Laser Radiation Influence on Viruses
Influence of the Laser Radiation on Infection Process by the Bacterial Viruses
Tech Area / Field
3 Approved without Funding
Tbilisi State University, Georgia, Tbilisi
Purposes of the investigation are explain the physico-chemical mechanisms of infection of bacterial viruses and the effects of laser beam radiation on the process of infection of healthy cells by virus particles. The final purposes are develop the technology and methods for cultivation of bacteriophages as a new vectors for biothechnology and new direction in the biomedicine-phagotherapie.
One of the most interesting and critical moments in phage infection is the mechanism of DNA injection into the host cell, in particular the energetics of this process. In the bacteriophage capsid the DNA is in a tightly packed condensed globular state of liquid crystals. Its removal from the capsid certainly requires a significant amount of work for unwinding and rearranging its highly compact conformation of liquid crystal into the standard double helix coils and moving it through the tiny exit channel, overcoming the friction with its walls. Nevertheless, this process is fast and proceeds with greatest efficiency. The energy source for this process is absolutely unclear. It cannot be the potential energy accumulated in the deformed DNA structure since whilst this might lead to expansion of the tightly coiled DNA, it would not result in its movement in a definite direction. The other possible source is the hydration energy that is released upon contact of the dehydrated DNA with water. We propose to investigate the energetics of this process and the role of water in this energetics by using microcalorimetric techniques.
The second part of the project concerned with the important role of laser irradiation and the packing and process of ejection of DNA from phage head and in the structural organization of viruses and naturaly in the process of infection of bacterial cells. These studies are intended to expand the investigations on the biological more realistic systems such as bacterial viruses. This super-molecular complexes should also be better models for the biological situation under which radiation is filtered by organelles and coats of protein before it hits the DNA. The such radiobiological investigations by means of laser beam are plan at first and including the problems of molecular biology, biophysics, virusology and biothechnology.
Under financial support of ISTC (the Project G-049) the high-power waveguide neodymium laser, radiating a series of giant pulses with frequency of following up to 100 KHz is created in laboratory of the Quantum electronics of the Tbilisi state university. The energy of giant pulses reaches a several GW. The flexibility of active elements enables to receive anyone beforehand specified distribution of a field of a laser radiation in a near zone. A large power of a radiation allowes to receive high harmonics, that is important for realization of researches on interaction of a laser radiation with biological objects and investigation of a kinetics of processes happening in presence of the bacterial phages. The application of fiber optics allowes to radiate the limited areas of bioobjects.
On the other hand, biological objects are hardly dispersing medium, that results in necessity to investigate processes of transfer of a laser radiation in a medium with allowance for repeated scattering on biological molecules. The methods of researching of process of transfer of a laser radiation in a hardly dispersing medium were developed by us in connection with studying of strong composite materials and dispersing aerosols.
The special interest represents study of a kinetics of DNA transition from a state of densely packed liquid crystal to state of a skein. These researches can be accomplished with the series of giant pulses, following one after another with a time interval of 10 mksec.
The importance of the investigation of physico-chemical effects of laser ionizing radiation on the genetic materials of mammalian cells is self-evident, but is not clear so far how to quantitative these effects. An excellent approach is via the characterization of the energetics of the helix-coil transition of the affected DNAs in the free state and inside the phages. Surpassingly the information on these highly significant reactions is utterly insufficient. Therefore laser-spectroscopic, heat capacity and hydrodynamic studies are not only timely, but also desirable. The best method to obtain quantitative energy parameters of biological polymers and complex biological systems as phages is by direct measurement of the temperature dependence of the partial specific heat capacity of the solution by high sensitivity microcalorimetry. Accurate determination of the heat capacity function of DNA and the phage suspension is extremely difficult, due to large heat capacity background of the aqueous solution. However, with the combined expertise of the participants of this proposal and the present day instrumentation there is an excellent chance to provide new solutions of the serious problem of low doses radiation damage including the double helix dynamic and relaxation properties.
The samples of various bacteriophages will be obtained from the Institute of Bacteriophages, Microbiology and Virology in Tbilisi. This is a very well known Research Institute that has an enormous collection of various bacteriophages. This Institute has a long-standing collaboration with Tbilisi State University and there will therefore be no problem in obtaining a broad range of various bacteriophages for the realization of the proposed project. We aim to start these experiments using the bacteriophage DDVI and Sd since we are well familiar with the thermal properties of DNA from this source.
The strategy of our proposal is based on the observation, that upon heating the bacteriophage it injects its DNA into solution at a quite specific temperature. This is a very intensive cooperative process that proceeds over a temperature range at about 48–70 °C, and results in significant increase in the viscosity and changes in optical properties of the solution due to unfolding of the tightly coiled DNA and the formation of its rigid double helical conformation. Upon further heating, the DNA duplex starts to unfold. The release of DNA from the capsid starts as a result of undefined changes in the proteins closing the outlet channel, but the capsid does not change noticeably at this temperature. Denaturation of proteins forming the capsid takes place at much higher temperatures.
We propose to measure calorimetrically the heat effects associated with the temperature-induced release of DNA from the phage capsid. Practically, this is not a simple experiment since the heat effects associated with injection of the DNA into solution should be small, whilst the process is associated with a very significant change in the viscosity of the phage solution. The result is that when the solution is heated at a constant rate, the sharp increase of viscosity can result in the change of the convection in the calorimetric cell and, consequently, the change of temperature gradients in the calorimetric cell. This might prevent measurements of the real heat effect of DNA release from the capsid. For such an experiment not only is a highly sensitive microcalorimeter needed but one which is insensitive to changes in the viscosity of the solution. Such an instrument has been developed by Privalov: this is the well-known DASM-4 which has been manufactured by the Russian Academy of Sciences. P.Privalov and coworkers recently built a scanning calorimeter of the next generation in the Biocalorimetry Centre at the Johns Hopkins University. Several DASM-4 instruments are in the Laboratory of Biophysics at the Department of Physics of Tbilisi University.
The experiments for studying the energetics of DNA release from the phage capsid consist of the following: the phage, in an appropriate solution, is placed in the cell of the scanning microcalorimeter and is heated at a constant rate. Parallel with the calorimetric experiment, the UV absorption and viscosity of the same solution are measured as a function of temperature. The UV absorption will be measured by spectrophotometer with automatic monitoring of the cell temperature. The viscosity of solution will be measured using a precise rotational viscometer, again with automatic temperature monitoring, which was designed in the Institute of Physics in Tbilisi. One such instrument is in the Laboratory of Biophysics of Tbilisi State University. The calorimetrically recorded temperature induced excess heat effects will be analyzed on the basis of spectrophotometric and viscosimetric information, which will permit the identification of the heat effect associated with DNA release from the phage capsid.
The novelty of investigation also including the determination of the water contribution to the energetics of DNA release from the phage capsid, is also based on calorimetric measurements. This method was first developed in Georgia with the active participation of Prof. Mrevlishvili. It is based on a determination of the amount of water bound by biological macromolecules, supramolecular structures or living tissues, by measuring calorimetrically the heat effect of freezing and subsequent melting of water in the aqueous solutions of biological molecules.
The Laboratory of Biophysics of Tbilisi State University has an adiabatic heat capacity calorimeter operating in the temperature range from -100 to 100 °C. This instrument was designed specifically for measurements of the thermal properties of aqueous solution and particularly the heats of freezing/melting of water in solutions of macromolecules. This is therefore probably the only place where such experiments could be carried out.
The new concept of bacterial viruses infection will be developed on the bases of study the fundamental physico-chemical properties (thermodynamical, reological and optical (Raman-spectroscopy) of bacterial suspensions, free genom material of viruses (ds-DNA) and protein coat of phages. It is the general problem of microbiology and molecular genetic of viruses and gave possibility developed the new strains of bacterial viruses with expected properties' as the vectors for biothchnology.
The results also gave possibility to design and developed therapeutic phage preparations useful in treating the current world-wide epidemic of antibiotic resistant infections in man, animals and plants.
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