Introduction
Bacteria are the very first life forms in the history of our planet. Since the beginning they were exposed to extreme changes of temperature, pressure, pH and nutrients; hence, to survive, they had to adapt themselves. One strategy of survival is to go into suspension or dormancy, by converting themselves into a seed form or spores. When conditions become normal, these spores germinate back to vegetative form. This property is also seen in pathogenic bacteria that infect higher animals such as humans. One such group of bacteria is from the Genus Bacillus.When exposed to stress, these bacteria go into dormancy by converting themselves to spores. These microorganisms produce a toxin during their vegetative cycle, which is fatal to humans. Due to its potent toxicity, it is also used in biological weapons (Mock, Fouet, 647). In this article, we will review the various protein structures of the spore, the genes responsible and their use in developing better vaccines to protect people from diseases.
Bacillus anthracis spore
The most widely studied spores are from B. subtilis and B. anthracis. In a spore, the DNA with its protective proteins known as small acidsoluble spore proteins (SASPs) makes the core. It is usually surrounded by layer of peptidoglycan known as cortex (Dowd, Orsburn & Popham 4541). The cortex is in turn covered by a coat – a multilayered protein shell, which forms the outermost covering of the spore. In some species of Bacillus, such as B. anthracis the whole spore is again engulfed in a layer called exosporium. The thickness of the exosporium differs among species; B megaterium has the thickest. The exosporium also has hair-like projections extending away from the exosporium. The space between the exosporium and the coat is called interspace and it is quite significant in the spore of B. anthracis. The exosporium plays a very important function in the adhesion of the bacteria to its host cells and also protects it from the host’s innate immune response. Macrophages interact with the spore via cell-surface integrin Mac-1 and the exosporial protein BclA.
There are several proteins and carbohydrate structures in the spore’sexosporium. BclA is the major structural protein component, especially in the hair-like projections, of the exosporium. Other proteins residing the exosporium include ExsFA or BxpB, its paralogueExsFB, CotY and ExsY. (Steichen, Kearney & Turnbough 5868). It also has orthologues enzymes like alanine racemase (AlR) and inosine hydrolase (IunH). The protein required for the assembly of the exosporium is CotE whereasBclB provides stability to it.
Exosporium protein ExsFA/ExsFB
Rebecca Giorno suggests that the exosprium assembly starts by deposition of a piece of basal layer close to the midpoint of the mother cell, which is directed by CotE. Then, the expansion of the exosporium, directed by ExsY, finishes the rest of the exosporium. BclA, the primary protein in the spore exosporium is assembled in anExsFA/BxpB dependent and independent manner but, ExsFA/BxpB is necessary forthe formation of BclA-projections. The main question that arises, is, that whether ExsFA/BxpB is necessary for causing infection in the B. anthracis host?When guinea pigs were infected with exsFA/bxpB mutant spores via intra-muscular route or by inhalation, it resulted in mortality similar to that of a wild-type spore strain. Thus, it is clear that defects in germination or morphology of the exosporium do not change the disease causing capacity of the spores. In addition, these results are consistent with other experiments in animal models of infection, where spores without BclA from the Sterne and wild-type strain were found to be equally virulent. Also, spores containing a mutation in the cotE gene showed similar virulent characters as wild-type spores (Giorno et al., 691). This observation suggests that the BclA-projections do not play a vital role in causing disease and that while developing new vaccines, ExsFA/BxpB should not be the only target, although they can be one of the subunits. The formation of B. anthracis spore is probably needed for its survival outside the host body. Thus, the function of BclA-projections lay in its survival under stressed conditions.More definitive studies have to be conducted to show the exact role of the exosporium and the various proteins that belong to the interspace.
The Cortex Protein SoaA
In the situation where conditions are right for the B. anthracis spore to germinate into vegetative bacilli, the inner membrane of the spore expands first, as much as twofold,with hydrolysis of the cortex and then there is activation of the metabolic machinery. Cote et al., in 2008 showed that a novel protein, SoaA, associated with the spore was found to be released during germination that affected the spore’sinitial interaction with macrophage.The SoaA protein is located in the area of cortex below the spore-coat and is incorporated in the forespore by the mother cell early in spore assembly. Cote et al., also demonstrate that SoaA protein is not important in resistance of the spore to extreme stress conditions such as temperature or pH.It has been demonstrated from time to time that both toxin and spore proteins have a protective role in the immune response to an B. anthracis infection (Enkhtuya et al., 3103). Opsonization is a critical step for an effective immune response and whether SoaA contributes significantly towards it, is the question.
The antigen presenting part of SoaA is a 6 kDa peptide, hydrophilic region which is exposed to the surface and readily detected by the IgG. Ungerminated spores which were gamma-irradiated also showed attraction towards this protein, suggesting its presence in the ungerminated stage along with BclA. The major protein in the wild-type spore against which vaccines are developed is the exosporium protein BclA (Steichen, Chen, Kearney & Turnbough 1903). Now, SoaA can alter the opsonic properties of BclA suggested by experiments with mutant spores. An allelicexchange mutation was made in the bclA gene giving it resistance to kanamycin - bclA : : Kan mutant strain of B. anthracis. When compared to wild-type spores, the bclA : : Kan mutant spores did not show opsonization against polyclonal antibodies IgG directed towardsBclA. Moreover, Similar results were obtained when mutation in the soaA gene produced soaA : : Kan mutant strain even though BclA was present in the spores. Thus, both SoaA and BclA contribute to the opsonization with polyclonal antibodies.
Vaccines are generally given to sensitize the immune system to the infection so that in the event of a real life exposure, the immune response launches a timely attack.There is a growing debate whether passive protection is efficacious against infection (Goossens, Sylvestre & Mock 301). In one study, anti-spore antibodies do not protect from infection, whereas Enkhtuya et al., showed that rabbit antibodies significantly protected the mice from an infection. To see whether passive immunization protects the host, mice were injected with rabbit IgG directed against irradiated spores or SoaA intraperitoneally. Cote et al showed that this passive immunization did protect the mice against infection. Overall, SoaA protein seems to a good target for vaccine development.
The Coat Protein BxpA
There are several coat proteins in B. anthracis, but the assembly and the cross linking of these proteins is not well understood. A better understanding of the mechanism can throw more light on the structure of the spore and use of such proteins give better vaccine quality. There are two main types of genes which control the assembly and cross linking of the coat proteins. Most of the proteolytic processing in controlled by yabG and some of the cross linking is controlled by transglutaminetgl. BxpA is one of the many proteins which is processed and assembled in the spore coat (Liu et al., 164). The BxpA is closely associated with the coat, is located below it and depends on YabG and Tgl for its modification. In Ames and Sterne strains of B. anthracis, when analyzing the proteins of the coat and exosporium by 12% Bis-Tris gel method of extraction, there was a 9 kDaband found in the Sterne strain. It was later identified as an exosporium component BxpA. To find out if bxpA gene encodes this protein in the Sterne strain only, an allelic mutant was developed in both Ames and Sterne strains. The western blot results revealed that the 9 kDa band appeared only in Sterne spores. However, BxpA is detected in Ames strains also as 11 and 14 kDa bands. To know whether BxpA is produced during spore formation or germination process, extracts from the Ames strain vegetative bacilli and purified spores were analyzed for BxpA. Vegetative bacilli did not show the BxpA band, thus, BxpA is synthesized and assembled during spore formation only. The size of BxpA detected in Ames and Sterne strains is higher – 27 and 29 kDa respectively, than the actual size which migrates to the coat (Steichen, Chen, Kearney & Turnbough 1903). To find out the possible reason and to see whether YabG has to do anything with it, Moddy et al., knocked out the yabG gene in Ames and Sterne strain that coded for coat proteases. The analysis of a western blot using anti-BxpA antibodies revealed that the bands with lower molecular weight reduced and that of higher molecular weight increased. Thus, it confirmed that YabG plays the role of a protease in processing and maturation of BxpA. The protease activity of YabG cleaves BxpA directly like trypsin. If it is so then the cleavage will require arginine residue in BxpA. This was true as the results of a western blot from spore extracts in which a plasmid containing bxpA 133R alanine was introduced showed more higher-molecular weight bands and less bands relating to mature BxpA.
Further to migration and maturation of the BxpA to the coat, it is also imperative to see what mediates the cross-linking of the same. Analysis of yabG showed that BxpA was present in multiple forms, some larger than the full-length protein. To explore whether BxpA is cross-linked by tgl gene product, it was inactivated in one of the study. When the BxpA mass was determined by western blot analysis, both bands corresponding to the native BxpA was detected as well as some higher molecular weight bands were also seen. Thus, this experiment does not conclusively say that Tgl is responsible for the cross-linking of BxpA, but as least it play some role in BxpA maturation and spore formation.
Discussion
Bacillus anthracis is a sporulating gram positive bacterium which causes the lethal disease anthrax in animals and humans. Once the spores get into the host, they germinate in to a vegetative form and release various toxins. The currently approved vaccine against anthrax is Biothrax – aluminum adsorbed supernatant protective antigens from a toxigenic strain of B. anthracis. But, due to resistance to antibiotics and genetic modifications, developing advanced vaccines against the anthrax bacteria have become ever more important. In order to discover novel vaccines, a deeper understanding of the spore biology is needed. The composition and structure of the spores reveal new avenues for vaccine development. Review of the current literature on spore proteins is one attempt to uncover such possibility. Various proteins from different parts of the spores are evaluated in this review to check their use in vaccine development.
Rebecca Giorno et al studied the ExsFA/ExsFB, the protein found in the exosporiumof B. anthracis that is responsible for BclA and hair-like projections. They studied the effect of ExsFA/ExsFB mutant spores in a guinea pig model of infection in comparison to wild-type spores. There was not change in the mortality pattern of the guinea pigs, suggesting that BclA is not critical for the spores to present themselves to the immune system. Thus antibodies against ExsFA/ExsFB are not a strong candidate for more advanced and effective vaccine development, although it can be used in a multi-unit vaccine.
Cote et al., studied the opsonizing protein SoaA, found in the cortex of the spore. They created mutation in the soaA with resistance to kanamycin (SoaA :: Kan) and studied them against wild-type Ames spores. The most intriguing results came from the studies using this mutant strain in the guinea pig animal model of infection. Independently, both mutant and wild-type strains caused infection and mortality in guinea pigs, but when mixed together, wild-type spores seemed to survive more efficiently. The explanation for such a behavior is possibly because the mutant strain germinates faster than the wild-type, exposing them more rapidly to the innate immune system. Further studies are required to probe into the biology of SoaA protein to make it a worthwhile candidate for vaccine development.
The third protein studied is the coat protein BxpA. To check the role of this protein in the virulence of B. anthracis, the mutant BxpA spores and wild-type Ames spores were injected in guinea pig and mice. The mortality pattern in mutant strain was similar to the wild-type strain suggesting that BxpA does not play a major role in virulence of the bacteria. Taken together, only the SoaA protein has potential to be studied extensively for developing an advanced vaccine which can prevent the disease and have minimum side effects.
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