**3. Exotoxins**

Exotoxins can be produced by gram positive and negative bacteria. A particular bacterium may secrete a one or more exotoxins. It also possesses a distinctive mechanism responsible for the stimulation of unique pathology, so the bacterial pathogenesis is related to specific exotoxin [10]. For example, *Corynebacterium diphtheriae* produces diphtheria toxins, whereas is being produced by *Vibrio cholera* produces cholera toxin which causes diphtheria and cholera respectively. Exotoxins differ in their cytotoxic potency as well as with respect to the host that can be intoxicated. For example an Exotoxin A (ETA) of *Pseudomonas aeruginosa* can intoxicates a number of species while some other exotoxins such as diphtheria toxin is more specie specific. Similarly, some bacterial toxins, for example pertusis toxin can intoxicate various types of cell, while some other toxins like clostridial neurotoxins intoxicates specific cells that are of neuronal origin [11].

## **3.1 Mechanism of action**

Exotoxins can consist of one or more than one polypeptides that act on various parts of the cells. Each exotoxin has a specific mode of action which causes a specific pathology. It also stimulate and involved in the specific chemical modifications in the host. These modifications may either start or stop the normal function of the target molecules in host cells to initiate a pathology [11]. Most important topic in this process is the need to plan framework that are useful in accessing cytoplasm while keeping the cell alive over the duration of examination. Such type of semi-unblemished cell framework can be maintained by treating cells with pore-shaping toxins. These are released as water solvent proteins and after addition in target host, attached to cell surface via receptors [12]. Exotoxins can be move across the eukaryotic membrane for intracellular activity. The needle like structure in gram-negative bacteria, (bacterial type II, III, or IV) injected their effector proteins via receptors. In some cases the phagocytic uptake of bacteria cells and receptors based endocytosis followed toxin secretion. Some unique receptors expressed during receptors based endocytosis that targets the special cells. When heteromeric toxin binds to the cell surfaces via receptors the process of endocytosis is initiated and involved in the translocation of effector protein through endosomal membrane to cytosol. As a result, toxin interact with the eukaryotic target protein that causes post-translational modifications in host. This modification responsible for causing inflammation and changes in cellular signaling cascades [13, 14].

In last decades, efforts have been made to discover the mechanism of action of various active toxins which are capable of inducing harsh symptoms in humans. The *Clostridium perfringens* toxin (α) is the first toxin having enzymes consist of phospholipase C activity at membrane surfaces [15]. Similarly, diphtheria toxin (DT) was firstly characterized having novel intracellular enzymatic activity [16, 17]. Hence, DT involved in the modification of EF-2 (elongation factor) by the process of ADP-ribosylation, which inhibits the process of synthesis and cell death. Among all, ADP- ribosylation was also identified as one of the active intracellular toxin. Various bacterial toxins involved in *An Overview of Bacterial Toxigenesis and a Potential Biological Weapon in Warfare DOI: http://dx.doi.org/10.5772/intechopen.114054*

the activation of enzymatic reactions can be utilized by other bacterial enzymes, such as protease glucosylase, RNase/DNase, with the intracellular target promoting effects [1].

Recent development in molecular field facilities the study of bacterial toxins in advanced level. Especially, complete genome analysis of bacterial toxins helps in studying their role and mechanism of action, proliferation and evolutionary process in bacterial cells. For example, previously it was assumed that the typhoid toxin was invaded in cells by *Salmonella typhi*, after genome sequencing and studying the their crystal structure revealed that that showed this toxin comprising two enzymatic subunits and five binding sites. It was also observed the typhoid toxin evolved from combination of several ancestral toxin genes that can be translocate to other bacteria, such as pertussis toxin and cytolethal distending toxins [18].

Additionally, bacterial toxins play an important role in various applied fields such as tools diagnosis, prevention process, and cure or therapy of bacterial toxin diseases. Now a days various diseases are diagnosed on the basis of toxin detection in environmental samples, like foods, water etc. In future other rapid and accurate in vitro methods such as mass spectrometry, ELISA, or fluorescent techniques will be used for detection of toxin [1].

#### **3.2 Classification**

Exotoxins are proteins that are soluble and released in the external medium by bacteria. Different protein molecules are being released by bacteria that help adhesion to or invasion of the host. Similarly, many others cause damage to host cells that may be physiological. Exotoxins differ in biological function, molecular structure, immunological properties and mechanism of secretion [19]. Bacterial toxins can be divided in various classes according to their nature and mechanism of action [20]. There are three classes of bacterial exotoxins on the basis of mode of action.

#### *3.2.1 Type I: cell surface-active*

Type I toxins adhere to a cell surface receptor and activate intracellular signaling mechanism. Its examples are as follow:

*Super antigens*; these are molecules produced by several bacteria. The common example is the *Staph aureus* and produces super antigens cause toxic shock syndrome in host [21].

*Heat-stable enterotoxins*; Some *Escherichia coli* (*E. coli*) strains produces heat stable enterotoxins. It is a small peptides capable to tolerate high temperature up to 100°C. Fe heat-stable enterotoxins also recognize the cell surface receptors and attached to it, therefore, affect different signaling mechanism intracellularly. The most common example is, STa enterotoxins attach and activate the membrane-bound guanylate cyclase that accumulate the cyclic GMP intracellular as a result effects on many downstream signaling pathways. As a result, water and electrolytes loss from intestinal cells.

#### *3.2.2 Type II: membrane-damaging toxins*

These are membrane disrupting toxins involved in the hemolytic or cytolytic activity. However, during infection the cell lysis induction may not be the key role of the exotoxins [21]. Membrane damaging toxins categorized into two main groups which are as follow;

*Channel forming toxins*; this type of toxins produced small pores in the host or target cell surfaces or membrane. These toxins are classified into two main classes such as cholesterol dependent and RTX toxins. The cholesterol dependent toxins extremely large sized pores i.e. 25–30 nm in diameter required the cholesterol for their action. These are released by type II secretion system [22] except pneumolysin that is secreted from the cytoplasmic region of *Streptococcus pneumonia* on bacteria lysis. While, RTX toxins comprised a unique tandem repeated nine amino acid residue sequence in protein. The prototype member of RTX toxin is haemolysin A of *E. coli*, RTX is also present in *Legionella pneumophila* [23].

*Enzymatically active toxins*; It includes *C. perfringens* (α) toxin having phospholipase activity and causes gas gangrene.

#### *3.2.3 Type III: intracellular targeting toxins*

These toxins are having a diverse virulence factors mainly comprised of covalent or non-covalent bound with the subunits (A and B). The A subunit having the enzymatic activity, while B subunits involved in the cell entry [22]. Type III exotoxins can be categorized by their mode of entry or heir mechanism of action inside the cell.

*By mode of entry*; these toxins directly access to the cytoplasm of target cells. Some bacteria transfer toxins directly released into host by a needle like structure. For example, the effectors proteins transferred by the type III secretion of Yersinia specie and another group of intracellular toxins is the AB toxins. B subunit binds to specific target regions on cell membrane. The subunit A is the active part and possesses the enzymatic reactions. It enters through the cell membrane and affects internal cellular bio mechanisms.

*By mechanism*; once exotoxins enter the cell via eukaryotic ribosomes (60S unit), and the most important difference between prokaryotes and eukaryotes is the structure of ribosome. Some exotoxins act directly at the ribosome to stop synthesis of protein (e.g. Shiga toxin) while some other act at elongation factor-2, e.g., diphtheria toxin, EF2 and Pseudomonas exotoxin. Some exotoxins not directly involved in the inhibition of protein synthesis for e.g., Cholera and Pertusis toxin.

#### **3.3 Structure**

Bacterial toxins are unconventional displayed in their structure. For example, different toxins may have similar structure and function but having diverse binding sites (Diphtheria toxin and Pseudomonas ExoA), or could have identical binding sites but different catalytic domains (Clostridial toxins) [24]. Basically, exotoxins composed of AB structure-function organization. A indicates the catalytic domain (effector) while the B is a receptor-binding domain and the translocation domain providing tropisim to specific cell types via receptors. The function of translocation domain is delivery of the catalytic A domain into an intracellular compartment of the host cell while B can have a single subunit or an oligomeric (B5) type. These two domains A and B may be associated by non-covalent interactions or linked by disulfide bond. A domain translocate the lipid bilayer through the channel or pore as made by the B [25]. Similarly, some toxins are multi-domain having complex proteins. The multi domin toxins interact with other protein considerably occupied a vast surface area as a result intra-molecular interactions having less space for interacting consequently compel toxins to be flexible in isolation. Additionally flexibility and secondary structure of bacterial toxins may have double-edged sword corelletion. The common example is Botulinum Neurotoxin,

## *An Overview of Bacterial Toxigenesis and a Potential Biological Weapon in Warfare DOI: http://dx.doi.org/10.5772/intechopen.114054*

(BoNT) having three domains; β-sheets (binding domin), α-helix (translocation domin) and α/β protein (catalytic domain) [26]. Alternatively, spider toxins, cry and three finger proteins having diversity in their structural and function resulted variations in secondary strutures of β-sheets and in their loops. It indicates a non linear relationship between flexibility and their secondary structure. The flexibility of any molecule is due variation in structure and dynamics [27].

The bacterial toxins having different types of virulence factors for e.g., the virulence factor of ADP-ribosylating exotoxins is related to enzymes involved in the ADP-ribosylation that includes diphtheria, cholera and *C. botulinum* (C2 and C3 toxin) [28].

## **3.4 Role of bacterial toxins in biological warfare or bioterrorism**

Biological warfare or Bioterrorism is a type of terrorism in which toxins are being used as biological weapons against crops, humans and animals. Diseases are the major effect of bioterrorism that contaminate the aquatic system, soil and food ultimately leads to death [29, 30]. These situations cause panic and fear on large scale publically, resulted in life and economic losses. Such conditions create inefficiencies in health care and emergency services [31–33].

Toxins are secreted by different microorganisms such as bacteria, fungi, and virus for their defensive purposes. These biomolecules cause deleterious effects on living matters by different ways like ingestion, absorption, inhalation and injection [34]. Many toxins are harmful for the nervous system, disintegrating the nerve impulses' conduction. While few toxins damaged the cell membranes which disturb or inhibit the cell function. They have irreversible effects that cause permanent damage to health [35–37]. Toxins have a significant role in the health and food sector causing food poisoning (e.g., staphylococcal enterotoxins) [34]. They are, however, extremely toxic due to their lethal doses. The lethal dose (LD50) is amount required to kill 50% of test animals (usually rats or mice). LD50 < 25 mg/kg represents very toxic; LD50 < 25 mg/kg to 200 mg/kg is toxic; LD50 < 200 mg/kg to 2000 mg/kg < LD50 is harmful, while substances of LD50 > 2000 mg/kg are not classified as toxic agents [38]. Human beings have been using biological toxins since long. In 1930s, Japanese Unit 731, in Manchuria used botulinum toxin as a biological weapon. While, United States developed mass-producing botulinum neurotoxin during World War II [39].

The reason behind the use of biological toxins in bioterrorism is due to simple culture technique and cheap and easily availability of extraction equipment's. Most of biological toxins disrupted the nerve transmission and affect the nervous systems, as a result the metabolic activities blocks and cell death occurred [30]. Moreover, there are psychological effects of bioterrorism such as long-term anxiety that may lead to panic attack, mass sociogenic illness, and widespread behaviors. Similarly, some anthrax spores are detected in mail envelopes causes disruption of the mail service, fear and destruction of US government to protect people [40]. Most potent biological toxins used in bioterrorism are Botulinum neurotoxins and Staphylococcal Enterotoxins. These two toxins contain high potential factors that cause disastrous diseases.
