An Overview of Bacterial Toxigenesis and a Potential Biological Weapon in Warfare

Abstract

Various bacterial cells releases proteinous compound known as toxins. These toxins damage or inhibit the physiological and cellular function of the healthy human cells as a result it can causes a lethal disease or death. Generally the bacterial toxins are classified according to mode of action at molecular level and the mechanism of targeting cells or organs. Some toxins are released within the bacteria called endotoxin and other secretes outside the cells delivered by gram positive and negative bacteria. These toxins encoded by bacterial genes, chromosome, phages or plasmids. The bacterial toxins such as botulinum, conotoxins, Clostridium perfrigens, epsilson toxin, saxitoxins, shigatoxins, tetrodotoxins, can be used in bioterrorism due to high toxicity and short incubation time. The promising role of nanomaterial’s in the detection of bacterial toxins have been summarized highlighting their advantages, need principles, and limitations in terms of sensitivity, accuracy, simplicity, sensitivity, cost effectiveness and multiplexing capability.

1. Introduction

The soluble protein antigen released by pathogenic gram positive and negative bacterial involved in the pathogenicity is known as toxins. These toxins involved in the balancing of cell capacity, targeting the specific host cell and modify their functions. The bacterial toxins are generally classified into two groups endotoxins and exotoxins. The endotoxins is a lipopolysaccride, an important constituent of outer membrane of gram negative bacteria [1]. Exotoxins are polypeptide diffusible proteins that are located on extra chromosomal genes. They are usually secreted by living bacterial species but also released on bacterial lysis and act locally or at far away from colonization site. They may enter the membrane at the cell surface to cause injury or damage and also involved in the binding to cell receptors or to facilitate/ interact with other bacterial cell types [2]. Bacterial toxins cause microbial infection by damaging or inhibiting the cellular mechanism in host. In this way bacterial toxins take over the control of vital processes of living organisms [3]. They are extremely diverse in their size ranges more or less from 15 to 2700 amino acids, secretion types, structure (mono, binary, ternary), enzymatic activity, and the receptor recognition or binding sites [1].

2. Endotoxin

The endotoxin is integral part of outer membrane in gram negative cell wall. The gram negative bacteria consists of the inner membrane, outer membrane and peptidoglycan layer. The inner membrane is permeable layer while outer membrane consists of lipopolysaccride layer and generally protects the cell from outer stress. So endotoxins are associated with the lipopolysaccharide (LPS). Generally endotoxin structure consist of three parts i.e., lipid A component made up of “disaccharide phosphates and fatty acids,” an O-specific polysaccharide side chain (O-antigen), and a core polysaccharide chain” weighing approximately 10 kDa (Figure 1). The core polysaccharide added to lipid A sequentially during the formation of the lipid polysaccharide chain. The O-antigen’s subunits are attached at the end of polysaccharide chain. But the most harmful’ harmful part is lipid A [4]. Various gram negative bacterial species having different endotoxin structure. The different in structure is due to composition of the O-antigen or Lipid A component, bacterial strains as well as environmental conditions. The O-antigen involved in the antigensity and Lipid A determines the toxicity and inflammation by detecting the myeloid differentiation factor II and toll IV receptors (MD2/TLR4) [5]. Endotoxins are heat stable, negatively charged and hydrophobic in nature. They also form aggregate in the form of vesicles or micelles with size ranges about 10–20 kDa in monomer) and more than 1000 kDa in vesicles [6].

Figure 1.

Structure of endotoxin.

2.1 Endotoxin toxicity

Endotoxins enter into living organisms via blood cells and causes several diseases including respiratory syndrome, fever, hypertension, intravascular coagulation and other related shock. It also causes sepsis, however the main mechanism of endotoxin mediated sepsis is still not clear. It depends on some of the genetic factor, the infection site, virulence factor, and the host response. The high concentration of enodotoxin involved in the activation of toll like receptors (TLR4), caspases and RAGE, that causes septic shock and ultimately death while low concentration causes inflammation and other less severe symptoms like rashes, fever etc. In the septic patient the endotoxin concentration (2.5–12 EU/mL) were detected in plasma. Similarly, meningococcal patient admitted in hosptital for septic shock having endotoxin 5 EU/mL level in their blood [7].

2.2 Mechanism of action of endotoxin

Endotoxin involved in the activation of inflammatory genes by activating the TLR4 receptors along with their MD2 co-receptor, resulting the activation of NF-κB transcription with cytokines (TNFα, IL-6 and pro-IL-1β). The Lipopolysaccharide binding plasma protein transfers to membrane-bound CD14, as a result LPS transfer to TLR4. The intracellular lipopolysaccharide directly involved in the activation of murine caspase-4, 5 and 11 in humans, which in turn cleavage and activate caspase-1 and then to cleave pro-IL-1β. The activation of caspase-1 and 11 can cleave and activate the gasdermin D. This process allows the IL-1β to get out from plasma membrane and cell death by pyroptosis [7]. The other recognition receptors are TREM2 RAGE, the macrophage scavenger receptors and the β2 integrins (CD11a/CD18, CD11b/ CD18 and CD11c/CD18) may involve in the clearing of LPS and bacterial LPS in tissues and blood. This promotes the inflammation and toxicity (Figure 2) [8].

Figure 2.

Mechanism of action of endotoxin.

2.3 Endotoxin entry in human body

The huge amount of negative bacteria present in the gut of the body system. In sometime the gut edge leak or break down as a result gram negative bacteria and their endotoxin directly enter into the blood streams. This translocation is major reason for septic shock in seriously ill patients. This is known as endogenous endotoxin. Other way of entry is bacterial infection due to cyst and catheter or wound infection. The external source of endotoxin includes the polluted fluids or equipment. This is known as exogenous endotoxinsGram negative bacteria causes infection in intravenous infusion fluids resulted septicemia. Along with theses glassware used in IV therapy, cardiopulmonary bypass and dialysis equipment’s also involved the endotoxin shock. After entry it causes pyrogen or can activate a number of signaling pathways of immuno competent cells that can cause to either swelling or programmed cell expire (apoptosis) of this cells. They are biologically essential even in the small amount [9].

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 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, (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, 32, 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, 36, 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.

3.4.1 Botulinum neurotoxins

This neurotoxin is produced by anaerobic, spore forming, gram-positive bacteria belong to genus Clostridium having more than 150 bacterial species. Botulinum neutoxin (BoNTs) consist of 7 serotypes (A-G) and possess more than 40 subtypes. Botulism disease is caused by Botulinum neurotoxin and A, B, C, E and F serotypes are responsible for causing this disease in human beings. In 2013 a new toxin was reported in human infant. This toxin was known as BoNT/H, BoNT/FA and BoNT/HA produces by bivalent Clostridium botulinum bacterial strain. Genetically it was analyzed that it has a toxin genes BoNT/H shares ≈ 84% resemblance with BoNT/A1 in its binding site, ≈ 80% with BoNT/F5 in its catalytic domain and exhibits a translocation domain identical to BoNT/F1. Further analyses revealed that this BoNT/H toxin neutralizes by available antisera. Though, its unusual toxicological effects, shows lower potency and development of botulism symptoms as compared to primary BoNTs. Various studies describe that there are some resemblances between BoNT/H (i.e., BoNT/FA or BoNT/HA) and BoNT/F or BoNT/A and this creates unanimity.about the position of the BoNT/H [41, 42].

The diversity of Botulinum neurotoxins was further explored by the using modern techniques such as sequencing and genomic analysis, bioinformatics and data-mining tools. BoNTs and its genes have been reported in Enterococcus feacium or Weisseria oryzae. These bacterial strains can produce BoNT toxins that depict same characteristics in the multidomain organization of BoNTs but with different toxicological features, specificity or operation principles [42]. BoNT/X is the first serotype of BoNTs identified by bioinformatics techniques and genomic sequencing. Toxin was identified in a Clostridium botulinum strain, which displayed the typical BoNTs characteristics including the residues forming a ganglioside binding pocket and metalloprotease consensus sequence or interchain disulphide bond. A significant trait of BoNT/X is its ability to cleave VAMP4 and atypical SNARE Ykt6. A new serotype, BoNT/X, BoNT/En and Weissella oryzae BoNT-like toxin (also known as BoNT/Wo) have been discovered in recent years. The bioinformatics analysis of bacterium Weisella oryzae has led to the recognition of an open reading frame 1, which has a strong sequence resemblance with both genes, but that does not contain the additional genes usually associated within the locus in Clostridia. The sequence similarity between BoNT/Wo and other BoNTs is ~14–16%. Moreover, the two cysteines that are part of essential inter-chain disulfide bond in BoNTs are not conserved in BoNT/Wo, depicting a different mode of action. A novel BoNT-like gene was identified in the genome of Enterococcus faecium. This BoNT called as BoNT/En composed of distinctive botulinum neurotoxins domain architecture, disulfide bond residues and conservation of the zinc peptidase HExxH motif. BoNT/En describes 29–38.7% similarity with the other BoNTs [43, 44].

Botulinum toxin mainly composed of metalloproteins (zinc-dependent endopeptidases). This toxin having single polypeptides cut with the help of clostridial or host proteases to its active form. The C-terminal heavy chain constitutes the binding and translocation domains of 50 kDa each, and is joined by a single disulfide bridge to the catalytic light chain making the N-terminal part [45]. This protein possesses three domains responsible for its biological functions. The binding domain is responsible for combining the toxin to the receptor on the surface of the cell membrane of the target the nerve cells. The translocation domain is responsible for the transport of toxin through the plasma membrane into the cell, while the enzymatic domain (with proteolytic activity) causes the detachment of peptide bonds in the SNARE transmembrane protein (SNAP-25, VAMP-1/2, syntaxin-1/2) that are integral to vesicular trafficking and neurotransmitters release. Depending on the type of toxin, the proteolytic effect is directed at different SNARE proteins. BoNT A and BoNT E cause hydrolysis SNAP-25 membrane protein. Type B, F and G carry out hydrolysis VAMP-1/2, while BoNT C conducts hydrolysis SNAP 25 and syntaxin-1/2 [46, 47]. The procedure of the toxic action of botulin toxin stops the release of neurotransmitters, including acetylcholine, within neuromuscular junctions, ultimately causing relaxation and paralysis of skeletal muscles. The first symptoms appear within a few to 36 h of poisoning with the toxin. Irrespective of the type, there are similar clinical indications of poisoning irrespective of the type. Primarily, the symptoms of speech and swallowing difficulties, double and blurred vision, anxiety, lack of saliva and tears appear followed by a loss of control over the body, and atrophy of the throat reflex. Afterwards, respiratory muscle paralysis lead to respiratory failure, and this is considered as main cause of death of infected patients [48, 49]. Botulinum toxin is considered the most toxic substance in the known world as the estimated mice LD50 for parenteral administration is 1 ng/kg [50]. It is the only biological toxin, that has been classified by the CDC in Atlanta as a Category A bio agent and the lethal dose for a human weighing about 70 kg is 0.7–0.9 μg of inhaled toxin, or 70 μg of poison ingested with food. This toxin is not resistant to chemical and physical agents. It is degraded at 85°C in 5 min, and is destroyed by sunlight within 1–3 h. Furthermore, it is instantly depurated by chloride or H₂O₂ [51]. Botulism is treated based on the quickest administration of botulinum antitoxin, preferably 24 h after the first symptoms, because antitoxin neutralizes only toxin molecules that are not yet attached with nerve endings. The antitoxin can cause the adverse reactions as well, such as post-surgical disease, anaphylaxis and hypersensitivity. Moreover, many cases require mechanically assisted respiration and supportive treatment (with a return to self-reliance in up to 2–3 months), [49, 52, 53]. There are two approaches that are being used for the development of vaccines against botulism. In first approach, a native BoNT is being used to develop chemically-inactivated toxoid and the second is using recombinant techniques to produce BoNT derivates [54]. During a bioterrorist attack, botulinum toxin may be deployed in aerosol form or by contaminating water and food during a bioterrorist attack. The efficiency of the neurotoxin is equally high, regardless of route of entrance, due to having almost similar disease symptoms. A bioterrorist attack with botulinum neurotoxin is difficult to identify and only an increase in the number of victims with symptoms of toxin poisoning could show its use in a bioterrorist attack [55, 56].

3.4.2 Staphylococcal enterotoxins (SEs)

These toxins are released by pathogenic bacterial strain that are responsible for food spoilage or poisoning. These are classified as staphylococcal enterotoxins and cholera toxins (AB5 group enterotoxins) Shiga toxins and heat-labile toxins (Escherichia coli) [57]. But the most significant among all enterotoxin is Staphylococcus aureus enterotoxins (SEs). SEs are known as super antigen, capable to activate T lymphocytes (~20–30%), as a result overproduction of cytokines is occurred, which are responsible for inflammation [58]. Staphylococcus aureus, is widely spread and rapidly growing toxin producing bacterium. In humans this bacterium is colonizes in the throat, nasal cavity, and crotch/anus of human body and usually resistant to antibiotics. It is also present in meat and dairy products [59, 60].

SEs having mass of 26–35 kDa proteins, containing a single polypeptide chains. Two unequal size domains are mainly composed of alpha (α) helices and beta |(β) strand separated by a deep shallow groove and having complex tertiary structure. These SEs proteins are highly solubilized in both salt and water. It is also resistant to proteolytic enzymes including papain pepsin, rennin and trypsin, thus remain unchanged in the gastrointestinal tract. These toxins are also resistant to dehydration, gamma radiation, and a wide pH range (2 < pH < 12) [30, 61, 62]. SEs gene coding region is present on plasmids, prophages, S. aureus pathogenicity islands and genomic island vSa, (SaPIs) [62]. These Super antigens are categorized in class II Major Histocompatibility Complex (MHC) molecules but the binding groove of MHC peptide-antigen is not involved. The SEs has four types A,B,C and D. A and D usually involved in natural food poisoning rarely by the B and C types [63, 64]. The most potent and lethal SEs enterotoxin is type B (SEB) and used in bioterrorism so termed as biological weapon. It is highly thermo stable heat resistant toxins and is detected even in sterilized food. In human it also cause toxic shock syndrome and severe food poisoning that leads to dehydration and ultimately death occur. If this toxin id enter into the body other via food or through inhalation, induces a septic response throughout the living organism. The toxicity levels for this type of poisoning are LD50 20 μg/kg and ED50 400 ng/kg [61, 65, 66].

4. Emergence of nanotechnology in bacterial pathogenesis

Nanotechnoly is an emerging field to develop reliable, accurate, cost effective diagnostic method for the detection of infectious agent. In this technology Nano scale particles (1–100 nm) having some novel characteristics such as small size, wide surface area along with some unique structural, electromagnetic, thermal, optical properties. Toxins are not transmitted among the humans as the bacterial infections, but their adverse effect on cell, tissue and other organs urges the scientist to develop the fast, reliable and accurate method of toxin detection. Moreover these toxin persist in environment long time after the death of pathogenic microorganism. Therefor the detection of toxins in the environmental samples are highly significant in the process of intoxication during the pandemic. Various methods such as ELISA, antibody microarrays, antibody-coated polystyrene microbeads, and western blotting etc. used for detection of toxin although these methods are reliable and sensitive but required purified and homogenous samples. Along with these another limitation is the determination of toxins via spectrophotometer or flurometer. To combat this limitation, LC MS liquid chromatography mass spectrometry and MudPIT (multidimensional protein identification), have been used. However, these are highly sophisticated and not user friendly so mostly avoided the broader use of these detection methods [67, 68].

The gold and silver are metallic nanoparticles having electronic and optical properties, coupled with ligands used in chemical sensors. Other gold nanparticles coupled with oligonucleotides used in the detection of cDNA strand [69]. Similarly carbon nanotubes and fluorescent quantum dots widely applied in detection of pathogenic strains, toxins and also detect DNA. Cholera toxins have been detected through molecular mimicry. In this method, the nanoparticles resembled the extracellular matrix of GM1 ganglioside at its terminal portion extracellular matrix which are present in the apex of intestinal epithelial cells and tissues. The UV spectrometer is generally used to detect and quantify the toxins concentration via nanoparticles. The nanoparticles suspension color changes from red to purple within 10 min having the detection limit up to 3 μg/mL. Similarly shiga toxin is also detected by modifying gold nanoparticles and molecular mimicry method. Gold nanoparticles are coupled to globotriose and shiga toxins are interact with these carbohydrate portion of modified nanoparticles that mimicked globotriaosylceramide (Gb3). This Gb3 is present on the renal epithelia and intestinal microvilli [70]. Toxins are recognized by the modified nanoparticles as a result absorption profile is changed, hence these nanoparticles potentially used in biorecognition methods [71].

5. Conclusion

Toxins are polypeptide diffusible proteins that are released by bacterial species that cause diseases due to drastic interactions of pathogen and host cell. Toxins may enter the cell membrane surfaces by damaging it and facilitate the interaction with other cells. Toxins are diverse in their size, mode of secretion, mechanism of action, structure, enzymatic activity, membrane/intracellular receptor recognition and spore forming activity. Various protein molecules are being released by bacteria that cause invasion of the host. Exotoxins differ in biological function, molecular structure, immunological properties and mechanism of secretion. Structure of bacterial toxins is based on rather unconventional strategies. The diversity in toxins is mainly due to structural organization and functions. These elemental variations play an important role in their flexibility and dynamics. Biological toxins are being used extensively in bioterrorism. Various biological toxins involved in the inhibition or disruption of nerve impulse transmission ultimately causing cellular death. These toxins having potential to use as biological weapons for terrorism or military use. These toxins are considered most suitable for bioterrorist attacks due to cost effective and ease of production along with the high toxicity and environmental stability. Various techniques have been used to identify the pathogens and toxins but these diagnostic methods are some limitation such as time consuming, laborious and bulky instrumentation etc. Among all, engineered naonoparticles is advanced, cost effective and sensitive technique for screening of toxins. The nanoparticles like polymerica and quantam dots, nanochips, carbon nanotubes, cantilevers etc. effectively used for the diagnosis of toxins. The flouresence molecules are also tagged in nanoparticles which help in detection process. Researchers are working on the designing of smart 2 or 3D nanohips that helps in diagnosis of toxin with minimum sample volume in less time. Microorganisms especially bacterial strains develops a resistance mechanisms against drugs, so the advancement in nanotechnology not only able to detect the toxins but also assess whether the bacterial strains are resistant to drugs.

5.1 Future prospective

Bacterial toxins can be used as antigens in the development of vaccines. These vaccines can induce an immune response without causing disease leading to immunity against the toxin-manufacturing bacteria. Some bacterial toxins have shown promise in cancer treatment by targeting and destroying cancer cells. Scientists are exploring the use of modified bacterial toxins as a part of targeted therapies. Considerate bacterial toxins are crucial for developing countermeasures against potential bioweapons. Certain bacterial toxins can be exploited for environmental purposes, such as the cleanup of contaminated areas. Scientists are exploring the potential of bacteria in breaking down pollutants. Bacterial toxins can serve as models for developing innovative drugs that target specific cellular processes. Investigate the mechanisms of action can inspire the design of therapeutic agents. Bacterial toxins can be associated with emerging infectious diseases. Current research is needed for surveillance, early detection, and prevention of epidemics. Advances in synthetic biology allow for the engineering of bacteria and their toxins for various applications, including the production of biofuels or other useful compounds.

Advances in technologies such as genomics, proteomics, and structural biology may contribute to a more comprehensive examination of enterotoxins and their effects on the host. Efforts may be directed towards the enhancement of vaccines targeting bacteria that produce toxins. Vaccination could be a preventive measure to reduce the incidence of diseases caused by these toxins. Techniques for preventing the spread of enterotoxin-producing bacteria may be enhanced including improvements in hygiene practices, water treatment processes and food safety regulations. Improvements in technology like nanotechnology may offer new chances for forming new methods to toxin related infections.

Conflict of interest

The authors declare no conflict of interest.