How do spores help bacteria survive

The appearance of these proteins in the sporulating cell in the form of monomers occurs an hour before the start of exosporia biosynthesis [89]. Manetsberger et al. These proteins form part of the outermost layer of B.

It was revealed that these proteins are a key component of the basal layer of the B. However, the complexity of the biochemical structure of exosporia does not end there. Previous studies conducted by V. Thompson et al. Exosporium structural proteins determine its properties [45]. The outer surface of the basal layer consists mainly of BclA glycoprotein, which plays a major role in protecting spores from macrophages [84,87]. Recently, it was shown that this protein mediates the mechanism of immune inhibition [86,97], which experimentally contributed to the preservation of spores in the lungs of mice.

Enzymes that make up exospores, inosine hydrolase, and alanine racemase [85,88,94] can inhibit premature spore germination [98,99]. However, the main protein that determines the structure of endosporium a long time was unknown. Jiang et al. According to the authors, the same protein determines the hexagonal crystal structure of exosporium by disulfide binding of cysteine residues within individual ExsY polypeptide chains and its subunits [18,28,63].

These disulfide bonds underlie the ability of ExsY and its homolog CotY to self-assemble into an ordered crystal lattice of high symmetry for stable assembly of exosporia in all species of Bacillus spp.

As is known, adhesion is an important factor of virulence in these types of bacteria [8,9,17]. It would seem that the question of basic proteins, which determines the structure of exosporium, is closed, however, in P. They found that during sporulation, all analyzed C. Despite the lack of experimental evidence, the authors suggested that each morphotype apparently plays a different role in the pathogenesis of the infection associated with C.

It is noteworthy that, despite the ultrastructural differences, the outer fleecy layer was found in both exospore morphotypes [9,10,69]. The same group of researchers, using a gel-free approach for analysis of the exosporium layer and combined extraction methods, revealed the presence of proteins in the exosporium layer [10,70]. In this group of proteins, special attention was paid to collagenlike exospore BclA proteins, which form hair filament structures [69,70,73].

Given the fact that BclA2 and BclA3 are widely present in most strains of these bacteria, it is possible that vaccines developed on their basis can provide immune protection against clinically significant isolates.

The absence of these proteins reduces the pathogenicity of C. These proteins are structural and turned out to be highly immunogenic [32,64,,]. Experimental data indicate that they are of interest for immune biotechnologies as potential antigens for the development of new types of vaccines.

For example, immunization of mice with CdeM protein demonstrated an IgG-specific immune response []. Vaccination of hamsters with the CdeM protein of the recombinant strain C. This is striking given that the CdeM protein is unique to C. Further experiments, for example, with the inclusion of adjuvants in the composition of the CdeM vaccine in order to more quickly form a pronounced and long-lasting specific immune response, may increase its protective effect [,]. Immunization of mice with cysteine rich CdeC protein caused a significant IgG immune response after three immunizations with IP recombinant CdeC [].

It was found that CdeC is an immunogenic protein [3,64]. The Role of Exosporium in the Infectious Process: The main object of research on the role of exosporium in the infectious process was the anthropozoonotic causative agent of anthrax, B. When pathogens spores enter the host organism, they are absorbed by macrophages and dendritic cells, which initiate clinical manifestations and the form of the disease pulmonary, skin, or gastrointestinal [89,90,].

In any form of the disease, phagocytes migrate by the lymph, generating an infectious process. Spores germinate inside phagocytes and dendritic cells, multiply and produce toxins. In the lymph nodes, cells are lysed and vegetative forms of bacteria are released, followed by invasion of the bloodstream, active reproduction and the production of toxin, which mediates the clinical manifestations of the infection and leads to death.

The stage of spore formation occurs only in the soil, under conditions of sufficient access of oxygen, which is an inducer of sporulation [,]. Thus, the key link in the infectious process is the binding of pathogen exospores to integrin and their phagocytosis. Oliva et al. Bozue et al. It was shown that ramnose deoxysaccharide, which is part of BclA, binds to CD14 and acts as a co-receptor when interacting with integrin Mas-1, promoting phagocytosis [78,79,].

The data presented indicate the leading role of the exosporium BclA glycoprotein in the pathogenesis of anthrax. The use of modern analytical methods from the arsenal of the concept of molecular microbiology of single cells atomic force microscopy, Raman spectroscopy, genetics in the study of these structures is likely to become the key to a more complete understanding of the morphological, nanomechanical and biochemical characteristics of exospores [15,,].

Recently, one of the most dynamically developing and actively used in scientific research analytical technologies is scanning probe microscopy. One of its best-known tools is the atomic force microscope, invented in as a scanning tunnel profilometer [Binning, ]. Since the 90s of the last century, Atomic Force Microscopy AFM has become most actively used in biomedical research, thanks to a simple sample preparation procedure and the possibility of submicron visualization of biological objects [30,].

For a short time, AFM as a method of three-dimensional visualization and research of local micromechanical properties has won leading positions in many fields of science, including microbiological studies, [30,,]. This analytical tool supplemented and expanded the capabilities of the main methods of visualization of microbiologists — light microscopy goes beyond its technical resolution , as well as electron microscopy, the main drawback of which is the complexity of preparation of preparations and the need for research in high vacuum [,,].

The attractiveness of using AFM in microbiology is associated with some specific technical features of this diagnostic method, based not on the properties of the lenses, but on the use of a special probe sensor that analyzes the surface of the sample using a needle on a thin and flexible elastic cantilever installed in the holder Figure 3.

This visualization tool combines microscopy in the usual sense with nanomolecular detection of the mechanical, immunochemical, adhesive and electrostatic properties of an object bacteria.

These capabilities have made AFM an extremely useful and indispensable tool of molecular microbiology, and prokaryotic cells, due to their size and properties, have become a favorable object for research [,,]. As a result of scanning, the AFM presents a digital three-dimensional topographic image of the surface of microorganisms in nanometer lateral and spatial resolution in any medium and at different temperatures [,].

In a short period of time, AFM has evolved from a topographic imaging method to an instrument for manipulating individual atoms and studying intermolecular interactions. Figure 3: The AFM optical imaging system consists of a laser beam directed to the reflecting surface of the console and a 4-section photodiode that fixes the vertical and lateral displacements of the probe A. In the process of scanning the spores of B. A computer converts information from a piezoscanner into a 3-dimensional image drawing and photo — authors.

Periodic atomic-force observations of bacterial endospores make it possible to track the dynamic processes associated with sporulation or germination in vegetative cells. Essential for microbiological studies of bacteria spores is the possibility of use of AFM in several modes. In this case, the force of interaction of the needle with the surface of the sample strongly depends on the distance between them and is determined by adhesion, as well as by van der Waals and capillary interactions.

The Z-position of the console reflects the topography height of the sample according to the type of geographic map, where the color of the image corresponds to the height of the relief [,,].

Being a method of force sensing, the AFM makes it possible to obtain not only an image of the surface topography and multilayer architecture of spores, but also their nanomechanical characteristics such as elasticity, viscosity, and adhesion [15, ]. For example, studies by R. Giorno et al. Chada et al. Zolock and his colleagues showed that, based on these morphological features, four closely related species of Bacillus spores can be differentiated [].

Visualization of spores in a fluid opens up wide prospects for monitoring dynamic processes, such as sporulation and germination of dormant cell forms. Interesting recent examples are studies of the dynamics of the germination of spores of Bacillus spp.

After 30 min of incubation at room temperature, an increase swelling in the size of the spores was observed, and after 5—6 hours, all spores grew into vegetative cells, which immediately formed a biofilm [,]. Li et al. Morisaku et al. Using a thermal probe AFM Thermal Scanning Microscopy, SThM , they developed a method for nanosurgical cutting of spores using a hard diamond tip, and a soft probe was used to visualize and characterize its internal structures on a nanometer scale.

It was found that the elasticity and adhesion indicators at elevated temperatures significantly varied in different areas of the spore cross section [], and a previously unknown peptidoglycan ultrastructure of the B. Over the nearly year history of the use of atomic force microscopy in molecular microbiology, it has become a powerful research tool in the study of spores, effectively complementing light microscopy, genetics, and biochemical methods traditionally used to analyze the structure of bacterial spores.

However, the potential use of this method in the study of bacterial dormant cell forms is sometimes underestimated and limited only by some of its functional capabilities [,].

Further use of AFM is associated with the use of dynamic multi-frequency, multi-harmonic, bimodal methods, which allow one to obtain faster, quantitative nanomechanical characteristics of a complex multilayer spore structure in higher resolution [,]. In addition, it is interesting and promising to develop and use AFM biosensors for the quantitative visualization of individual molecules of structural proteins of endospores and exosporium using G-quadruplex DNA technologies or functionalized probes with individual chemical groups or single molecules immobilized on them, especially in combination with other analytical instruments, such as Raman spectroscopy [].

The development of highly sensitive and rapid methods for the detection of bacterial spores is an urgent problem for medical diagnostics, epidemiology, and also solving biosafety problems. The public outcry caused by the bioterrorist attack in , which used B. The material needed to be germinated in nutrient media to vegetative cell forms, biomass accumulation, and, finally, molecular genetic studies to identify the 16S rRNA gene specific for this type of pathogen.

In addition, similar hours-long studies with other suspicious powders were required to determine the scale of the terror attack [79,,]. As a result, awareness has come of the need to search for new informative and much faster detection methods of infectious agents. Early information on the identification of a pathogen could accelerate the onset of specific prophylaxis, etiopathogenetic treatment and minimize the tragic consequences of the attack associated with contamination and death of people [29,,].

The problem of quick identification is complicated by a number of aspects related to the minimum amount of test material — inhaling only endospores of B.

Moreover, it is necessary to differentiate a pathogenic material from a similar but harmless to eliminate of panic fear that accompanies epidemic outbreaks of dangerous infections [99,,]. Therefore, the new method of indication should be not only fast, but also highly specific, reliably differentiate dangerous bacterial endospores from other biological objects, in any environmental objects. Another method, freeze-drying, involves drying the microorganisms under a vacuum.

This preserves most bacteria for many years. Researchers do not completely understand the mechanisms that contribute to cell survival under adverse conditions. But maintaining the proteins of the cell in an active form is clearly critical to survival. Keeping a very low level of water inside the cell appears to be likewise essential to long-term survival.

Spores, endospores, cysts and desiccated cells all have low water content. Freezing itself does not usually harm cellular components.

Ice crystals, however, are lethal to living cells. Therefore, removing waterespecially while the cells are cold, as is done in freeze-dryingwill usually keep the proteins active.

Many bacteria can also be stored frozen at ultracold degrees Celsius temperaturesthe secret is to add a substance like glycerol that prevents the formation of ice crystals. Although scientists do know something about the survival mechanisms of organisms that have been studied for many years, we still have a long way to go.

Indeed, for the vast number of bacteria that cannot be cultured in the laboratoryincluding many that live in microbial communities, which may offer them some protectionwe know very little about the means by which they persist in the often hostile microbial world. Sign up for our email newsletter.

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