MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 10. INFECTION AND THE INFECTIOUS PROCESS

PATHOGENICITY AND VIRULENCE OF MICROORGANISMS

A defining property of pathogenic microorganisms is their ability to trigger an infectious process—a complex of physiological and pathological, adaptive and reparative reactions that arise and develop within the macroorganism during its interaction with pathogens, disrupting the internal environment and physiological Functions.

The infectious process is one of the most intricate biological phenomena in nature, while infectious diseases remain threatening and destructive factors for humanity, causing immense damage.

The onset, clinical course, and outcome of an infectious process depend on The properties of the microorganism, the state of the host Organism, and environmental conditions, all of which fundamentally influence the interaction between the pathogen and the host.

The infectious process develops within the macroorganism only under specific conditions and according to certain biological laws. These conditions take into account the pathogen's virulence potential, its infectious dose, The rate of reproduction within the macroorganism, and the reactivity of the macroorganism itself.

Pathogenicity (from Greek *pathos* - disease, *genos* - birth) is a species-specific trait of microorganisms that is genetically fixed and characterizes their ability to cause disease.

Pathogenicity is characterized by Specificity, meaning The ability to induce pathomorphological and pathophysiological changes typical of a given pathogen species in specific Tissues and Organs under natural routes of infection. This manifests in the corresponding pathogenetic and clinical types of infection, such as purulent, respiratory, or intestinal infections.

Based on pathogenicity, microorganisms are categorized into non-pathogenic (Saprophytes), opportunistic (conditionally pathogenic), and pathogenic.

Opportunistic microorganisms are most often natural inhabitants of various human body biotypes and cause disease only under conditions of a sharp decline in general and/or local Immunity and/or an increase in their own pathogenic potential.

Virulence (from Latin *virulentes* - poisonous) reflects the degree of pathogenicity among various isolates or strains of a specific pathogenic species. Some pathogenic species may include highly virulent strains capable of causing the death of an animal from just a few microbial Cells, as well as strains with reduced virulence, whose lethal doses amount to hundreds of thousands or millions of units. For instance, There are two distinct types of the tularemia agent (*Francisella tularensis*), Type A and Type B, which differ significantly in virulence. Type A strains cause more severe forms of human disease and are pathogenic to rabbits, where the administration of even a single microbial Cell proves fatal. Type B Bacteria cause milder forms of human disease and require the Introduction of 1 billion cells to cause the death of rabbits.

Virulence is a quantitative measure or degree of pathogenicity, measured in special units such as DLM and LD₅₀. 1 DLM (*Dosis Letalis Minima*) represents the minimum lethal dose, defined as the smallest number of microbial cells that, under a specific route of infection, causes the death of 95% of susceptible animals of a given species, weight, and age within a specified timeframe. LD (Lethal Dose)₅₀ causes the death of 50% of affected animals. In practice, LD₇₀, LD₇₅, and LD₉₀ are also utilized.

When a minimal number of pathogenic microorganisms enters the body (which is most common), they are typically eliminated effectively by the body's defense mechanisms. For a disease to develop, the pathogen must possess sufficient virulence, and its quantity (infectious dose) must exceed a certain threshold determined in each specific case by the pathogen's virulence and the host's resistance state. To quantify this, the Infectious Dose (ID) is used, representing the minimum number of pathogen cells capable of causing disease in a given proportion of laboratory animals. Similarly to lethal doses, ID₅₀ and ID₇₅ are determined. For example, cholera develops when a person ingests 10¹¹–10¹² *V. cholerae*, typhoid fever requires 10⁶–10⁹ *S. typhi*, dysentery requires up to 100 cells of *S. dysenteriae*, and brucellosis requires 10–100 bacterial cells. The infectious dose for the exact same pathogen can vary depending on the route of entry into the macroorganism. For instance, when *F. tularensis* enters The Human Body via microtraumas of the Skin or through inhalation, 10–50 microbial bodies are sufficient to cause disease, whereas alimentary infection requires about 10⁸ microbial cells. Naturally, these differences in the infectious dose are dictated by the specific reactivity of the organism within the context of the respective portals of entry.

Reproduction rate. The likelihood and severity of an infectious process are heavily influenced by the reproduction rate of the pathogen. For example, the plague bacillus (*Y. pestis*) multiplies so rapidly within the body that The Immune System practically fails to "respond" in time with protective reactions. Conversely, the causative agents of tuberculosis and leprosy (*M. tuberculosis* and *M. leprae*) develop very slowly, inducing tolerance in the host's immune system, which accounts for the chronic nature of these infections.

Portals of entry. Many pathogens exhibit a distinct tropism (from Greek *trope* - direction) for specific tissues. For instance, the gonococcus causes typical lesions upon contacting the mucous membranes of the genital organs or eyes, whereas the dysenteric amoeba targets the intestinal mucosa. On the other hand, tubercle and plague bacilli can cause disease regardless of the route of entry, leading to polymorphic lesions that vary depending on the site of penetration. Such pathogens are characterized by pantropism.

Reactivity of the organism. The state of the macroorganism's general biological reactivity determines not only the possibility and character of the infectious process, but also the likelihood of its manifestation as an overt clinical disease. It is worth emphasizing that regardless of how a pathogen acts upon the macroorganism, all physiological systems of the host participate in the response reactions to varying degrees, not just the immune system. These Reactions of the organism as a unified whole are governed by its reactivity—defined as the capacity of the organism to mobilize physiological mechanisms aimed at the inactivation, disruption, and elimination of the pathogen and its associated substances, as well as the compensation of impaired functions.

Dynamics of the infectious process. The Development of an infectious process typically comprises several stages:

- entry of the infectious agent, its adaptation to the internal environment of the body, and colonization of the tissues for which the microorganism exhibits tropism;

- production of metabolic products (toxins, exoenzymes) that exert a damaging effect and lead to disruptions in the body's Homeostasis;

- dissemination of the infectious agent from the primary site of entry. Most commonly, microbes spread via the lymphatic and/or Blood Vessels.

This state may be accompanied by clinical manifestations or proceed entirely without symptoms. Frequently, microorganisms may circulate in the bloodstream temporarily, entering the blood under conditions of heavy physical exertion or stress (prolonged Sleep deprivation, hypothermia, or overheating).

Clinically pronounced stages usually develop when microorganisms invade the bloodstream As a result of trauma, medical Procedures, or from a localized infectious focus. Transient or sustained bacteremia is characteristic of many viral (Influenza, hepatitis B) and bacterial (typhoid fever, meningococcal infection, rickettsiosis) infections. The Circulation of pathogens in the bloodstream is a crucial and obligatory stage in the Pathogenesis of vector-borne infections transmitted via arthropod bites (plague, epidemic typhus), which sustains the pathogen's circulation in nature and thereby preserves it as a species.

The actual state of microorganism circulation in the blood is termed bacteremia. A reduction in the microbicidal properties of blood allows pathogenic microorganisms to multiply within the bloodstream, leading to severe generalized conditions known as Sepsis (septicemia) (from Greek *sepsis* - putrefaction). Typically, this situation is the consequence of continuous or periodic seeding of the bloodstream by pathogens. Conditions in which pathogenic microorganisms not only multiply in the blood but also establish new, distant foci of purulent inflammation in various tissues and organs are known as septicopyemia. If the pathogenesis of an infectious disease is driven by intoxication resulting from the circulation of exo- or endotoxins in the blood, this condition is designated as toxinemia.

In response to the invasion of a microorganism and/or its toxin, the body mounts defense reactions aimed at inactivating the pathogen and restoring homeostasis. The natural termination of an infectious process is the recovery of the patient, the death of the host, or the transition of the infection into a chronic form. In the majority of infectious cases, recovery leads to the development of specific resistance against re-infection by the same pathogen.

The forms of an infectious process can vary significantly. Not all stages of its development always occur, because when a pathogen enters an insensitive (immune) organism, the infection process is aborted at the very beginning.

Characteristics of infectious diseases. Infectious diseases are characterized by such features as specificity, contagiousness, and cyclicity.

Specificity is due to the fact that each infectious disease is caused by a specific pathogen. However, certain infections (e.g., purulent-inflammatory processes) are known to be caused by various opportunistic microorganisms. On the other hand, a single pathogen, such as Streptococcus, is capable of causing lesions in various organs and systems.

Contagiousness refers to the ability of a pathogen to be transmitted from one person to another and the rate of its spread within a susceptible population. To quantify contagiousness, the contagiousness index is proposed—the percentage of individuals in a population who have contracted the disease over a specific period. Highly contagious diseases include cholera and plague; moderately contagious include brucellosis; and non-contagious include botulism, tetanus, and staphylococcal enterotoxin poisoning.

Cyclicity means that the development of a specific infectious disease is time-limited and proceeds in cycles marked by distinct clinical stages.

Incubation period (Lat. incubatio - lying, sleeping somewhere). Typically, between The entry of an infectious agent into the body and the appearance of clinical signs, There is a certain period characteristic of each disease—the incubation period, typical only of exogenous infections. During this period, the pathogen multiplies, and both the pathogen and its secreted toxins accumulate up to a certain threshold, beyond which the organism begins to respond with clinically manifested reactions. The duration of the incubation period can range from hours and days to several years. For example, the incubation period for cholera ranges from a few hours to 2–3 days, for dysentery 1–7 days, for brucellosis 3 weeks (acute form) to several months, and for leprosy 3–5 years.

Prodromal period (Gr. prodromos - running before). As a rule, the initial clinical manifestations of infectious diseases do not present any pathognomonic (Gr. pathos - disease, gnomon - indicator, sign) features specific to a particular illness. Typical manifestations include weakness, headache, and a general feeling of malaise. This stage of an infectious disease is called the prodromal period or the "stage of precursors." Its duration generally does not exceed 24–48 hours.

Period of disease development. This phase exhibits either disease-specific traits or general features common to many infectious processes, such as fever, inflammatory changes, etc. In the clinically pronounced phase, one can distinguish the stages of symptom escalation, peak illness, and the subsiding of manifestations.

Convalescence (Lat. re - repetition of an action, convalescentia - recovery). The recovery period, or convalescence, as The final stage of an infectious disease, can be acute (crisis) or gradual (lysis), or it may be characterized by the transition of the infection into a chronic state. In favorable cases, clinical symptoms usually disappear faster than the morphological damage to organs and tissues is normalized and the pathogen is completely eradicated from the body. Recovery can be complete or accompanied by the development of complications (e.g., affecting the Central Nervous system, Musculoskeletal System, or Cardiovascular system).

Pathogenicity factors. Pathogenicity and virulence are determined by a complex of microbial properties formed during adaptation to parasitism within a macroorganism. The Emergence of pathogenicity in microbes is associated with acquiring a set of traits that ensure the ability to penetrate and spread within the macroorganism, resist its defense mechanisms, and cause damage to vital systems. The primary factors of pathogenicity and virulence include the ability of microorganisms to adhere and colonize, invasiveness, toxigenicity, and the capacity for persistence.

Ability to adhere and colonize. The multiplication of microorganisms at the primary infection site is preceded by adhesion (Lat. ad-haesio - attaching to something), i.e., the binding of bacteria to The Cell surface, which essentially marks the beginning of the infectious process. Attachment to cell surfaces (e.g., to the epithelium of mucous membranes) is mediated by adhesins or colonization factors (Proteins, lipopolysaccharides (LPS), lipoteichoic acids). Adhesion molecules are located directly On the surface of the bacterial cell and are part of The Cell wall, microvilli, pili, or capsules.

Adhesins that ensure the attachment of the pathogen to host cells are extremely diverse. Their unique Structure is characteristic of specific species and even strains, determining the high specificity of this process. This explains why certain microorganisms adhere to and colonize primarily the respiratory epithelium, others the intestinal tract, and still others the Urinary System.

The adhesins of many Gram-negative bacteria are associated with various types of pili, which are designated by numbers or symbols. Colonization factors, representing specific pilus Antigens, are also distinguished. For example, type 1 pili are found in many bacteria; type 4 pili in *Proteus*, *Pseudomonas*, *Vibrio cholerae*, *Neisseria*, *Salmonella*, and enteropathogenic and enterotoxigenic *Escherichia*; P-pili (Pyelonephritis associated pili) in nephritogenic strains of *E. coli*; and CFA/I, CFA/II, CFA/III (Colonization Factor Antigens) in certain enterobacteria.

The adhesive function in Gram-negative bacteria is also performed by the capsule, the capsule-forming envelope, and outer Membrane Proteins of the cell wall. For example, the surface antigen Yad of *Y. enterocolitica* ensures the attachment of the microorganism to Extracellular matrix components—Fibronectin, Collagen, and Laminin—while the surface antigen complex of mycobacteria, known as the BCG85 complex, ensures microbial attachment to fibronectin.

The interaction between an infectious agent and epithelial cells occurs via several types of bonds that differ in nature and specificity. These include bonds based on electrostatic forces, those driven by surface hydrophobic properties, and Ligand-receptor interactions.

Certain bacteria are capable of pre-conditioning a site for subsequent multiplication. For example, neuraminidase facilitates the penetration of *Vibrio cholerae* through the mucus layer, thereby ensuring contact with sialic acid-containing receptors of the intestinal epithelium. Microorganisms are also capable of adhering to bacteria that have already colonized The surface of mucous membranes or tooth enamel.

Receptors on human tissue cells that specifically interact with bacterial adhesins are also heterogeneous in composition. They are classified as native, induced, and acquired.

Native receptors are located on epithelial cells under normal conditions and participate in the adhesion of corresponding bacteria and their entry into The Eukaryotic Cell. For example, the Lewis^b blood group antigen expressed by gastric epithelial cells facilitates the adhesion of *H. pylori*. *E. coli* penetrates macrophages via receptor-mediated endocytosis involving CD48, whereas *S. flexneri* enters enterocytes following interaction with the CD44 receptor.

Induced receptors form on the cell surface only after the entry of Viruses (e.g., influenza virus). Subsequently, staphylococci and other bacteria can adhere to the surface of such cells. This is because viral hemagglutinin, which becomes embedded in the cytoplasmic membrane of virus-infected epithelial cells, serves as a receptor for these bacteria. This fact is crucial for understanding the mechanisms underlying secondary bacterial infections during primary viral diseases, such as influenza.

Acquired receptors appear under specific conditions. They act as "bridges" linking epithelial and Bacterial cells and consist of IMMUNOGLOBULINS of various classes, albumins, fibronectin, and Other Compounds capable of interacting with complementary bacterial adhesins.

Colonization is The process of microbial multiplication at the site of adhesion. This stage ensures the accumulation of microorganisms to a critical concentration capable of causing pathological damage. To successfully colonize the primary infection site, bacteria must withstand the action of numerous diverse microbicidal factors of the host organism. To defend against them, microorganisms actively utilize various structures (e.g., capsules) and synthesized substances (e.g., Enzymes).

Inactivating enzymes can also be classified among the factors that ensure successful colonization of mucosal surfaces by bacteria. Microorganisms synthesize various enzymes that degrade humoral defense factors. For example, many pathogens, especially those parasitizing mucous membranes (gonococci), secrete proteases that cleave IgA (IgA proteases). Bacterial enzymes are also capable of altering the pH of the surrounding environment to render it favorable for multiplication. For instance, *H. pylori* secretes urease, which hydrolyzes urea to produce ammonia, thereby alkalinizing the acidic environment of The Stomach.

Invasiveness. The virulence properties of pathogens can manifest in the ability of some to invade mucosal surfaces and penetrate inside epithelial cells, macrophages, or lymphocytes. The terms "invasion" and "penetration" are synonyms; however, some authors define invasion (Lat. invasio - to enter, attack) as the passage of microorganisms through mucosal and Connective Tissue barriers into underlying tissues, and penetration as entry into the interior of Eukaryotic cells.

Shigellae and enteroinvasive *Escherichia* penetrate epithelial cells and multiply within them, whereas mycobacteria and listeria parasitize inside macrophages, and so forth. Penetration begins after bacteria enter the intercellular space, where they interact with cell membrane binding proteins. Binding to these proteins induces Conformational Changes in microtubules and membrane ruffling, as a result of which the bacteria are internalized into the cell.

The ability of bacteria to multiply intracellularly is linked to their resistance to lysosomal enzymes. Intracellularly replicating bacteria can spread to neighboring cells without entering the extracellular environment (as seen in *Shigellae*). In the process, the epithelial cells are destroyed,

accompanied by damage to the integrity of the epithelial lining of the affected organ or cavity and the onset of a pathological process.

The primary factors driving invasiveness and penetration are motility (enabling movement through the intercellular space) and substances that degrade the extracellular matrix, facilitating entry into epithelial cells via endocytosis. Certain microorganisms breach the epithelial barrier either through active invasion or passive entry via various skin lesions.

Invasion is facilitated by The production of such exoenzymes as hyaluronidase, neuraminidase, lipases, collagenase, Elastase, and mucinase.

Hyaluronidase is produced by C. perfringens, certain species of Streptococcus, Staphylococcus, Brucella, and others. This enzyme specifically cleaves hyaluronic acid, a component of the intercellular substance, thereby increasing the permeability of mucous membranes and connective tissue. Of particular interest is the ability of certain bacteria (genus Streptococcus) to disrupt their own capsule during tissue invasion through the synthesis of hyaluronidase.

Neuraminidase is produced by Vibrio cholerae, Corynebacterium diphtheriae, and other bacteria. The enzyme neuraminidase cleaves derivatives of neuraminic acid—sialic acids—from various Glycoconjugates (Glycoproteins, Glycolipids, and Oligosaccharides), which is why this enzyme is also referred to as sialidase. Utilizing neuraminidase, pathogens can pass not only through the mucous membrane but also penetrate inside cells and spread within the intercellular space.

Lipases facilitate adhesion and tissue penetration. Specifically, these enzymes are capable of disrupting sebaceous "plugs," thereby easing the entry of staphylococci into Hair follicles.

The collagenase of C. perfringens (κ-toxin) induces the Hydrolysis of connective tissue collagen.

Elastase, produced, for example, by Pseudomonas aeruginosa, degrades Elastin, casein, Hemoglobin, fibrin, immunoglobulins, Complement system components, and other proteins.

Mucinase liquefies mucus and facilitates the access of Vibrio cholerae to the epithelial cell surface.

It should be noted that the Classification of pathogenicity factors into specific groups is somewhat arbitrary, as the same factors perform multiple functions and can be assigned to different categories. For instance, the ability of gonococci to secrete IgA1 protease, firstly, facilitates the attachment of gonococci to epithelial cell receptors (factors promoting adhesion) and, secondly, protects microbial cells from antibody-mediated phagocytosis (factors promoting persistence). Similarly, the presence of a capsule simultaneously protects bacteria against antimicrobial phagocytosis and promotes epithelial adhesion, among other functions. We will discuss specific factors within the groups where, in our view, they play The most significant role in manifesting the pathogenic potential of bacteria.

Capacity for Persistence. Aggression. To ensure their long-term persistence within the host organism, bacteria employ various strategic approaches sharing a common goal: to overcome the resistance of the host immune system. In this interplay, the aforementioned mechanisms of invasion are paramount, promoting the rapid dissemination of bacterial cells throughout the host. Alongside the enzymes responsible for bacterial invasive properties mentioned earlier, fibrinolysin—produced by streptococci and certain other bacteria—deserves special note. Fibrinolysin activates plasminogen, leading to The formation of plasmin and the dissolution of fibrin clots, which allows the pathogen to spread within the body. When a pathogen undergoes rapid dissemination, the immune system is unprepared to eliminate the microorganisms during the Initial Stages of infection. Once bacteria colonize susceptible tissues, another pathogenic strategy comes into play, aimed at shielding bacterial cells from host immune defense mechanisms. At this stage, enzymes such as staphylocoagulase, which induces Blood Plasma clotting, become crucial. This enzyme does not interact directly with fibrinogen; rather, it promotes the formation of a Thrombin-like substance that presumably interacts with prothrombin. The resulting fibrin film acts as a sort of supplementary capsule, protecting the bacteria and enabling them to evade direct contact with the immune system.

Virulence factors that suppress the body's defenses are called aggressins, as they possess the ability to directly inhibit the non-specific and specific immune Responses of the host organism. These comprise substances of diverse chemical nature that constitute the Surface structures of the bacterial cell: Polysaccharides, proteins (capsules), staphylococcal protein A, streptococcal M protein, lipopolysaccharides (LPS) of the cell wall of Gram-negative bacteria, as well as exoenzymes and exotoxins.

The capsule (or its less distinct analog, the slime layer) inhibits the initial stages of defense reactions—recognition and engulfment. Capsules "shield" cell wall antigens that activate The Complement System and can otherwise be recognized by immunocompetent cells. For example, the layer of capsular substance protects staphylococcal Teichoic Acids from binding by opsonins. Bacterial capsules are

hydrophilic, which impedes the engulfment of bacteria by phagocytes, while the capsular substance itself protects the bacterium against the action of lysosomal enzymes and toxic oxygen radicals released by phagocytic cells. The easy shedding of capsules or the slime layer from the bacterial surface is of great importance. When encapsulated bacteria (such as Pseudomonas aeruginosa) are engulfed, the bacterium easily "sheds" its capsule and avoids direct contact with the phagocyte. Furthermore, capsular components, such as fractions V and W antigens of Yersinia pestis, directly exhibit antiphagocytic properties.

Bacterial evasion of host defense mechanisms is also achieved through the action of their own exoenzymes. These include IgA protease, protease of the C3b and C5a complement components, lecithinase, and alkaline protease.

The IgA protease of members of the genus Neisseria acts extracellularly, disrupting bonds in the heavy chains of IgA and cleaving the IgA molecule at the hinge region. IgA1 protease is synthesized by gonococci that parasitize mucous membranes.

The enzyme lecithinase, produced by anaerobic infection agents, staphylococci, and other microorganisms, induces the hydrolysis of lecithin, a constituent of membranes in various cell types, including leukocytes.

Alkaline protease, synthesized in large quantities by Pseudomonas aeruginosa, hydrolyzes A wide variety of proteins (including γ-IFN).

A prerequisite for the prolonged persistence of microorganisms is the ability to survive and actively replicate within damaged or metabolically inactive host phagocytes. Furthermore, bacteria can evade destruction by translocating from phagosomes into the Cytoplasm within macrophages. For instance, Listeria monocytogenes cells escape from phagosomes by secreting the enzyme listeriolysin O, which disrupts the membrane of these Organelles. Other pathogens, such as Mycobacterium leprae, are capable of inducing their own uptake by cells that typically lack phagocytic functions and possess insufficient antibacterial activity. In such cases, microbial cells cannot be destroyed by activated phagocytes or other bactericidal mechanisms until they are released from the cells where they find "shelter." Their release is facilitated by cytotoxic T lymphocytes, which disrupt infected cells. Mycobacterium tuberculosis contains a "cord factor" that inhibits the fusion of Lysosomes with phagosomes; additionally, mycobacteria disrupt the proton "pump" that acidifies the phagosomal content, preventing the pH within phagosomes from dropping. Pathogenic bacteria can secrete catalase, which degrades hydrogen peroxide produced by phagocytes. The bacterial enzyme superoxide dismutase may also be involved in inactivating toxic oxygen products of phagocytes. Mycobacteria are likewise capable of secreting lipoarabinomannan, which blocks the ability of macrophages to mount an activation response to IFN-γ.

Toxigenicity. Toxins (from the Greek toxikon meaning poison) are major pathogenicity factors produced by microorganisms that frequently drive the pathogenesis of infectious processes. The Role of microbial toxins in the pathogenesis of infectious diseases was first demonstrated by E. Roux and A. Yersin (1888), who separated the "poisonous principle" of the diphtheria pathogen from bacterial cells and successfully reproduced the clinical picture of the disease in guinea pigs using it.

Toxic substances synthesized by bacteria are chemically classified primarily as proteins and LPS. Peptidoglycan, teichoic acids, and cell wall proteins of Gram-positive bacteria (such as staphylococcal protein A) also possess toxic properties.

THE SPECTRUM OF toxin activity is extremely broad, ranging from substances that facilitate tissue dissemination to metabolites that selectively impair The activity of specific cells.

Bacterial toxins are traditionally divided into endotoxins and exotoxins (Table 10.1). In a broad sense, endotoxins can encompass any Components of the bacterial cell: peptidoglycan and teichoic acids found in the cell wall of Gram-positive bacteria, lipopolysaccharide of Gram-negative bacteria, and bacterial DNA. Bacterial DNA is no exception in this regard. The direct effector of DNA is the cytosine-guanine dinucleotide. In the human body, such pairs are methylated, whereas in bacteria they are unmethylated; consequently, they easily penetrate eukaryotic cells and, as a result, trigger the release of pro-inflammatory cytokines that regulate the development of inflammation. Peptidoglycan exerts a direct toxic effect. For instance, the peptidoglycan of the gonococcal cell wall directly damages the urethral epithelium.

In a narrower sense, endotoxins generally refer to LPS, which is an integral component of the cell wall of Gram-negative bacteria. Most of these are released only after the bacterial cell dies. The toxic properties of endotoxin are determined by the entire LPS molecule rather than its individual fractions: polysaccharides or lipid A.

Endotoxins, unlike protein-based toxins, are more heat-stable, less potent, and moderately specific. Various LPS preparations, when administered to experimental animals, induce

a more or less uniform response, regardless of which gram-negative bacteria they are derived from.

Class="center">Table 10.1. Comparative characteristics of exo- and endotoxins

*Compared to strychnine (strychnine activity is conventionally set at 1).

Administration of endotoxins in small doses causes minor stimulation of various Links of the immune system: stimulation of phagocytosis, elevated body Temperature, and mild toxic symptoms.

When endotoxins are administered in high doses, animals exhibit the opposite reaction: suppression of phagocytosis, lowered body temperature, pronounced toxic symptoms accompanied by weakness, intestinal disorders (diarrhea), hypotension, and cardiovascular collapse.

In humans, the entry of endotoxins into the bloodstream causes fever due to their effect on Blood Cells (granulocytes, monocytes), which release endogenous pyrogens.

Endotoxemia is characterized by hypotension due to the release of increased amounts of serotonin and kinins into the blood, as well as impaired organ Blood supply and acidosis. LPS activates the complement system via The alternative pathway, leading to a decrease in serum C3 component levels and the accumulation of BIOLOGICALLY ACTIVE SUBSTANCES (C3a, C3b, C5a, etc.). A large amount of endotoxin entering the blood leads to the development of toxic-septic Shock.

Lipopolysaccharides are relatively weak immunogens. The blood serum of animals immunized with pure endotoxin does not possess high antitoxic activity and is unable to completely neutralize its toxic properties.

Protein toxins (exotoxins) are secretory protein substances that exhibit enzymatic activity and modify intracellular "targets" in such a way that normal cell function is disrupted, sometimes leading to cell death. Exotoxins are frequently the primary virulence factor of a microorganism, acting at a distance (far from the infection site) and responsible for the clinical manifestations of the infection (e.g., enterotoxins cause diarrhea, neurotoxins cause paralysis and other neurological symptoms). Botulinum toxin ranks first in toxicity, followed by tetanus and diphtheria toxins.

To date, more than 80 bacterial toxins have been described, differing from one another in chemical structure, specificity of action, and biological activity. Some of them are thermolabile, while others are thermostable. For instance, the thermolabile diphtheria histotoxin is disrupted at 60 ⁰C within 1 h, and tetanus toxin within 20 min. Thermostable toxins of Clostridium botulinum, Escherichia coli, and staphylococci can withstand brief boiling.

Bacterial exotoxins vary in structural Organization and Mechanisms of action, but they share certain general Principles of structure and function. Bacterial toxins are divided into two broad categories based on their MECHANISM OF ACTION on the target cell: enzymes with enzymatic activity and pore-forming toxins (Pore-Forming Toxins (PFT)). Enzyme toxins have a rather limited spectrum of activity, yet they can modify diverse cellular targets, ultimately leading to various consequences at the level of individual cells, tissues, organs, and the organism as a whole. Many enzyme toxins are constructed on the A+B principle, meaning they typically have one A subunit (active) and one or several B subunits (binding) (Table 10.2). Subunit A possesses enzymatic activity and modifies a specific intracellular target. Subunits B ensure the binding of the entire toxin to corresponding receptors on the surface of the eukaryotic cell and help transport the active subunit A into the Cytosol. The binding of subunit B is highly specific. For example, Shiga toxin binds to the Gb3 glycolipid, and choleragen to the GM1 ganglioside. The enzymatic activity of subunit A varies among different toxins from ADP-ribosylating activity (choleragen, pertussis and diphtheria toxins) to proteolytic activity (clostridial neurotoxins). However, the domains with enzymatic activity are structurally conservative among different toxins since they perform similar functions. For instance, at least five toxins with ADP-ribosylating activity (choleragen, E. coli heat-labile toxin, pertussis and diphtheria toxins, P. aeruginosa exotoxin A) share the same NAD-binding site. Enzyme toxins with NAD-glycohydrolase activity catalyze the Cleavage of NAD into adenosine phosphoribose and nicotinamide, while simultaneously transferring the ADP-ribose moiety to specific eukaryotic cell target proteins. Such proteins can include G-proteins (from GTPase) that, for example, regulate adenylate cyclase activity, elongation factor 2 (E2F), and Rho Cytoskeleton proteins. Such biochemical modification of intracellular target proteins by toxins leads to the disruption of their activity. Thus, ribosylation of elongation factor 2 by diphtheria toxin or P. aeruginosa exotoxin A results in impaired factor function and blockade of cellular Protein Synthesis. Ribosylation of the Gs protein (which stimulates adenylate cyclase activity) by choleragen leads to increased activity and, consequently, an elevated cAMP level in small intestinal epithelial cells, causing the loss of Water and electrolytes. Ribosylation of the Gi protein (which inhibits adenylate cyclase activity) by pertussis toxin also leads to increased adenylate cyclase activity, elevated intracellular cAMP content, and the loss of water and electrolytes by the cell, but on a much smaller scale than in the case of choleragen action.

Proteolytic toxins include clostridial neurotoxins and the lethal factor (LF) of the anthrax complex toxin. All of these toxins are Zn²⁺-dependent Endopeptidases. Clostridial neurotoxins cleave synaptobrevin proteins located in the membranes of neurotransmitter endosomes in nerve endings, which are required for neurotransmitter exocytosis and Nerve Impulse transmission. Tetanus and botulinum toxins share identical mechanisms of action but operate in different regions of The Nervous System, which accounts for the differences in the clinical signs of tetanus and botulism. Tetanus toxin blocks the function of Spinal Cord inhibitory interneurons, whereas botulinum toxin blocks motor Neurons. Therefore, the main clinical manifestation of the former is tetanic Muscle contractions (opisthotonos), and of the latter, muscle relaxation (paresis). B. anthracis LF cleaves the MAPKK protein, which is essential for cell survival and proliferation signaling, so the disruption of this protein leads to target cell death.

Table 10.2. Structure and mechanisms of action of A-B toxins

A major group of bacterial toxins consists of pore-forming toxins (Pore Forming Toxins). The primary mechanism of toxin action is the formation of a pore in The Plasma Membrane of the cell, through which the cell loses water and electrolytes. Consequently, the target cell dies. Such toxins include E. coli hemolysin (Hly), S. aureus α- and γ-toxins, and streptolysin-S and streptolysin-O of streptococci. An interesting example of such toxins is listeriolysin-O, which listeria use to escape from phagosomes into the cytosol.

A special category of toxins includes exfoliatins and erythrogenic toxins, synthesized by Staphylococcus aureus and Streptococcus pyogenes, respectively. These toxins disrupt desmosomal contacts between epidermal cells and lead to clinical signs such as "scalded skin syndrome," frequently observed in infants infected with certain S. aureus strains, and skin redness in erysipelas induced by S. pyogenes.

Among bacterial toxins, there is a distinct group of toxins with superantigen properties. These toxins include exfoliatins, erythrogenic toxins, staphylococcal enterotoxins, and staphylococcal toxic shock syndrome toxin (TSST-1). A characteristic feature of these toxins is their ability to induce polyclonal activation of the host organism's lymphocytes. Due to their unique structure, these toxins bind Major Histocompatibility Complex class II (MHC II) molecules on the surface of antigen-presenting cells and β-chains of T-cell receptors. This results in massive activation and proliferation of T cells accompanied by the release of A number of pro-inflammatory cytokines (interleukin-1, -2, -6, tumor necrosis factor, interferon-γ). The clinical manifestations of these processes are a significant increase in body temperature, blood pressure drop, and erythematous skin rashes.

The immunogenic properties of protein toxins manifest in their ability to elicit an Immune Response from the macroorganism, specifically by inducing the Synthesis of specific antibodies—antitoxins that neutralize the corresponding toxin.

Another characteristic feature of a number of protein toxins, such as tetanus, diphtheria, and certain others, is their ability under the action of formalin to lose their toxicity while retaining their immunogenic properties. Such toxins are called toxoids. They are used as vaccine components for the Prevention of corresponding diseases.

Many bacteria produce not one, but several protein toxins with different effects: lethal, dermonecrotic, cytotoxic, and hemolytic. For example, B. anthracis produces a toxin containing three components: protective antigen (PA), corresponding to the B subunit, and two components corresponding to the A subunit—edema factor (EF) and lethal factor (LF). Some bacteria simultaneously produce both protein toxins and endotoxins, such as Escherichia coli, Vibrio cholerae, etc.

Toxin activity is measured in the same units used to evaluate virulence—DLM and LD50.

All bacterial pathogenicity factors act in unison. For instance, adhesion and invasion factors ensure close contact between the bacterial and eukaryotic cells, enabling targeted delivery of the toxin to the target cell.

Bacterial secretion systems. Extracellular protein secretion is one of the MAIN MECHANISMS OF pathogenicity in bacterial infections. Both gram-positive and gram-negative microorganisms possess the ability to secrete proteins extracellularly. In gram-positive microorganisms, secretion occurs via the primary secretory apparatus using the Sec (Secretin) protein system. In gram-negative microorganisms, extracellular protein transport is organized more complexly than in gram-positive ones, as it involves translocation across two cell wall layers known as the inner and outer membranes. This has led to the emergence of a wide variety of structurally and functionally distinct secretion systems in gram-negative microorganisms.

To date, five types of major secretion systems (I to V) have been described in gram-negative bacteria.

Systems I and III secrete proteins in an unfolded state and do not utilize the Sec system.

Secretion systems II, IV, and V (autotransporters) utilize the Sec system.

Examples of type I secretion systems include the export of hemolysin (HlyA) by E. coli, metalloprotease by Erwinia chrysanthemi, leukotoxin by Pasteurella haemolytica, and adenylate cyclase by Bordetella pertussis. A type I secretion system consists of three proteins: an inner membrane ATPase (ATP-Binding Cassette (ABC)-transporter) functioning as a transport protein, a chimeric protein anchored in the inner membrane and spanning the periplasmic space, and an outer membrane pore-forming protein. The secretion process begins with the attachment of the protein to be secreted, via its C-terminal specific sequence, to the ABC-transporter. This signal sequence is specifically recognized by the ABC-transporter, ensuring that the transport protein exclusively translocates corresponding substrates. However, the pore-forming outer membrane protein lacks such substrate specificity and can transport various proteins from the periplasmic space to the extracellular environment.

The type II secretion system is typically exemplified by pullulanase (PulA) in Klebsiella oxytoca. The type II secretion system is Sec-dependent. The Sec system enables the secretion of proteins across the inner membrane and consists of the ATPase SecA, several integral membrane proteins (SecD, SecE, SecF, SecG, SecY) spanning the inner membrane, and a signal peptidase. In the cytoplasm, the chaperone SecB directs the transported protein to the Sec-translocon. Transport occurs in two stages. Proteins destined for transport are synthesized as precursor proteins (preproteins) featuring a typical N-terminal signal sequence. Following Transport Across the inner membrane, this sequence is cleaved off by a signal peptidase. In Gram-positive bacteria, these events are sufficient to release secreted proteins to the cell exterior. In Gram-negative bacteria, after the removal of the N-terminal signal peptide and release into the periplasmic space, proteins cross the outer membrane in a separate step. Within the periplasm, the secreted protein folds into its native conformation, a process facilitated by specific chaperones such as Disulfide bond isomerase (DsbA). Transport across the outer membrane requires the additional activity of 12-16 accessory proteins that make up the extracellular protein secretion (Eps) apparatus. Proteins secreted via the type II pathway include proteases, cellulases, pectinases, phospholipases, lipases, the heat-labile enterotoxin of E. coli, V. cholerae toxin, Legionella pneumophila toxin, and P. aeruginosa toxin. Primarily, these proteins are involved in damaging host Cells and Tissues. The expression of the genes encoding these proteins, as well as the components of the secretion systems themselves, is subject to quorum-sensing control—meaning it is tightly regulated by microenvironmental conditions. This regulatory mechanism ensures that virulence factors are secreted only when bacteria reach specific microenvironments and attain a critical population density.

The type III secretion system comprises 20 proteins that form a needle-like structure spanning the inner and outer membranes of the bacterial cell. The type III secretion system exists to translocate proteins across a third membrane, enabling Gram-negative bacteria to secrete and inject pathogenic proteins directly into the cytosol of eukaryotic cells. Naturally, this mode of protein translocation requires close cell-to-cell contact. Type III-mediated translocation is observed in extracellular pathogens during interaction with the cytoplasmic membrane, as well as in intracellular pathogens upon interacting with the phagosomal membrane. Proteins secreted by this system contain a specific N-terminal sequence of approximately 20 Amino Acids. This secretion mechanism is evolutionarily conserved across widely divergent animal and plant pathogens, such as Yersinia pestis, Erwinia spp., Salmonella enterica, Shigella flexneri, Escherichia coli, Pseudomonas syringae, Ralstonia solanacearum, and Chlamydia trachomatis.

The type IV secretion system involves the coordinated interaction of 9-10 proteins associated with the inner and outer membranes. This system is also capable of translocating proteins directly into host eukaryotic cells. Some of the best-characterized systems include the toxin secretion systems of Bordetella pertussis, Brucella suis, Legionella pneumophila, and Helicobacter pylori (encoded by the cagA Gene). Interestingly, unlike Other toxins, the B. pertussis toxin is secreted directly into the extracellular space.

Compared to other systems, the type V secretion system (autotransporters) has one of the simplest organizations. All proteins secreted via this system share a unified structure consisting of an amino-terminal leader peptide (for Sec-mediated inner membrane translocation), the mature secreted protein itself (or passenger domain), and a C-terminal domain that forms a pore in the outer membrane through which the passenger domain reaches the cell surface. All requirements for outer membrane translocation are met by the single polypeptide molecule, making the secretion an energy-independent process. While this mechanism appears to be the simplest form of secretion, it possesses a number of biophysical peculiarities. Typically, the leader and C-terminal components are highly conserved among certain proteins, whereas the passenger domain is specific and performs a distinct function in each individual case. Adhesins, hemagglutinins of Escherichia, the VacA toxin and adhesin of Helicobacter pylori, the IgA protease of Neisseria, and the mucinase and proteolytic toxin of Shigella flexneri are all secreted via the type V system.

A bacterial cell may simultaneously harbor multiple secretion systems of different types, as well as several distinct systems of the same type.