See also Antibiotic Resistance Mechanisms
Videos: Anthrax Anthrax2 (types) Anthrax (endospores) E. coli (virulent strain)
C. Difficile (sniffing dogs) Endotoxins (description) Inside the Phagosome (e.g., TB)
Tetanus (spores) Tetanus (Brigham; symptoms, treatment)
Introduction:
In order to successfully colonize the gut and cause infection or disease, bacteria (or pathogenic bacteria) have evolved multiple virulence factors in interaction with the human microbiome that enable them to counteract colonization resistance.
Bacteria are extremely complex at the molecular level, composed of thousands of proteins, large quantities of nucleic acids and a great many types of small organic molecules. However, the host is oblivious to the vast majority of these molecules. Only a handful of them (e.g., LPS, lipopeptides, lipoteichoic acid, flagellin and unmethylated DNA) incite an innate immune response.
The gene products produced by microorganisms to establish themselves within a particular
eukaryotic host to cause disease are known as virulence factors. Bacterial virulence
factors can be expressed as being membrane associated, secretory, or cytosolic in nature. The membrane-associated cell surface receptors mediate bacterial attachment,
aiding the bacterium in adhesion to the host cell. Secreted factors include bacterial
capsules that can protect a cell from ingestion and destruction by host immune cells
through phagocytosis and hydrolytic enzymes that can contribute to the pathogenicity.
Mobility and chemotaxis:
Flagella:
Flagella are highly complex bacterial organelles which are unusually well conserved among diverse bacterial species. Over 50 genes are involved in the synthesis and function of flagella, suggesting that their preservation and role in chemotaxis and motility are important in the survival of many organisms. However, flagella are known to be highly immunogenic and in certain settings Fla+ bacteria may be more readily cleared than Fla- organisms. In the model of acute P. aeruginosa pulmonary infection, one model of infection proposed is (1) in an initial phase of infection, P. aeruginosa from any aqueous setting are inadvertently inhaled or otherwise deliverd to the upper respiratory tract. It is at this stage where the expression of functional flagella is critical, providing chemotaxis toward desirable substrates, such as mucin, and the motility essential for widespread dissemination. In the normal host, the presence of defensions and additional antimicrobial peptides, the affinity of flagella components for mucin glycopeptides and the process of microciliary clearnce are likely to eradicate the transiently inhalled organisms from the respiratory tract before any epithelial immune response is elicited. () in an aimmunostimulatoyr phage of infection, patietns with CF and probably in those with nosocomial P. aeruginosa pneumonia, there is a seocnd stage of P. aeruginosa respiratory tract infection characterized by the development of airway inflammation in response to the organisms. The long flagella may function as a ether, recognizing acccessbile GM1 moieites on the apical surface of respiratory epithelial cells. (3) in an adaption stage and in CF, there are enough bacteria within the airway to allow for the slection of mutants which can adapt. To avoid clearance by the host phagocytic cells, Fla- mutants proliferate by deletions of genomic DNA. Accoringly, while flagellated organisms are likely to be involved in the earliest settings of infection, there is eventual selection of Fl1- mutants in response to host immune pressure. (Prince, “Role of Flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection” Infection & Immunity, 1988, p. 43-51).
Flagellar motility and chemotaxis help bacteria to find sources of nutrients and localize suitable ecological niches for growth, improving the efficiency of bacterial environmental colonization. Falgellar mediated chemotaxis can assist bacteria to reach specific sites in new hosts, facilitating its colonization or invasion as well as growth and maintenance. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023).
Some studies have reported that essential oils and ethanolic extracts have been confirmed to reduce the invasion of Campylobacter jejuni on INT407 epithelial cells and motility through modulation of its LuxS system. Flagellar motility and its medaited chemotaxis are essential for bacterial colinization and thus the reduction of the flagellar motility of bacteria could b e a target for anti-colonization. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)
Adhesion and invasion:
Adhesion of pathogens to host cells is a key step during the establishment of an infection. The need for viruses to subsequently be internalized into host cells is evident as these pathogens fully depend on them for replication In the case of bacteria, adhesion to the host cell surface may avoid mechanical clearance. Internalization allows persistence in a shielded niche, concealed from circulating antibodies. Entry in non-phagocytic cells also allows tissue invasion. To this end two main mechanisms may be used: expression of surface proteins that functionally ‘mimic’ natural ligands of host receptors, or the use of secretion systems, for example the type III secretion system, which directly inject proteins that will induce membrane and cytoskeleton rearrangements, triggering internalization. Depending on their mechanisms, internalization processes have been classified as zipper or trigger.
Adhesion and invasion are key steps in the colonization and infection of bacteria within the host. This procss requires the help of functional adhesins such as fibronectin-binding and laminin-binding proteins (Lmb). Fibronectin-binding proteins A and B is also important for persistent colonization of Staphylococcus aureus in the nose and intestine. Pili, especially type IV pili also play an important role in bacterial adhesion, ecological niche selection and establishment. Disruption of bacterial pilus-mediated adhesion processes can be achieved through inhibition of bacterail pilus production, adhesion inhibition, and adhesion based vaccines and antibodies. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023).
Bacterial adhesion is a process that allows bacteria to attach to host tissues and is
a crucial step in the bacterial pathogenesis. During this process, the bacterial surface
protein adhesins bind with complementary polyhydroxylated glycan or glycosaminogly
can (GAG) receptors located on the host cell surface.
Pili:
Pili are hairlike filamentous appendages which extend several micrometers from the bacterial surface and have long had an important role in the pathogenesis of gonococcal infections. Fresh isolates of meningococci are also pilliated, but the role of pili in pathogenesis has been less extensively studied than that of gonococcal pili, and consideration of their interation with host cells is complicated by the additional presence of hydrophilic capsule on the surface of the bacteria. Nevertheless, pili do mediate adhesion of meningococci to nasopharyngeal cells. (Heckels, “Structure and Funciton of pili of pathogenic Neisseria species” Clinical Microbiology Reviews, 1998)
The type IV pilus of Neiseria meningitidis is the major factor for meningococcal adhesion to host cells. Type IV pili are long, hair-like filaments that are helical polymers of one major subunit, pillin (Pile), produced by the N-temrinal cleavage of prepillin by prepilin papetidase PiLD. A mutant of N. meningitidis pilV, a minor pilin protein, internalized less efficiently to human endothelial and epithelial cells than the wild-type strain. Matrix-assisted laser desorption ionization–time of flight mass spectrometry and electrospray ionization tandem mass spectrometry analyses showed that PilE, the major subunit of pili, was less glycosylated at its serine 62 residue (Ser62) in the pilV mutant than in the pilV strain, whereas phosphoglycerol at PilE Ser93 and phosphocholine at PilE Ser67 were not changed. (Takahashi, “Meningococcal PilV potentiates Neisseria meningitidis type IV pilus-mediated internalization in to human endothelial and epithelial cells” Infeciton & Immunity, 2012).
Type VI Section System:
The type VI secretion system (T6SS) is not a type of pilus; rather, it is a complex molecular machine, often described as a bacteriophage tail-like structure, that injects toxic effector proteins into target cells. Type IV pili (T4Ps) are often involved by facilitating the close cell-to-cell contact necessary for the T6SS to function and deliver its toxins.
The type VI secretion system (T6SS) is used in gram-negative bacteria and can deliver toxic substances to competitors and kill them or alter some of their key functions, which provides an advantage for the pathogen to conquer the colonization resistance of the host commensal bacterial. This promotes its colonization and persistence. Effective inhibition of T6SS can reduce the invasion of commensal microbiome and niche dominance by exogenous pathogens. A mixture of organic acids and plant extracts was reported to down-regulate the expression of T6SS related genes and reduce the virulence and cecum colonization of Camplobacter jenuni and Camplylobacter coli. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)
Fimbriae:
Fimbriae are short, numerous hair-like appendages primarily responsible for bacterial adhesion and colonization, while pili are generally longer, fewer in number, and can have various functions, including transferring genetic material through conjugation (sex pili) or facilitating cellular motility.
Among the many factors that contribute to the adhesion of Salmonella to host cells,
fimbriae are some of the most important and have been extensively studied. Fimbriae, also called “attachment pili,” are extracellular appendages that can be found on
many bacteria. Fimbriae are thinner and shorter than flagella and measure 2–8 nm across
and 0.5–10 μm in length. Salmonella fimbrial gene clusters (FGCs) usually are composed
of between 4 and 15 genes and encode for the structural element, their assembly, and
the regulatory proteins required to synthesize and export extracellular fimbriae. So far,
more than 30 different FGCs have been identified
Cellular Pumps and enviromental condition tolerance:
The intracellular pH of Gram-negative bacteria, including Salmonella, is relatively constant at pH 7.6–7.8. For Salmonella to survive the low pH, as well as many organic acids and digestive enzymes that are present in the stomach, they utilize approaches to keep their intracellular pH relatively constant even as the extracellular pH can change dramatically. The maintenance of
intracellular pH of Gram-negative bacteria, including Salmonella, occurs through cellular
pumps and the sodium/potassium-proton antiporter system that extrudes protons from
the bacterial cytoplasm in low pH settings. Acid adaptation helps Salmonella survive
in the stomach, and higher levels of acid tolerance have been associated with more
highly virulent Salmonella.
Outer Membrane Vesicles are small, spherically bilayered (100–300 nm) vesicles released into extracellular milieu from the OM of Gram-negative bacteria. Several bacterial species have been reported to produce OMVs, such as Escherichia coli, Pseudomonas aeruginosa, Shigella, Helicobacter pylori, ampylobacter jejun and others.
Study of OMVs reveal abundance of OM proteins (OMPs; OmpA, OmpC, and OmpF), periplasmic proteins (AcrA and alkaline phosphatase) and a series of virulence factors involved in the adhesion and invasion of host tissues.
Outer Membrane Vesicles are speculated to modulate many physiological and pathological procedures. Exploiting their physiological characteristics, delivery of a series of therapeutic cargos (siRNA, microRNA, and proteins) to tissues has been already achieved. (Jan, “Outer Membrane Vesicles (OMVs) of Gram-negative bacteria: a perspective update” frontiers in microbiology, 2017)
Disruption of Human Gut Microbiome:
Introduction: The human gut microbiome is well known to be an improtant reservoir of ARG and likely to be a key factor in regualting the emergency and spread of ARB. Antibiotic therapy for example can have a large impact on the human gut microbiome and its resistome, which eliminates not only pathogenic but also beneficial bacterial. The impact of antibiotics is influenced by the type of antibiotic, the route of adminsitration and the microbiome status of the patient. Antibiotics, expecially broad spectrum antibiotics, may generally have a negative impact on the diversity of the gut microbiome and promote the expansion of antibiotic specific ARG in humans. Appropritate routes of adminsitraiton combined with antibiotics with less impact on the gut microbiome can better reduce antibotic resistance while minimizing damage to the gut microbiome. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)
Fecal microbiota transplantation (FMT): the delivery of feces form a healthy donor to the recipient’s intestine via enema or oral capsule, can rapidly reverse diseases associated with intestinal flora dysbiosis, enhance Cr, and limit increasing antibiotic resistance. FMT is theoretically the replacement of ARB in the recipient by a high abundance of non-antibiotic resistant bacteria.
Benefits of Probiotics: Probiotics and prebiotic regulate the intestinal microbiota to enhance its CR intestinal barrier function. Some probiotic organisms (like lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Bifidobacterium animalis Bb-12 and Saccharomyces crevisia boulardii have been reported to enhance nonspecific cellular immune response and miantain the gut homeostasis.
A healthy gut microbiome is critical for maintaining its CR and reducing exogenous bacterial colonization. Dietary fiber can promote the production of SCFA (including acetate, propionic acid and butyric acid) by relevant bacterial. SCFA plays an important role in maintaining the integrity of the gastrointestinal barrier and the loss of SCFA may lead to a decrease of CR against pathogenic bacteria. For instance, in patients receiving antimicrobila therapy, the abundance of the SCFA producing commensal bacteria decreased, accompanied by the expansion of carbopenem-resistant Enterobacteriaceae . SCFA inhibited the growth of antibiotic-restant E. coli and K. pneumoniae. Conversely, a high sugar, high fat, and high protein diet promoted colonizaiton by exogenous bacteria and promoted ARG expansion and transfer. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)
Polyphenols like pomegranate ellagitannins, green tea polyphenols, resveratrol, juice, blueberry and mango pup polyphenols and vitamins (A dn D) and Omega-3 fatty acids, also play an essential role in regualting microbiota and strenghtening barrier function. Polyphenols expand the population of beneficial species such as Bifidobacterium and Lactobacillus. and significantly enhance intesting related bacterial produciton of SCFA, which enhance intestinal barrier function.
Interaction with Immune system: Under normal conditions, the immune system communes with commensal bacteria—the so-called good bacteria—on the skin and elsewhere in the body while keeping pathogens out. Although some strains of S. aureus are pathogenic, others are commensals, a vital member of the skin microbiome. S. aureus, for example, is a common commensal colonizer of the nasal passages. For example, children with the genetic condition known as recessive dystrophic epidermolysis bullosa—RDEB—have immune responses and clinical severity shaped by skin-adapted S. aureus. S. aureus exists in a multitude of strains, some antibiotic resistant and more dangerous than others. Scientists classify strains based on their genetic composition, whether they repel antibiotics or produce toxins. In RDEB, S. aureus causes blisters. The more dangerous the strain, the more complicated the condition of the affected child. RDEB causes blisters and wounds because of a mutation in the COL7A1 gene of affected children. S. aureus exploits the mutation, ensuring its survival as it attacks and colonizes the mucous membranes and other sites throughout the body, especially the skin. Children with RDEB develop blisters of the mouth and esophagus, which can lead to difficulty with eating and swallowing. Under normal conditions, the immune system communes with commensal bacteria—the so-called good bacteria—on the skin and elsewhere in the body while keeping pathogens out. Although some strains of S. aureus are pathogenic, others are commensals, a vital member of the skin microbiome. S. aureus, for example, is a common commensal colonizer of the nasal passages. Children with severe RDEB displayed a distinct immune signature marked by elevated quantities of CD4+ T cells and mucosal-associated invariant T—MAIT—cells that expressed interleukin-17A, an inflammatory signaling molecule. See Jamet
Colonization:
Urease:
Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymatic activity, urease has a toxic effect on human cells. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. (Kaca “Bacterial Urease and its Role in Long-Lasting Human Diseases” Current Protein and Peptide Science, 2012, 13, 789-806).
Ureolytic activity is often observed in pathogenic bacteria. Such a feature is characteristic of pathogenic Staphylococcus strains. Over 90% of clinical methicillin resistant Staphylococcus aureus strains are capable of urea hydrolysis. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).
In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as molecular mimicry, leads to the appearance of autoantibodies, which take part in host molecules destruction. Detection of antibodies-binding motives (epitopes) in bacterial proteins is a complex process. However, organic chemistry tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serologic investigations. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).
Urease is capable of urea hydrolysis. This compound is widespread: it is found in the natural environment (water and soil) and in human body, where its occurrence is connected with protein degradation. In humans, urea is a factor of normal functions of kidneys. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).
A healthy adult excretes about 30 g of urea per day. However, it is present not only in urine, but also in blood serum, sweat and even in stomach.
Urease is a nickel-containing enzyme, which requires activity of a few additional proteins for acquisition of its hydrolytic properties. This process involves genes coding structural enzyme polypeptides as well as genes coding accessory proteins, located in a joint cluster.
The role of urease in bacterium surviving in unfavorable microenvironment in the host’s body is especially noticeable in case of H. pylori, a causative agent of gastritis and peptic ulceration. Ureolytic activity is essential for surviving M. tuberculosis, an etiologic factor of tuberculosis, a long-lasting inflammatory lung disease. Bacteria infect macrophages. They reside in phagosome, where alkalization due to ureolytic activity and subvert phagosome maturation takes place. Additionally, urease activity enables bacterium to exist in the environment where nitrogen sources are limited to urea.
Exterior Protection:
Cell Wall:
The cell wall of Gram-positive bacteria such as S. pyogenes assist the organism in its survival. Not only does the cell wall provide a major passive function as a rigid exosckeleton in protection of the cytoplasmic membrane against hypotonic death of the bacteria, but it also serves actively in binding proteins, carbohydrates, and lipids that are displayed on the bacterial surface. In turn, these surface exposed molecules are involved in growth and division of the bacterial cells and also serve as adhesins and invasins for eukaryotic host cells. (Castellino, “The M protein of Streptococcus pyogenes Strain AP53 retains cell surface functional plasminogen binding after inactivaiton of the sortase A gene” J. Bacteriology, 2020).
The mycobacterial bacillus is encompassed by a remarkably elaborate cell wall structure.
The mycolyl-arabinogalactan-peptidoglycan (mAGP) complex is essential for the viability
of Mycobacterium tuberculosis and maintains a robust basal structure supporting the upper
“myco-membrane.”
Outer Membrane:
Gram-negative bacterial cell envelope consists of two lipid bilayers, an inner membrane (IM) and an outer membrane (OM), separated by a periplasmic space which contains a rigid layer of peptidoglycan. Unlike the symmetric IM, the OM is largely asymmetric, with the outer leaflet composed predominately of lipopolysaccharide (LPS) and an inner leaflet consisting of glycerophospholipids (GPL). The asymmetry of the OM serves as a selective barrier, permitting the uptake of necessary nutrients while blocking the entry of toxic compounds. The OM is a complex organelle containing pore-forming -sheet membrane proteins that contribute to the selective barrier multiprotein organelles, including secretion machineries and flagella that
bridge the membranes and lipoproteins that can form a covalent link from peptidoglycan to the OM. See Fang
Endospores:
Bacterial spores contain genes in the form of DNA within the spore’s core, protected by specialized layers and proteins. While the spore itself is dormant, it holds the complete genetic blueprint of the cell and also contains mRNA transcripts for some housekeeping genes, allowing for a rapid response once conditions become favorable for germination and growth.
Endosporulation is characterized by the morphogenesis of an endospore within a mother cell. Based on the genes known to be involved in endosporulation in the model organism Bacillus subtilis, a conserved core of about 100 genes was derived, representing the minimal machinery for endosporulation.
Spores formed by bacteria of the genera Clostridium and Bacillus provide a uniquely effective means of surviving environmental stress. they act as the infectious agent in pathogens such as Bacillus anthracis, Clostridium botulinum and Clostridium difficile. In the anaerobic clostridia, they are essential for survival in air. Despite the early evolutionary divergence of the genera Clostridium and Bacillus, a number of the genes responsible for regulation and morphogenesis in sporulation are coserved. (Bullough, “Architecture and self-assembly of Clostridium sporogenes and Clostridium botulinum spre surfaces illustrate a general protective strategy across spore formers”. Molecular Biology and Physiology, 2020).
–Sporulation is the process by which a vegetative cell undergoes a developmental change to form a metabolically inactive and highly resistant endospore. For most species, sporulation is caused by conditions unfavorable for growth, such as nutrient depletion. Both internal and external signals can signal the vegetative cell to cease growth and form a less metabolically demanding and more resistant state that can survive nutrient poor conditions. Under these conditions, a decrease in guanosine triphosphate (GTP) levels is known to be an internal signal inducing sporulation. Externally, cell density is a factor that has been shown to play a role in the induction of sporulation, as B. subtilis, for example, possesses a sporulation-linked quorum-sensing system to detect both nutrient limitation and high cell density. It is also important to appreciate that not all cells in a population sporulate, as sporulation is controlled by a complex set of feed-forward and feedback loops which modulate the number of cells entering sporulation.
The outermost layer of the mature spore, the balloon-like exosporium, is found in only a select number of spore-forming species, including Bacillus cereus and Bacillus anthracis, as well as C. difficile. Located below the exosporium is the spore coat, which is made up primarily of protein in a composition that differs greatly between species. Beyond providing resistance, the spore coat is also able to sense and respond to external environmental signals, allowed by the coat’s flexibility and species-specific ridges formed during sporulation. Located beneath the spore coat is the outer membrane, which has little known function in resistance or germination, but has been shown to be important in sporulation. The spore cortex is composed of a thick layer of peptidoglycan (PG), which plays an important role in germination, sporulation, and wet heat resistance, the latter most likely by the cortex’s involvement in reducing spore core water content. The germ cell wall, located underneath the spore cortex, is also a PG layer. Unlike the cortex, the structure of the germ cell wall PG is identical to that of vegetative cells. The inner membrane (IM) of the spore, located below the germ cell wall and surrounding the spore core, has a fatty acid and phospholipid composition rather similar to that in growing cells. The center of the mature spore, called the core, contains ~25% of its dry weight as CaDPA, a 1:1 chelate of pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA) and Ca2+, and CaDPA accumulation from the mother cell contributes to the core’s low water content, as low as 25% of wet weight.
Spore germination is the process by which the dormant spore is converted into a vegetative cell. Germination can be divided into three stages: activation, Stage I of germination and Stage II of germination; completion of germination is followed by outgrowth leading to production of a vegetative cell. Notably, activation and Stage I of germination requires no ATP or macromolecular synthesis, and this is most likely also true of Stage II. However, outgrowth requires resumption of metabolism and synthesis of new RNAs and proteins. Despite being metabolically inactive, spores are capable of sensing the external environment and returning to life during germination. Since germination is an irreversible process, appropriate signals to commit to germination are important to ensure conversion of most spores into growing cells. Within the gut, bile acids and the hosts’ nutritional intake are the major source of germinants for the sporobiota.
In contrast with the pathogenic species, many spore-forming species have been implicated as commensal and are even central to a healthy gut microbiome. Commensal spore-formers include members of the Clostridium clusters XI and XIVa, such as Clostridium leptum, Clostridium scindens, and C. innocuum. Bacterial spores, specifically those of the Bacillus genus, have been used as probiotics in humans since the 1960s to treat various disorders linked to the gastro-intestinal tract. B. subtilis spores of the variety Natto are consumed in Japan, as they are the main microbial species in the fermented soybean product Natto.
Spores can also be genetically manipulated to carry immunogenic peptides; thus, acting as vaccine vehicles. The temperature stable nature of spores offers a shelf-stable and therefore economically sound alternative to vaccine vectors such as attenuated viruses or RNA, which often need to be stored at freezing temperatures. Commensal spore-forming bacteria might be a safe alternative to viral vaccine vectors, which have the rare potential to convert or cause unwanted hyperimmune reactions.
Capsule:
Polysaccharide capsules are found on the surface of a wide range of bacteria. With gram-negative bacteria, the capsule lies outside the outer membrane and is composed of highly hydrated polyanionic polysaccharides. Capsules have a significant role in determining access of certain molecules to the cell membrane, mediating adherence to surfaces, and increasing tolerance of desiccation. Furthermore, capsules of many pathogenic bacteria impair phagocytosis and reduce the action of complement-mediated killing.
The polysaccharide capsule of Streptococcus pneumoniae is the dominant surface structure of the organism and plays a critical role in virulence, principally by interfering with host opsonophagocytic clearance mechanisms. The capsule is the target of current pneumococcal vaccines, but there are 98 currently recognised polysaccharide serotypes and protection is strictly serotype-specific. Widespread use of these vaccines is driving changes in serotype prevalence in both carriage and disease.