of Sciences 1880-2010

PROGRAM FRIDAY, APRIL 23, 2010 REGISTRATION FOR ACADEMY, Lobby of Lecture wing, Olin Hall Aeronautics and Space Science, Session A, Olin 249 Aeronautics and Space Science, Session B, Olin 224 Chemistry and Physics, Section A, Chemistry, Olin A Collegiate Academy, Biology Session A, Olin B Collegiate Academy, Chemistry and Physics, Session A, Olin 324 Biological and Medical Sciences, Session A, Olin 112 Biological and Medical Sciences, Session B, Smith Callen Conference Center Chemistry and Physics, Section B, Physics, Planetarium History and Philosophy of Science, Olin 325 Junior Academy, Judges Check-In, Olin 219 Junior Academy, Senior High REGISTRATION, Olin Hall Lobby NWU Health and Sciences Graduate School Fair, Olin and Smith Curtiss Halls Junior Academy, Senior High Competition, Olin 124, Olin 131 Aeronautics and Space Science, Poster Session, Olin 249 Teaching of Science and Math, Olin 325 MAIBEN MEMORIAL LECTURE, OLIN B Dr. Mark Greip, Vice-Chair, Department of Chemistry, University of Nebraska-Lincoln LUNCH, PATIO ROOM, STORY STUDENT CENTER (pay and carry tray through cafeteria line, or pay at NAS registration desk) Aeronautics Group, Conestoga Room Anthropology, Olin 111 Biological and Medical Sciences, Session C, Olin 112 Biological and Medical Sciences, Session D, Smith Callen Conference Center Chemistry and Physics, Section A, Chemistry, Olin A Chemistry and Physics, Section B, Physics, Planetarium Collegiate Academy, Biology Session A, Olin B Collegiate Academy, Biology Session B, Olin 249 Collegiate Academy, Chemistry and Physics, Session A, Olin 324 Junior Academy, Judges Check-In, Olin 219 Junior Academy, Junior High REGISTRATION, Olin Hall Lobby Junior Academy, Senior High Competition, (Final), Olin 110 Earth Science, Olin 224 Junior Academy, Junior High Competition, Olin 124, Olin 131 NJAS Board/Teacher Meeting, Olin 219 Junior Academy, General Awards Presentations, Smith Callen Conference Center BUSINESS MEETING, OLIN B SOCIAL HOUR for Members, Spouses, and Guests First United Methodist Church, 2723 N 50th Street, Lincoln, NE ANNUAL BANQUET and Presentation of Awards and Scholarships First United Methodist Church, 2723 N 50th Street, Lincoln, N

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date