Infection & Immunity Tutorials (BBS3024) 2024-2025 PDF

Infection & Immunity BBS3024 Academic year: 2024–2024 Basic Challenge 3 Evolution of viruses Faculty of Health, Medicine and Life Sciences Bachelor Biomedical Science  2017 Maastricht University, Faculty of He alth, Medic...

Infection & Immunity BBS3024 Academic year: 2024–2024 Basic Challenge 3 Evolution of viruses Faculty of Health, Medicine and Life Sciences Bachelor Biomedical Science  2017 Maastricht University, Faculty of He alth, Medicine and Life Sciences. Nothing in this publication may be reproduced and/or made public by means of printing, offset, photocopy or microfilm or in any digital, electronic, optical or any other form without the prior written permission of the owner of the copyright.  Faculty of Health, Medicine and Life Sciences The Trick. "Any virus that can cause disease in humans must have at least one immune evasion mechanism—at least one immune evasion “trick.” Without the ability to evade the immune system, a virus is usually harmless. Understanding immune evasion by a virus is frequently important for understanding the pathogenesis of the virus, as well as understanding challenges faced by the adap ve immune system and any candidate vaccine." Literature: Se e A, Cro y S. Adap ve immunity to SARS-CoV-2 and COVID-19. Cell. 2021;184(4):861-880. doi:10.1016/j.cell.2021.01.007 Case COVID 19. A 48-year-old man presents to the emergency department, referred by his family doctor due to fever and significant shortness of breath. He has experienced flu-like symptoms for the past three days—fever, muscle aches, sore throat, runny nose, and cough—coupled with progres-sive shortness of breath that has not responded to oral an bio cs (amoxicillin). Notably, he reports a recent loss of smell and taste. The pa ent's medical history reveals obesity (body mass index: 31 kg/m²) and a long history of smoking—one pack of cigare es per day for 25 years, along with vaping for the last three years. He has not received vaccina ons for influenza A and B or for SARS-CoV-2. In his community, several individuals are exhibi ng similar flu-like symptoms. On physical examina on, he has fever and exhibits signs of dyspnea and tachypnea, with a rapid pulse and normal blood pressure. Ausculta on reveals bilateral lung involvement con-sistent with pneumonia. He requires supplemental oxygen at 5 liters per minute via nasal can-nula to maintain acceptable oxygen satura on levels. A nasopharyngeal swab tests posi ve for SARS-CoV-2 via polymerase chain reac on (PCR), with a cycle threshold (CT) value of 22, while tests for influenza and parainfluenza viruses are nega ve. Laboratory results indicate signs of systemic inflamma on. Imaging studies, includ-ing chest X-ray and CT scan, reveal diffuse bilateral pneumoni s without lung emboli. The medical team diagnoses him with severe COVID-19 pneumonia. They ini ate treatment with intravenous dexamethasone and tocilizumab to manage the inflammatory response. Ad-di onally, they deliberate on further therapeu c op ons, including remdesivir, nirma-trelvir/ritonavir, molnupiravir, monoclonal an bodies, or convalescent plasma. They also plan to assess his blood serum for the presence of an bodies against SARS-CoV-2, which may in-form his immune status. BBS3024 Infection & Immunity 2024-2025 2 BBS3024 2022-2023 2  Faculty of Health, Medicine and Life Sciences Case HIV A 32-year-old woman is referred by her family doctor to the emergency department because of high fever, flu-like symptoms, cervical lymphadeni s, and a skin rash. Her previous medical history is unremarkable. On physical examina on she appears ill. Temperature 39.0⁰ Celsius, blood pressure 120/80 mmHg and pulse rate 94/minute. Her tonsils are enlarged. Cervical, axillary and inguinal lymph nodes are enlarged and elas c on palpa on. Blood results show lymphocytopenia. Serology is posi ve for an acute HIV (human immunodeficiency virus) infec on, Fiebig stage 3. The consulted infec ous diseases specialist recommends the pa ent to start combina on an retroviral therapy (cART) immediately. However, the pa ent is in doubt and wonders why she can’t wait to see how the infec on evolves and if her immune system can cure the infec on. Next to this, she wants to know if HIV, like covid-19, also has a lot of variants. Compare and constrast: SARS-CoV2 and HIV BBS3024 Infection & Immunity 2024-2025 3 BBS3024 2022-2023 3 Tutorials Infection & Immunity BBS3024 Academic year: 2024–2025 Basic Challenge 1 Commensal Microbes – with a little help from our (little) friends Faculty of Health, Medicine and Life Sciences Bachelor Biomedical Science  2017 Maastricht University, Faculty of He alth, Medicine and Life Sciences. Nothing in this publication may be reproduced and/or made public by means of printing, offset, photocopy or microfilm or in any digital, electronic, optical or any other form without the prior written permission of the owner of the copyright.  Faculty of Health, Medicine and Life Sciences Basic Challenge 1 Commensal Microbes – with a little help from our (little) friends Samira is a healthy and ambitious student who is fascinated by the tiny living organisms residing on and inside of us – microbes. Which microbes do you recognize in the figure below? What do you still know about the structure and classification of bacteria? Do microbial ecosystems differ across body sites? Why? Figure 33.2 (Prescot’s Microbiology 12th edition) BBS3024 Infection & Immunity 2024-2025 2 BBS3024 2022-2023 2  Faculty of Health, Medicine and Life Sciences She is particularly intrigued by the many functions that all these billions of microbes have on us as a host and how we can manipulate them for our own well-being. To maintain her own balanced and healthy microbiota, she puts quite some effort in a healthy lifestyle. She finds this even more important after her recent delivery of her first child. Unfortunately, Samira had to take several courses of antibiotics during pregnancy and labor and finally had to undergo a cesarean section. She is now very worried that her newborn might not be colonized by a healthy microbiota on its different body sites due to these artificial circumstances. She is afraid that the microbiota function might be impaired and her child might develop dysbiosis-related diseases or that her child’s commensal microbes cannot keep opportunistic or strict pathogenic microbes at bay. Therefore, she is even more eager to allow her newborn child to have a lot of contact with nature and provide optimal nurture for a positive impact on the microbial communities to be developed across the different body sites. Still, she is wondering if there was an additional option to compensate for the antibiotics and the artificial way of delivery. Her curiosity led her to delve into the literatur where she found evidence on different interventions, such as probiotics and vaginal swabs or fecal microbiota transplantation (FMT). The table below depicts several different bacteria and infections they can cause. Could you reason whether they should be considered opportunistic or true pathogens and why? Which infections might be the result of a dysbiotic microbiome? Bacterium/bacterial Infec on Opportunis c or Related to TCDB community true pathogen? dysbiosis? exotoxins > - sporulation Clostridioides difficile An bio c-induced coli s opp? Salmonella enterica serotype foodborne salmonellosis Typhimurium true E. coli urinary tract infec on opportunistic hemolytic Streptococcus pneumoniae Secondary bacterial ? capsule - macrophage resistant pneumonia following respiratory viral infec on opp Bifidobacterium (formerly bacterial vaginosis Gardnerella) vaginalis opportunistic BBS3024 Infection & Immunity 2024-2025 3 BBS3024 2022-2023 3  (sporulation bacterial cells endospores -dormant in number of -no increase cells 1. What are the Different Types of Bacteria (Gram-Positive vs. Gram-Negative)? nutrient Overview: depletion struct are o Bacteria are categorized into Gram- Ngramt positive and Gram-negative based on their cell wall spheres · structure, a classification established by the Gram rods staining technique. This distinction reflects differences · spiral shapes in their chemical composition, structure, pathogenic potential, and antibiotic susceptibility. by transpeptidase ↓ penicillin > - Gram-Positive Bacteria: binding proteins ·thin filaments o Cell Wall Structure: Nage Nam Peptidecross-linking ains fimbriae adhesion ▪ Thick peptidoglycan layer (20-80 nm): This rigid > - > - filaments pilli (sex pillus long > structure provides physical support and - · types pilli-movement > protection. The thickness of the peptidoglycan - · flagella movement - layer is what enables Gram-positive bacteria to retain the crystal violet stain, giving them a capsule thick binds topeptide a - purple color under the microscope. teichoic outer layer adherence to each > - ▪ Teichoic and lipoteichoic acids: These acids are lipoteichoic other embedded in the cell wall and contribute to its in plasma rigidity. They also help bacteria adhere to host memprane 70S ribosomes tissues, initiate infections, and elicit immune 305550S responses. cytoplasm/membrane ▪ Absence of outer membrane: Without an outer no nucleus membrane, Gram-positive bacteria are more supercoiled DNA susceptible to certain antibiotics (e.g., penicillin, > - nucleoid/plasmid which targets peptidoglycan synthesis). plasmidshotals is o Examples: ▪ Staphylococcus aureus: Commonly found on the et skin and nasal passages; can cause a variety of circular/linear antibiotic resistance have - can be shared  infections, from superficial skin infections to life- threatening diseases like endocarditis. ▪ Staphylococcus epidermidis: Part of the skin microbiome, generally non-pathogenic but may cause infections in individuals with weakened immune systems or invasive medical devices (e.g., catheters). ▪ Lactobacillus species: Play a vital role in the gastrointestinal and genitourinary tracts by producing lactic acid, which maintains an acidic environment and inhibits pathogen growth. o Function in Microbiomes: ▪ In environments like the skin, Gram-positive bacteria form a protective barrier against pathogens, assist in wound healing, and contribute to the skin’s acidic pH. ▪ In the vagina and gut, Lactobacillus species produce lactic acid to maintain low pH, inhibiting pathogens and protecting against infections such as bacterial vaginosis. to antibiotics Gram-Negative Bacteria: more resistant o Cell Wall Structure: ▪ Thin peptidoglycan layer: Located between the inner and outer membranes, this thin layer does not retain the crystal violet stain and thus appears pink or red after counterstaining. lipid accharide ▪ Outer membrane with lipopolysaccharides (LPS): The LPS component is a t powerful endotoxin that can cause strong immune responses. LPS is a major virulence factor, and in severe infections, it can lead to septic shock.  hydrophilic Porin channels: Embedded in the outer ▪ molecules enter membrane, these channels regulate the entry of can · periplasmic space nutrients and antibiotics, often contributing to antibiotic resistance. o Examples: ▪ Escherichia coli: A key member of the gut microbiome, assisting in nutrient absorption, vitamin synthesis, and metabolism. However, pathogenic strains (e.g., E. coli O157 ) can cause foodborne illnesses and severe urinary tract infections. ▪ Bacteroides fragilis: Predominantly anaerobic, Bacteroides species are crucial in bile acid metabolismand complex carbohydrate breakdown. Though typically commensal, they can cause abscesses if they escape the gut environment. ▪ Pseudomonas aeruginosa: An opportunistic pathogen known for its role in hospital-acquired infections, particularly in immunocompromised patients or those with cystic fibrosis. o Function in Microbiomes: ▪ Gut microbiome: Many Gram-negative bacteria are essential for carbohydrate fermentation, SCFA production, and modulation of immune responses. ▪ Colonization resistance: By occupying ecological niches in the gut, Gram-negative bacteria help prevent colonization by pathogenic bacteria. 2. Opportunistic vs. True Pathogens vs. Commensal Bacteria  Overview: o Bacteria in the human body interact with the host in varying ways, which are influenced by the host’s immune status, microbiome composition, and environmental conditions. These interactions can classify bacteria as true pathogens, opportunistic pathogens, or commensal bacteria. True Pathogens: o Characteristics: ▪ True pathogens possess inherent virulence factors, enabling them to cause disease in healthy individuals, regardless of immune status. ▪ They actively invade host tissues, evade immune responses, and can disseminate to various organs. o Examples: ▪ Mycobacterium tuberculosis: Causes tuberculosis, an airborne infection where the bacteria survive and replicate within host macrophages, evading immune detection. ▪ Salmonella typhi: Causes typhoid fever; can survive gastric acidity and penetrate the intestinal mucosa, entering the bloodstream and lymphatic system. o Mechanism: ▪ These pathogens often possess specialized factors, like toxins or invasion proteins, that directly damage host tissues or impair immune defenses. Opportunistic Pathogens: o Characteristics: ▪ Normally non-pathogenic and may even benefit the host. However, when host immunity is  compromised (due to factors like stress, antibiotic use, or underlying disease), opportunistic pathogens can exploit these conditions to cause disease. o Examples: ▪ Staphylococcus epidermidis: A common skin commensal that can cause infections in immunocompromised patients or individuals with indwelling medical devices. ▪ Escherichia coli: A gut commensal; certain strains can cause urinary tract infections (UTIs) when they enter the urinary tract. o Role in Microbiome and Disease: ▪ Opportunistic pathogens are implicated in antibiotic-associated colitis (e.g., Clostridium difficileovergrowth following antibiotic therapy), bacterial vaginosis (overgrowth of anaerobes like Bifidobacterium vaginalis due to disrupted vaginal pH), and UTIs. Commensal Bacteria: o Characteristics: ▪ Commensals are naturally occurring, generally non-harmful bacteria that coexist with the host and often provide benefits, such as aiding digestion or preventing pathogen colonization. ▪ Through colonization resistance, commensals occupy niches and compete for nutrients, helping prevent the establishment of pathogens. o Examples: ▪ Lactobacillus in the vaginal and intestinal microbiomes helps maintain a low pH, creating  an environment hostile to pathogens. ▪ Staphylococcus epidermidis on the skin provides a natural barrier against pathogenic bacteria. o Clinical Importance: ▪ The disruption of commensal bacteria can lead to dysbiosis, where a reduction in commensals can facilitate pathogen overgrowth, contributing to conditions like bacterial vaginosis and certain gastrointestinal infections. 3. Where are They Located, and Why? Skin Microbiome: o Location: Found on the outer epidermis, sebaceous glands, and hair follicles. o Environment: Relatively dry and mildly acidic with regions of high lipid content (sebum). o Primary Bacteria: Staphylococcus epidermidis, Propionibacterium acnes. o Role: ▪ Produces antimicrobial compounds (e.g., bacteriocins) that prevent pathogenic colonization. ▪ Helps regulate the skin’s immune response and maintains a low pH, inhibiting harmful bacteria like Staphylococcus aureus. Respiratory Tract Microbiome: o Location: Nasal passages, throat, and lungs. o Environment: Moist with variable pH and oxygen levels. o Primary  Bacteria: Streptococcus and Neisseria species. o Function: ▪ Helps trap inhaled pathogens and particles, with mucociliary clearance supporting bacterial removal. ▪ Respiratory commensals can modulate immune responses, reducing inflammation and protecting lung tissue. Gastrointestinal Tract Microbiome: o Location: Stomach, small intestine, and colon. o Environment: Nutrient-rich with a gradient in pH (acidic stomach, neutral intestines) and a low-oxygen environment in the colon. o Primary Bacteria: Bacteroides, Lactobacillus, Escherichia coli, Bifidobacterium. o Role: ▪ Facilitates carbohydrate and protein fermentation, producing short-chain fatty acids (SCFAs) like butyrate, propionate, and acetate, liver which provide energy to colon cells and maintain ↓ gut health. Gile acids primary ▪ Plays a role in bile acid metabolism, breaking emodified by microbes ↳ secondary file acids down bile acids that regulate lipid absorption and ↳ modulatethe microbes have antimicrobial properties. ▪ Aids in vitamin synthesis (e.g., vitamins B and K) and bolsters immune defenses against pathogenic bacteria. Genitourinary Tract Microbiome: o Location: Primarily the vagina and, to a lesser extent, the urethra.  o Environment: Acidic pH (especially in the vagina), largely anaerobic. o Primary Bacteria: Lactobacillus and, in cases of bacterial vaginosis, overgrowth of Bifidobacterium vaginalis. o Role: ▪ Lactobacillus species maintain an acidic pH, which prevents colonization by potential pathogens. ▪ An imbalance can lead to bacterial vaginosis, which is characterized by an overgrowth of anaerobic bacteria. 4. What is the Function of the Gut Microbiome (Bacterial Metabolism and Immunity)? The gut microbiome is integral to maintaining metabolic and immune balance in the human body. It performs complex functions that support digestion, produce essential metabolites, influence immune development, and guard against pathogens. Metabolic Functions: o Short-Chain Fatty Acid (SCFA) Synthesis: ▪ Process: SCFAs, primarily acetate, propionate, and butyrate, are produced during the fermentation of dietary fibers and complex carbohydrates by gut bacteria, particularly by members of Bacteroidetes and Firmicutes phyla. ▪ Role: ▪ Energy Source: Butyrate serves as the primary energy source for colonocytes, aiding in intestinal barrier integrity. ▪ Anti-inflammatory Effects: SCFAs influence immune cells, promoting anti-inflammatory  cytokine production and suppressing pro- inflammatory pathways. Butyrate, in particular, inhibits histone deacetylases, which has been linked to reduced inflammation. ▪ Systemic Health: SCFAs can enter circulation and impact peripheral tissues, including the liver, where they influence lipid metabolism, glucose homeostasis, and insulin sensitivity. o Carbohydrate and Protein Fermentation: ▪ Process: Through fermentation, gut bacteria break down non-digestible carbohydrates, proteins, and peptides into gases and organic acids. ▪ Role: ▪ Gaseous Byproducts: Hydrogen, carbon dioxide, and methane gases are generated, which may affect gut motility and contribute to the gut’s microbial community structure. ▪ Protein Metabolism: Certain gut bacteria degrade amino acids into byproducts like indole, ammonia, and hydrogen sulfide. These can exert toxic effects on the gut lining if overproduced, as seen in dysbiosis. o Bile Acid Metabolism: ▪ Primary and Secondary Bile Acids: Primary bile acids are synthesized in the liver and modified by gut bacteria into secondary bile acids (e.g., deoxycholic acid). ▪ Role: ▪ Fat Digestion: Bile acids are crucial for emulsifying dietary fats, allowing  absorption. ▪ Antimicrobial Action: Secondary bile acids create an environment that prevents overgrowth of pathogenic bacteria. Additionally, specific bacteria like Bacteroides regulate bile acid modification, impacting lipid metabolism methanogenesis and glucose regulation in the host. ▪ Gut Integrity: Bile acids also signal to gut epithelial cells to maintain mucosal integrity and balance inflammation. Immune Functions: o Colonization Resistance: ▪ Mechanism: The microbiome provides “colonization resistance” by competing with pathogens for nutrients and attachment sites and by producing antimicrobial compounds like bacteriocins. ▪ Example: In the gut, Bacteroides and Lactobacillus species prevent colonization by pathogens such as Clostridium difficile, which can overgrow if commensals are diminished, as seen in antibiotic- associated colitis. o Modulation of the Immune System: ▪ Development of Immune Tolerance: Early-life interactions between gut bacteria and the host’s immune cells aid in “educating” the immune system to tolerate beneficial microbes while remaining responsive to pathogens. ▪ Regulation of Inflammation: The microbiome helps maintain a balance between pro-  inflammatory and anti-inflammatory responses. Bifidobacterium and Lactobacillus pro mote regulatory T-cells, which suppress excessive immune responses, reducing risk of inflammatory bowel disease (IBD) and allergies. ▪ Pathogen Defense: Certain microbes stimulate the production of mucins, antimicrobial peptides, and IgA antibodies that collectively enhance the gut’s defense barrier. 5. How Do We Acquire Our Commensal Bacteria? The process of acquiring a stable microbiome begins at birth and continues to evolve through interactions with diet, environment, and lifestyle. Birth Mode: o Vaginal Birth: Infants born through the vaginal canal are exposed to maternal vaginal and intestinal microbiota, such as Lactobacillus, Bacteroides, and Bifidobacterium. This initial exposure provides infants with protective bacteria that colonize the gut and support early immunity. o Cesarean Section (C-Section): C-section-delivered infants primarily acquire skin-associated bacteria, including Staphylococcus, Corynebacterium, and Propionibacterium, which may lack some of the beneficial gut-colonizing species found in vaginal births. Studies have shown that C-section delivery can lead to delayed microbial diversity, with increased risks for immune-mediated conditions such as allergies and asthma. Infant Feeding: o Breastfeeding: Breast milk contains oligosaccharides  that act as prebiotics, selectively nourishing beneficial bacteria like Bifidobacterium infantis. Breast milk also contains immune factors (e.g., IgA, lactoferrin) that help shape a protective microbiome and prevent pathogenic colonization. o Formula Feeding: While formula lacks the unique oligosaccharides and antibodies in breast milk, many formulas are now enriched with prebiotics and probiotics to support a more balanced microbiome. Environmental Exposure: o Early-Life Interactions: Exposure to family members, pets, and other environmental factors plays a significant role in microbiome diversification. Children raised in rural or farm environments tend to develop more diverse microbiomes with reduced allergy and asthma risks, potentially due to increased microbial exposure. o Antibiotic Exposure: While sometimes necessary, early antibiotic use can disrupt the natural microbial colonization process, sometimes leading to reduced microbial diversity and a higher susceptibility to infections and allergic diseases. Dietary Transitions and Lifestyle: o Introduction of Solid Foods: Introducing a varied diet with fibers promotes the growth of bacteria involved in complex carbohydrate digestion, including Bacteroides and Firmicutes, enhancing SCFA production. o Lifestyle Factors: As individuals age, lifestyle factors such as diet, exercise, stress, and travel continue to influence the microbiome’s composition and health- related functions.  6. How Can We Manipulate Our Commensal Microbiome for Our Well-Being? The composition and function of the microbiome can be modified to enhance health, reduce disease risk, and manage certain conditions. Dietary Modifications: o Fiber-Rich Diets: Diets high in dietary fiber from fruits, vegetables, legumes, and whole grains promote the growth of SCFA-producing bacteria like Bifidobacterium and Lactobacillus and increase butyrate production, which has anti-inflammatory effects. o Polyphenols and Fermented Foods: Foods rich in polyphenols (e.g., berries, green tea) and fermented foods (e.g., yogurt, kimchi) support the growth of beneficial bacteria, enhancing gut barrier integrity and reducing inflammation. Prebiotics: symbolis o Definition and Function: Prebiotics are non-digestible fibers that selectively stimulate the growth of beneficial bacteria. Common prebiotics include inulin, fructooligosaccharides (FOS), and galactooligosaccharides (GOS). o Health Benefits: Prebiotics enhance the growth of Bifidobacterium and Lactobacillus, leading to increased SCFA production, improved immune modulation, and reduced risk of metabolic disorders. Probiotics: o Definition: Probiotics are live microorganisms that, when ingested in adequate amounts, confer health benefits by promoting a healthy balance of gut bacteria.  oCommon Strains: Lactobacillus acidophilus, Bifidobacterium bifidum, and Saccharomyces boulardii. o Health Effects: Probiotics are used to treat conditions like antibiotic-associated diarrhea, irritable bowel syndrome (IBS), and inflammatory bowel disease (IBD) by reducing inflammation, enhancing gut barrier function, and competing with pathogens. Fecal Microbiota Transplantation (FMT): o Process: FMT involves transferring stool from a healthy donor to a patient with severe dysbiosis, most commonly for recurrent Clostridium difficile infection. o Benefits: FMT can restore microbial diversity and resilience in the gut, reducing symptoms and preventing recurrence of infection. 7. How is Our Commensal Microbiome Disrupted (Dysbiosis)? Dysbiosis is a disruption in the balance and diversity of the microbiome, often associated with various diseases. It can be triggered by factors such as antibiotic overuse, diet, and stress. Causes of Dysbiosis: o Antibiotic Use: Antibiotics, especially broad-spectrum types, can indiscriminately kill beneficial bacteria, leading to reduced diversity and a shift in microbial balance. This can increase susceptibility to infections, including overgrowth of antibiotic-resistant pathogens like Clostridium difficile. o Poor Diet: Diets high in sugar and low in fiber can reduce beneficial bacteria like Bifidobacterium and Lactobacillus, increasing inflammation and promoting the growth of pathobionts (disease-associated microbes).  Stress and Lifestyle: Chronic stress and lack of sleep o can influence gut barrier integrity and reduce microbial diversity, promoting conditions like irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Diseases Associated with Dysbiosis: o Metabolic Syndrome: Dysbiosis is linked to obesity, insulin resistance, and inflammation due to decreased SCFA production and changes in bile acid metabolism. o Inflammatory Bowel Disease (IBD): Loss of beneficial species and overgrowth of pro- inflammatory bacteria contribute to chronic gut inflammation. o Colorectal Cancer: Dysbiosis may promote carcinogenesis through increased production of toxic metabolites and chronic inflammation. motility different microdes in different places bacteria spread > too slow - overgrowth of > - to wrong places -too fast > - under fed bacteria  bacteriocins microsius colicins Infection & Immunity BBS3024 Academic year: 2024–2025 Basic Challenge 2 Evolution of bacteria - the thin line between commensals and pathogens - Faculty of Health, Medicine and Life Sciences Bachelor Biomedical Science  2017 Maastricht University, Faculty of He alth, Medicine and Life Sciences. Nothing in this publication may be reproduced and/or made public by means of printing, offset, photocopy or microfilm or in any digital, electronic, optical or any other form without the prior written permission of the owner of the copyright.  Faculty of Health, Medicine and Life Sciences Basic Challenge 3: Evolution of bacteria - the thin line between commensals and pathogens What do you still recall from your previous courses? Figure 1 - Source: Prescott’s Microbiology, 12th edition Figure 2 - Source: Wikimedia commons BBS3024 Infection & Immunity 2024-2025 2 BBS3024 2022-2023 2  Faculty of Health, Medicine and Life Sciences “Good and evil are so close as to be chained together in the soul.” ― Robert Louis Stevenson, The Strange Case of Dr. Jekyll and Mr. Hyde conjugationa transformation Figure 3 - Source: Balasubramanian et al. Trends in Microbiology 2022 [in press] https://doi.org/10.1016/j.tim.2022.02.003. Figure 4 - Source: Balasubramanian et al. Trends in Microbiology 2022 [in press] https://doi.org/10.1016/j.tim.2022.02.003. BBS3024 Infection & Immunity 2024-2025 3 BBS3024 2022-2023 3  Faculty of Health, Medicine and Life Sciences Ready - set - fire ……Type III, IV and VI secretion systems Figure 5 - Summary of known bacterial secretion systems. In this simplified view only the basics of each secretion system are sketched. HM: Host membrane; OM: outer membrane; IM: inner membrane; OMP: outer membrane protein; MFP: membrane fusion protein. ATPases and chaperones are shown in yellow. Source: Tseng, TT., Tyler, B.M. & Setubal, J.C. Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol 9 (Suppl 1), S2 (2009). https://doi.org/10.1186/1471-2180-9-S1-S2 Studying bacterial evolution Figure 6 - Genetic map of the UPEC strain 536 chromosome. The two inner circles represent all putative genes (pangenome), depending on ORF orientation. The third circle from the center gives the scale. The fourth circle from the center shows the G + C distribution. Regions with a highly aberrant G + C content (greater than or less than 2-fold standard deviation) are highlighted in red. The outermost circles show the result of a three-way genome comparison with the UPEC strain CFT073 and E. coli MG1655 (K-12) genomes: blue, backbone genes (core genome) found in all three strains; red, genes present in 536 and CFT073 but absent from MG1655; green, genes found in 536 only; orange, genes of 536, which are present in CFT073 but located in a different genomic region. Pathogenicity and genomic islands (GEIs) are highlighted; flanking tRNAs are given in brackets. Source: Brzuszkiewicz E., et al. PNAS 2006:103(34)12879-12884 https://doi.org/10.1073/pnas.0603038103 BBS3024 Infection & Immunity 2024-2025 4 BBS3024 2022-2023 4  1. How do the different types of antibiotics work, and how do bacteria resist antibiotics? Mechanisms of Antibiotic Action Antibiotics target specific processes critical for bacterial survival and replication. Understanding how each type works provides insights into their effectiveness and limitations: 1. Inhibition of Cell Wall Synthesis: o Beta-lactams (e.g., penicillins, cephalosporins): ▪ These antibiotics inhibit the synthesis of peptidoglycan, an essential component of the bacterial cell wall. They target penicillin-binding proteins (PBPs), enzymes involved in the final stages of assembling the cell wall and reshaping it during cell growth and division. ▪ Without a properly formed cell wall, bacteria cannot maintain osmotic balance, leading to cell lysis. ▪ Example: Streptococcus pneumoniae and Staphylococcus aureus are susceptible to beta-lactams unless resistant mechanisms are present. o Glycopeptides (e.g., vancomycin): ▪ Vancomycin binds directly to the D-Ala-D-Ala termini of peptidoglycan precursors, preventing cross-linking. It is particularly effective against Gram-positive bacteria like MRSA but ineffective against Gram-negative bacteria due to its large size and inability to penetrate the outer  membrane. 2. Inhibition of Protein Synthesis: o Aminoglycosides (e.g., gentamicin, streptomycin): ▪ These bind irreversibly to the 30S ribosomal subunit, causing misreading of mRNA, leading to defective proteins. The action is bactericidal, especially against aerobic Gram-negative bacteria. o Tetracyclines: ▪ Tetracyclines bind reversibly to the 30S ribosomal subunit, blocking the attachment of aminoacyl- tRNA to the A site of the ribosome. This prevents protein elongation. They are broad-spectrum antibiotics used against Gram-positive and Gram- negative bacteria, as well as atypical pathogens like Chlamydia and Mycoplasma. o Macrolides (e.g., erythromycin, azithromycin): ▪ These target the 50S ribosomal subunit, specifically inhibiting translocation by blocking the exit tunnel of the growing peptide chain. This prevents elongation of the protein chain, effectively halting protein synthesis. Macrolides are commonly used for respiratory infections and are effective against Gram-positive bacteria and some Gram-negative bacteria. o Chloramphenicol: ▪ Inhibits the peptidyl transferase activity of the 50S ribosomal subunit, preventing peptide bond formation. Though effective, its use is limited due to potential serious side effects like aplastic anemia. 3. Inhibition of Nucleic Acid Synthesis:  o Fluoroquinolones (e.g., ciprofloxacin, levofloxacin): ▪ These antibiotics inhibit bacterial DNA gyrase (topoisomerase II) and topoisomerase IV, enzymes crucial for supercoiling and uncoiling DNA during replication and transcription. The inhibition of these enzymes leads to DNA strand breaks and ultimately bacterial cell death. They are effective against a broad range of Gram- negative bacteria and some Gram-positive bacteria. o Rifamycins (e.g., rifampin): ▪ Rifamycins bind to bacterial RNA polymerase, blocking the initiation of RNA synthesis. This prevents transcription, effectively inhibiting bacterial replication and protein production. Rifampin is commonly used to treat tuberculosis and bacterial infections with Neisseria meningitidis. 4. Disruption of Metabolic Pathways: o Sulfonamides and Trimethoprim: ▪ These antibiotics inhibit enzymes involved in the bacterial folic acid synthesis pathway, which is crucial for nucleotide biosynthesis. ▪ Sulfonamides compete with para-aminobenzoic acid (PABA) for the enzyme dihydropteroate synthase, preventing the synthesis of dihydrofolic acid. ▪ Trimethoprim inhibits dihydrofolate reductase, blocking the conversion of dihydrofolic acid to tetrahydrofolic acid, which is necessary for DNA synthesis. ▪ These drugs are often used in combination (e.g.,  cotrimoxazole) to enhance effectiveness and prevent resistance. Mechanisms of Antibiotic Resistance Bacteria have evolved multiple strategies to evade the effects of antibiotics. These mechanisms include both inherent resistance and acquired resistance through genetic mutations or horizontal gene transfer. 1. Enzymatic Degradation or Modification: o Beta-lactamases: ▪ These enzymes hydrolyze the beta-lactam ring of penicillins and cephalosporins, rendering them inactive. There are several types of beta- lactamases, including extended-spectrum beta- lactamases (ESBLs) that confer resistance to a broader range of beta-lactams, including third- generation cephalosporins. ▪ Carbapenemases (e.g., KPC, NDM) can degrade even carbapenems, which are often used as a last resort for multidrug-resistant infections. o Aminoglycoside-Modifying Enzymes: ▪ These enzymes add chemical groups (acetyl, phosphate, adenyl) to aminoglycosides, preventing their binding to the ribosome. 2. Modification of Target Sites: o Alteration of PBPs: ▪ In Staphylococcus aureus, resistance to methicillin (MRSA) is due to the acquisition of a modified PBP2a, encoded by the mecA gene, which has a low affinity for beta-lactams. o Mutations in Ribosomal RNA or Proteins: ▪ Mutations in the 23S rRNA or ribosomal proteins  prevent the binding of macrolides and other ribosome-targeting antibiotics. 3. Efflux Pumps: o Multidrug efflux pumps actively transport antibiotics out of the bacterial cell, reducing intracellular concentrations to sub-lethal levels. These pumps can confer resistance to a wide range of antibiotics, including tetracyclines, fluoroquinolones, and macrolides. o Example: The AcrAB-TolC efflux system in E. coli. 4. Reduced Permeability: o Changes in porin proteins reduce the uptake of antibiotics, particularly in Gram-negative bacteria. For example, mutations in OmpF porins can confer resistance to beta-lactams and fluoroquinolones by limiting drug entry. 5. Biofilm Formation: o Bacteria in biofilms are embedded in a protective extracellular matrix that restricts antibiotic penetration. Within biofilms, bacteria can exhibit a slower growth rate and metabolic activity, making them less susceptible to antibiotics that target rapidly dividing cells. 6. Target Bypass: o Some bacteria acquire alternative pathways or redundant enzymes that bypass the inhibited pathway. For example, resistance to trimethoprim can arise through the acquisition of a plasmid-encoded alternative dihydrofolate reductase enzyme. 7. Horizontal Gene Transfer (HGT): o Resistance genes can spread through bacterial  populations via transformation, conjugation, and transduction: ▪ Transformation: Uptake of free DNA from the environment. ▪ Conjugation: Transfer of plasmids or integrative conjugative elements through direct cell-to-cell contact. ▪ Transduction: Transfer of genetic material by bacteriophages. These mechanisms allow bacteria to adapt rapidly to antibiotic pressure, posing a significant challenge to clinical management of bacterial infections. Vesiceaction residuction 2. How do horizontal gene transfer and mobile elements work? Horizontal gene transfer (HGT) is a critical process in bacterial evolution, allowing the transfer of genetic material between organisms without the need for reproduction. This mechanism significantly contributes to genetic diversity and the rapid spread of traits such as antibiotic resistance. Mechanisms of Horizontal Gene Transfer (HGT): 1. Transformation: o Transformation involves the uptake of naked DNA fragments from the environment by a competent bacterial cell. Competence refers to the cell's ability to take up and incorporate exogenous DNA. o Natural Competence: Some bacteria, such  as Streptococcus pneumoniae and Bacillus subtilis, are naturally competent. They possess specific proteins that facilitate DNA uptake and integration into their genomes. o Induced Competence: In other bacteria, competence can be induced under laboratory conditions using treatments like calcium chloride or electroporation. o Mechanism: ▪ DNA binding proteins on the bacterial surface capture DNA fragments. ▪ The DNA is transported into the cell and integrated into the bacterial chromosome by homologous recombination. ▪ This process can introduce new traits, such as antibiotic resistance or virulence factors, into the bacterial population. same replication 2. Conjugation: enot compatible o Conjugation is a process of direct transfer of DNA plasmids · replicate between two bacterial cells, typically mediated by a · move conjugative plasmid. · regulate (limited copies o F Plasmid (F Factor): The F plasmid in Escherichia per bacterial plasmid encodes for coli is a well-studied example. It carries the Tra pilus operon, which encodes the proteins required for the formation of a sex pilus and DNA transfer. o Mechanism: ▪ A donor cell (F+) containing the F plasmid forms a conjugation pilus to connect with a recipient cell a secretion type (F-). system ▪ The pilus retracts, bringing the two cells into close contact, forming a conjugation bridge. relaxosome ▪ A single strand of the F plasmid is transferred to  the recipient cell and replicated, converting the F- cell into an F+ cell. o Hfr Conjugation: ▪ Sometimes the F plasmid integrates into the bacterial chromosome, forming an Hfr (high- frequency recombination) cell. ▪ During conjugation, Hfr cells transfer chromosomal genes along with plasmid genes to the recipient, allowing for genetic recombination. 3. Transduction: o Transduction is the process of DNA transfer mediated by bacteriophages (viruses that infect bacteria). o Generalized Transduction: ▪ In the lytic cycle, a phage infects a bacterial cell and accidentally packages fragments of the host's DNA into new phage particles. ▪ These defective phages can inject the bacterial DNA into a new host cell, where it can recombine with the recipient's genome. o Specialized Transduction: ▪ Occurs in the lysogenic cycle when a temperate phage integrates into the host genome as a prophage. ▪ Upon excision, the phage may take adjacent bacterial genes with it, transferring them to a new host upon infection. o Lytic vs. Lysogenic Cycle: ▪ Lytic Cycle: The phage replicates inside the host, leading to cell lysis and release of new phages. ▪ Lysogenic Cycle: The phage DNA integrates into the host genome, replicating along with it until  induced to enter the lytic cycle. Mobile Genetic Elements: 1. Insertion Sequences (IS): o The simplest type of mobile genetic elements. o Comprise a gene encoding transposase, flanked by inverted repeat sequences. o Transposase catalyzes the movement (transposition) of the IS element within the genome. o Function: They can disrupt genes or regulatory regions when inserted, leading to mutations or altered gene expression. 2. Transposons: o Larger than IS elements and can carry additional genes, such as antibiotic resistance genes. o Composite Transposons: Consist of two IS elements flanking a central region containing other genes. o Unit Transposons: Do not have IS elements but carry genes for transposase and additional functions. o Transposons can move within the genome or between DNA molecules, such as plasmids and chromosomes. 3. Integrative and Conjugative Elements (ICEs): o Hybrid elements that can integrate into the bacterial genome and excise to transfer between cells via conjugation. o Combine features of transposons (integration and excision) and conjugative plasmids (transfer between cells). o Example: ICEs can carry genes for antibiotic resistance, virulence, or metabolic pathways. 4. Recombinases:  o Enzymes like transposases, integrases, and resolvases facilitate the movement and integration of mobile genetic elements. o Transposases: Catalyze the excision and insertion of transposons. o Integrases: Mediate the integration of prophages into bacterial genomes during lysogeny. o Resolvases: Resolve cointegrates formed during transposition events. Impact of HGT and Mobile Genetic Elements on Bacterial Evolution: 1. Genetic Diversity: o HGT introduces new genetic material, increasing variability and allowing bacteria to rapidly adapt to environmental changes, such as the presence of antibiotics. 2. Spread of Antibiotic Resistance: o Resistance genes are often located on mobile genetic elements like plasmids, transposons, or ICEs, facilitating their transfer across different bacterial species. o This can lead to the rapid emergence of multidrug- resistant strains, posing significant challenges to treatment. 3. Virulence Factors: o Genes encoding toxins, adhesion factors, and secretion systems can be transferred via HGT, enhancing the pathogenicity of recipient bacteria. 4. Comparative Genomics: o Analysis of genomic islands, including pathogenicity islands, reveals horizontal transfer events and the  acquisition of adaptive traits. o Core genome and pangenome studies highlight the genetic diversity within bacterial species, distinguishing essential genes from accessory genes acquired through HGT. In summary, horizontal gene transfer and mobile genetic elements play pivotal roles in bacterial evolution, adaptation, and the spread of important traits like antibiotic resistance and virulence. Understanding these processes is crucial for developing strategies to combat bacterial infections and prevent the dissemination of resistance genes. Direct repeats sticky ends * simple replicative transposition : vs. * look into conjugation , FT , , , F F ets. depends on how long connected 3. How Can You Study the Evolution of Bacteria? Studying bacterial evolution provides crucial insights into how bacteria adapt to changing environments, develop resistance to antibiotics, and enhance their virulence. This involves examining genetic changes, horizontal gene transfer, and the role of mobile genetic elements. Several methodologies and concepts are essential in understanding bacterial evolution: 1. Comparative Genomics Comparative genomics involves comparing the genetic material of different bacterial strains or species to identify evolutionary relationships and functional differences. Core Genome vs. Pangenome: o Core Genome: The set of genes shared by all strains of a species. These genes are essential for basic cellular functions and survival. o Pangenome: Includes the core genome plus accessory genes that are present in some but not all strains. The pangenome reflects the genetic diversity within a species and includes genes acquired through horizontal gene transfer (HGT), which may confer advantageous traits like antibiotic resistance or virulence. Genomic Islands: o These are large segments of DNA acquired via HGT, often containing genes that provide adaptive advantages such as resistance to antibiotics, virulence factors, or metabolic capabilities. o Pathogenicity Islands are a specific type of genomic island that carry genes contributing to a bacterium’s ability to cause disease, such as toxins, adhesion factors, and secretion systems. G+C Content Analysis: o Bacterial genomes have characteristic G+C content (the percentage of guanine and cytosine bases). Variation in G+C content can indicate the acquisition of foreign DNA, as horizontally transferred genes often retain the nucleotide composition of their original host. 2. Horizontal Gene Transfer (HGT) and Its Role in Evolution HGT plays a significant role in bacterial evolution by allowing the transfer of genetic material between different organisms, bypassing the traditional vertical transmission from parent to offspring. Mechanisms of HGT: o Transformation: Uptake of free DNA from the environment, integrating it into the genome. o Conjugation: Direct transfer of DNA between bacteria via cell-to-cell contact, often mediated by conjugative plasmids. o Transduction: Transfer of DNA by bacteriophages, which can inadvertently package bacterial DNA during their replication cycle. HGT accelerates bacterial evolution by introducing genetic variability, enabling rapid adaptation to environmental pressures such as antibiotic treatment or immune system attacks. 3. Mobile Genetic Elements Mobile genetic elements such as plasmids, transposons, and integrative conjugative elements (ICEs) contribute to the dynamic nature of bacterial genomes. Plasmids: Circular DNA molecules that replicate independently of the bacterial chromosome. They often carry genes for antibiotic resistance or virulence factors. Transposons and Insertion Sequences (IS): These elements can move within and between genomes, spreading genes that confer advantageous traits. Integrative and Conjugative Elements (ICEs): These elements integrate into the host genome and can excise to transfer to other bacteria, combining the mobility of plasmids and the integration ability of transposons. 4. Phylogenetic Analysis Phylogenetic analysis helps trace the evolutionary relationships among bacterial species or strains by comparing genetic sequences.  16S rRNA Gene Sequencing: o The 16S rRNA gene is highly conserved across bacterial species, making it a valuable tool for reconstructing phylogenies and identifying evolutionary lineages. Whole-Genome Sequencing: o Provides a comprehensive view of genetic differences, revealing evolutionary relationships and the impact of horizontal gene transfer. Molecular Clocks: o By estimating mutation rates, researchers can infer divergence times and evolutionary rates, providing insights into how quickly bacteria evolve under different environmental pressures. 5. Adaptation to Antibiotics and Environmental Pressures Antibiotic Resistance Evolution: o Studying how bacteria acquire and spread resistance genes sheds light on evolutionary pressures exerted by antibiotic use. o Comparative studies of resistant and susceptible strains can identify genetic changes associated with resistance. Environmental Adaptation: o Bacteria often acquire genes enabling them to survive in diverse environments. Studying such adaptations can reveal how bacteria evolve in response to specific ecological niches or host environments. 6. Experimental Evolution Laboratory Evolution Studies: o Researchers can observe bacterial evolution in real- time by subjecting bacteria to controlled  environmental pressures, such as antibiotics or nutrient limitations, over many generations. o Lenski’s Long-Term Evolution Experiment (LTEE): A famous example where E. coli populations have been evolved for tens of thousands of generations, revealing insights into adaptation, mutation rates, and genetic diversification. 7. Genetic and Functional Studies Mutagenesis and Gene Knockout Studies: o By creating mutations or knocking out specific genes, researchers can determine their role in bacterial survival, adaptation, and virulence. Transcriptomics and Proteomics: o Studying changes in gene expression (transcriptomics) and protein production (proteomics) under different conditions can reveal how bacteria adapt to environmental stresses and antibiotics. 8. Analyzing Evolutionary Pathways and Common Ancestors Ancestral Gene Reconstruction: o Computational methods can infer the sequences of ancestral genes, providing insights into the evolutionary pathways and functions of modern genes. Tracing Common Ancestors: o By comparing genetic sequences across multiple strains or species, researchers can identify common ancestors and trace the evolutionary history of specific traits, such as virulence or resistance. Conclusion Studying bacterial evolution involves a combination of comparative genomics, experimental evolution, and molecular techniques. It reveals how bacteria adapt, acquire resistance, and evolve new functions. Understanding these processes is crucial for developing strategies to combat antibiotic resistance and predict bacterial responses to environmental changes. 4. What Are the Types of Virulence Factors and How Do They Function? Virulence factors are specialized molecules or structures that enable bacteria to colonize a host, evade or suppress the immune system, obtain nutrients, and cause disease. These factors are often encoded on mobile genetic elements like plasmids, transposons, and pathogenicity islands, which facilitate their horizontal transfer among bacterial populations. Understanding virulence factors is crucial for developing strategies to prevent and treat bacterial infections. 1. Secretion Systems Bacterial secretion systems are specialized apparatuses used to transport proteins or other molecules across bacterial membranes into host cells or the surrounding environment. These systems play critical roles in bacterial pathogenicity by delivering effector proteins that manipulate host cellular processes. Type III Secretion System (T3SS): o Found in Gram-negative bacteria like Salmonella, Shigella, and Yersinia. o Resembles a molecular syringe that injects effector proteins directly into the host cell cytoplasm. o These effector proteins can alter host signaling  pathways, disrupt the cytoskeleton, and suppress immune responses, facilitating bacterial invasion and survival. Type IV Secretion System (T4SS): o Used by bacteria like Helicobacter pylori and Legionella pneumophila. o Capable of transferring both DNA and proteins. In H. pylori, the T4SS injects the CagA protein into gastric epithelial cells, promoting inflammation and potentially leading to cancer. Type VI Secretion System (T6SS): o Found in pathogens like Pseudomonas aeruginosa and Vibrio cholerae. o Functions as a molecular spear, delivering toxic effector proteins into competing bacteria or eukaryotic host cells. o T6SS can disrupt host immune cells or outcompete other microbes in the same niche. 2. Adhesion Factors Adhesion is the first step in the establishment of many bacterial infections. Adhesins are surface molecules that enable bacteria to attach to host cells or tissues, facilitating colonization and subsequent infection. Pili and Fimbriae: o These are hair-like structures on the bacterial surface that mediate attachment to host cell receptors. o Type I fimbriae in E. coli enable adherence to urinary tract epithelial cells, a key factor in urinary tract infections. Adhesins: o Non-pilus adhesins like the MSCRAMMs (Microbial  Surface Components Recognizing Adhesive Matrix Molecules) bind to extracellular matrix proteins such as fibronectin and collagen. o Example: Staphylococcus aureus produces protein A, which binds to host immunoglobulin G (IgG), disrupting opsonization and phagocytosis. 3. Invasion and Dissemination Factors Once attached, some bacteria invade host cells or tissues to establish infections. This process often involves enzymes and other proteins that break down host barriers or evade immune detection. Invasins: o Bacterial proteins that trigger host cell uptake, facilitating intracellular survival. o Example: Listeria monocytogenes produces internalin, which induces its uptake by epithelial cells. Enzymes Facilitating Spread: o Hyaluronidase: Degrades hyaluronic acid in the extracellular matrix, allowing bacteria to penetrate tissues. o Collagenase: Breaks down collagen, aiding in tissue invasion. o Streptokinase: Converts plasminogen to plasmin, dissolving blood clots and allowing bacteria to spread through tissues. 4. Biofilm Formation Biofilms are structured communities of bacteria encased in a self-produced extracellular matrix that adheres to surfaces and protects the bacteria from environmental stresses. Biofilm Advantages: o Enhanced resistance to antibiotics and immune  responses. o Example: Pseudomonas aeruginosa forms biofilms in the lungs of cystic fibrosis patients, contributing to chronic infections. Biofilm Components: o Exopolysaccharides form the primary matrix, trapping nutrients and shielding bacteria from immune attacks. o Biofilms can harbor persister cells that are dormant and highly tolerant to antibiotics, making infections difficult to eradicate. 5. Iron Scavenging Mechanisms Iron is essential for bacterial growth, but in the host, free iron is limited due to sequestration by host proteins like transferrin and ferritin. Siderophores: ATP o High-affinity iron-chelating molecules secreted by production bacteria to capture iron from host proteins. o Example: Enterobactin produced by E. coli binds iron requires iron tightly and is then re-imported into the bacterial cell. Direct Iron Acquisition: o Some bacteria have receptors that directly bind host iron-carrying proteins, extracting iron for bacterial use. 6. Toxins Bacterial toxins are categorized into exotoxins and endotoxins based on their structure, function, and release mechanism. Exotoxins: o Soluble proteins secreted by bacteria that can cause damage to host cells by various mechanisms. o AB Toxins:  ▪ Consist of two components: the A (active) subunit and the B (binding) subunit. ▪ Example: Diphtheria toxin (produced by Corynebacterium diphtheriae) inhibits protein synthesis by ADP-ribosylating elongation factor- 2 (EF-2), leading to cell death. o Membrane-Disrupting Toxins: ▪ Example: Hemolysins produced by Streptococcus species lyse red blood cells to release iron. o Superantigens: ▪ These toxins cause an excessive immune response by non-specifically activating T-cells. ▪ Example: Toxic shock syndrome toxin-1 (TSST- 1) from Staphylococcus aureus leads to a massive release of cytokines, causing toxic shock. Endotoxins: o Components of the outer membrane of Gram-negative bacteria, specifically the lipopolysaccharide (LPS)layer. o The Lipid A component of LPS is the toxic portion, which, when released during bacterial lysis, triggers a strong immune response. o Endotoxins can cause septic shock, characterized by fever, hypotension, and multiple organ failure. 7. Antiphagocytic Factors Bacteria have evolved various mechanisms to avoid being engulfed and destroyed by host phagocytes. Capsules: o Polysaccharide layers that cover the bacterial surface, preventing phagocytosis.  oExample: Streptococcus pneumoniae uses its capsule to evade detection and destruction by the immune system. Protein A: o Found in Staphylococcus aureus, it binds the Fc region of antibodies, preventing opsonization and phagocytosis. Conclusion Virulence factors are diverse and multifaceted, enabling bacteria to effectively colonize hosts, evade immune defenses, and cause disease. These factors include secretion systems for delivering effector molecules, adhesion molecules for attachment, enzymes for tissue invasion, biofilm formation for protection, iron acquisition systems for survival, and toxins that directly damage host tissues. Understanding these mechanisms is crucial for developing targeted therapies and preventive measures against bacterial infections. resistance : intrinsic / acquired 5. What Genetic Mechanisms Do Bacteria Develop to Counteract Antimicrobial Processes? Bacteria have evolved a variety of sophisticated genetic mechanisms to resist the effects of antimicrobial agents. These mechanisms involve both intrinsic resistance and the acquisition of resistance genes through horizontal gene transfer (HGT) and mutations. Let’s delve into the detailed mechanisms by which bacteria develop and propagate resistance to antibiotics. 1. Enzymatic Degradation or Modification of Antibiotics Beta-lactamases: o Beta-lactamases are enzymes that hydrolyze the beta- lactam ring found in penicillins, cephalosporins, and carbapenems, rendering these antibiotics ineffective. There are various classes of beta-lactamases, including: ▪ Class A Beta-lactamases: Include extended- spectrum beta-lactamases (ESBLs), which can hydrolyze third-generation cephalosporins. ▪ Class B Beta-lactamases (Metallo-beta- lactamases): These enzymes require zinc for activity and can hydrolyze carbapenems (e.g., New Delhi Metallo-beta-lactamase, NDM-1). ▪ Class C Beta-lactamases: Also known as AmpC beta-lactamases, which confer resistance to a broad range of beta-lactam antibiotics. Aminoglycoside-Modifying Enzymes: o These enzymes add chemical groups (acetyl,  phosphate, or adenyl) to aminoglycosides, such as gentamicin and tobramycin, preventing them from binding to their target ribosomal subunit. Chloramphenicol Acetyltransferase (CAT): o This enzyme acetylates chloramphenicol, preventing it from binding to the 50S ribosomal subunit and thereby inactivating it. 2. Target Modification Bacteria can develop mutations or acquire genes that alter the target site of antibiotics, reducing the drugs’ binding affinity and effectiveness. Alteration of Penicillin-Binding Proteins (PBPs): o In methicillin-resistant Staphylococcus aureus (MRSA), the acquisition of the mecA gene leads to the production of PBP2a, a penicillin-binding protein with a low affinity for beta-lactams, thereby conferring resistance. Ribosomal Modifications: o Mutations in 16S rRNA confer resistance to aminoglycosides by preventing the drug from binding to the 30S ribosomal subunit. o Methylation of 23S rRNA by erm genes prevents macrolides from binding to the 50S ribosomal subunit. DNA Gyrase and Topoisomerase IV Mutations: o Mutations in genes encoding DNA gyrase (gyrA) and topoisomerase IV (parC) confer resistance to fluoroquinolones by reducing drug binding to these enzymes, which are essential for DNA replication. 3. Efflux Pumps Efflux pumps are membrane proteins that actively expel antibiotics from the bacterial cell, reducing intracellular concentrations to sub-lethal levels. Major Efflux Pump Families: o Resistance-Nodulation-Division (RND) Family: Found mainly in Gram-negative bacteria, e.g., the AcrAB-TolC system in E. coli, which pumps out a wide range of antibiotics, including tetracyclines, fluoroquinolones, and beta-lactams. o ATP-Binding Cassette (ABC) Transporters: Use ATP hydrolysis to export antibiotics out of the cell. o Major Facilitator Superfamily (MFS): Includes the TetA pump, which confers resistance to tetracyclines by expelling them from the cell. 4. Reduced Permeability Bacteria, particularly Gram-negative species, can modify their outer membrane to reduce the uptake of antibiotics. Porin Modifications: o Porins are channels that allow small molecules, including antibiotics, to pass through the outer membrane of Gram-negative bacteria. o Decreased expression or mutations in porins (e.g., OmpF in E. coli) reduce the uptake of beta- lactams and fluoroquinolones, leading to resistance. 5. Biofilm Formation Biofilms are structured communities of bacteria embedded in a self-produced extracellular polymeric matrix. This matrix protects bacteria from antibiotics and immune responses. Biofilm Characteristics: o Bacteria within biofilms are in a metabolically slow- growing state, reducing the efficacy of antibiotics that target actively dividing cells. o The extracellular matrix acts as a physical barrier,  preventing antibiotic penetration. o Biofilms facilitate the horizontal transfer of resistance genes among bacteria within the community. 6. Target Bypass Bacteria can bypass metabolic pathways inhibited by antibiotics by acquiring alternative enzymes or pathways. Sulfonamide and Trimethoprim Resistance: o Resistance arises from the acquisition of alternative dihydropteroate synthase and dihydrofolate reductase enzymes encoded by plasmid-borne genes (sul and dfr), which are not inhibited by these drugs. Vancomycin Resistance in Enterococcus (VRE): o Vancomycin-resistant Enterococcus spp. acquire genes (vanA, vanB) that alter the peptidoglycan precursor from D-Ala-D-Ala to D-Ala-D-Lac, which has a reduced affinity for vancomycin. 7. Horizontal Gene Transfer (HGT) The rapid spread of antibiotic resistance is often facilitated by horizontal gene transfer, which allows bacteria to acquire resistance genes from other organisms. Conjugation: o Transfer of plasmids carrying resistance genes through direct cell-to-cell contact. For example, conjugative plasmids like the F plasmid in E. coli can carry multiple resistance genes. Transformation: o Uptake of free DNA from the environment, including fragments carrying resistance genes. Transduction: o Bacteriophages can inadvertently package and transfer  bacterial DNA containing resistance genes during the lytic or lysogenic cycle. 8. Mobile Genetic Elements Plasmids: o Circular DNA molecules that replicate independently of the bacterial chromosome. They often carry multiple resistance genes and can be transferred between bacteria via conjugation. Transposons and Insertion Sequences: o Transposons (e.g., Tn3) can move resistance genes between DNA molecules, including plasmids and chromosomes. o Integrons: Genetic elements that can capture and express genes, particularly resistance genes, through site-specific recombination mediated by an integrase enzyme. Pathogenicity Islands and Genomic Islands: o Large DNA segments acquired through horizontal gene transfer that often carry clusters of resistance genes. These islands are typically integrated into the bacterial chromosome and may be associated with virulence factors. 9. Mutation and Adaptive Evolution Spontaneous Mutations: o Random mutations in bacterial genomes can lead to resistance. For instance, mutations in the rpoB gene confer resistance to rifampin by altering the binding site on RNA polymerase. Adaptive Evolution: o Under selective pressure, such as antibiotic exposure, resistant bacteria are more likely to survive and  propagate. Over time, this leads to the predominance of resistant strains in a population. Conclusion Bacteria deploy a wide array of genetic mechanisms to counteract antimicrobial agents, including enzymatic degradation, target modification, efflux pump activity, reduced permeability, biofilm formation, and horizontal gene transfer. These mechanisms allow bacteria to survive and thrive in the presence of antibiotics, contributing to the global challenge of antimicrobial resistance (AMR). Understanding these mechanisms is crucial for developing new strategies to combat bacterial infections and curb the spread of resistance. Infection & Immunity BBS3024 Academic year: 2024–2024 Basic Challenge 3 Evolution of viruses Faculty of Health, Medicine and Life Sciences Bachelor Biomedical Science  2017 Maastricht University, Faculty of He alth, Medicine and Life Sciences. Nothing in this publication may be reproduced and/or made public by means of printing, offset, photocopy or microfilm or in any digital, electronic, optical or any other form without the prior written permission of the owner of the copyright.  Faculty of Health, Medicine and Life Sciences The Trick. "Any virus that can cause disease in humans must have at least one immune evasion mechanism—at least one immune evasion “trick.” Without the ability to evade the immune system, a virus is usually harmless. Understanding immune evasion by a virus is frequently important for understanding the pathogenesis of the virus, as well as understanding challenges faced by the adap ve immune system and any candidate vaccine." Literature: Se e A, Cro y S. Adap ve immunity to SARS-CoV-2 and COVID-19. Cell. 2021;184(4):861-880. doi:10.1016/j.cell.2021.01.007 Case COVID 19. A 48-year-old man presents to the emergency department, referred by his family doctor due to fever and significant shortness of breath. He has experienced flu-like symptoms for the past three days—fever, muscle aches, sore throat, runny nose, and cough—coupled with progres-sive shortness of breath that has not responded to oral an bio cs (amoxicillin). Notably, he reports a recent loss of smell and taste. The pa ent's medical history reveals obesity (body mass index: 31 kg/m²) and a long history of smoking—one pack of cigare es per day for 25 years, along with vaping for the last three years. He has not received vaccina ons for influenza A and B or for SARS-CoV-2. In his community, several individuals are exhibi ng similar flu-like symptoms. On physical examina on, he has fever and exhibits signs of dyspnea and tachypnea, with a rapid pulse and normal blood pressure. Ausculta on reveals bilateral lung involvement con-sistent with pneumonia. He requires supplemental oxygen at 5 liters per minute via nasal can-nula to maintain acceptable oxygen satura on levels. A nasopharyngeal swab tests posi ve for SARS-CoV-2 via polymerase chain reac on (PCR), with a cycle threshold (CT) value of 22, while tests for influenza and parainfluenza viruses are nega ve. Laboratory results indicate signs of systemic inflamma on. Imaging studies, includ-ing chest X-ray and CT scan, reveal diffuse bilateral pneumoni s without lung emboli. The medical team diagnoses him with severe COVID-19 pneumonia. They ini ate treatment with intravenous dexamethasone and tocilizumab to manage the inflammatory response. Ad-di onally, they deliberate on further therapeu c op ons, including remdesivir, nirma-trelvir/ritonavir, molnupiravir, monoclonal an bodies, or convalescent plasma. They also plan to assess his blood serum for the presence of an bodies against SARS-CoV-2, which may in-form his immune status. BBS3024 Infection & Immunity 2024-2025 2 BBS3024 2022-2023 2  Faculty of Health, Medicine and Life Sciences Case HIV A 32-year-old woman is referred by her family doctor to the emergency department because of high fever, flu-like symptoms, cervical lymphadeni s, and a skin rash. Her previous medical history is unremarkable. On physical examina on she appears ill. Temperature 39.0⁰ Celsius, blood pressure 120/80 mmHg and pulse rate 94/minute. Her tonsils are enlarged. Cervical, axillary and inguinal lymph nodes are enlarged and elas c on palpa on. Blood results show lymphocytopenia. Serology is posi ve for an acute HIV (human immunodeficiency virus) infec on, Fiebig stage 3. The consulted infec ous diseases specialist recommends the pa ent to start combina on an retroviral therapy (cART) immediately. However, the pa ent is in doubt and wonders why she can’t wait to see how the infec on evolves and if her immune system can cure the infec on. Next to this, she wants to know if HIV, like covid-19, also has a lot of variants. Compare and constrast: SARS-CoV2 and HIV BBS3024 Infection & Immunity 2024-2025 3 BBS3024 2022-2023 3 1. Types of viruses recap 1. Based on Genetic Material: Viruses are distinguished by whether their genome consists of DNA or RNA, and whether this genetic material is single or double-stranded. DNA Viruses: o Double-Stranded DNA (dsDNA) Viruses: These viruses have double-stranded DNA similar to human DNA, which allows them to use the host's cellular machinery efficiently. ▪ Examples: ▪ Herpesviruses (e.g., HSV-1 and HSV-2) cause cold sores and genital herpes. They can remain latent and reactivate under certain conditions. ▪ Adenoviruses cause respiratory infections and conjunctivitis. ▪ Poxviruses (e.g., Variola virus) cause smallpox. They are unique because they replicate in the cytoplasm rather than the nucleus. o Single-Stranded DNA (ssDNA) Viruses: These viruses contain only one strand of DNA and often require the host cell to synthesize a complementary strand. ▪ Examples: ▪ Parvoviruses (e.g., B19 virus) cause conditions like Fifth disease in children and  can infect animals too. ▪ Circoviruses cause diseases in pigs and birds and are circular ssDNA viruses. RNA Viruses: o Positive-Sense Single-Stranded RNA (ssRNA+) Viruses: The viral RNA can act directly as messenger RNA (mRNA), allowing for immediate protein synthesis upon infection. ▪ Examples: ▪ Picornaviruses (e.g., Poliovirus, Rhinovirus) cause diseases like polio and the common cold. ▪ Coronaviruses (e.g., SARS-CoV-2) cause respiratory diseases, including COVID-19, and have a distinctive spike protein for cell entry. ▪ Flaviviruses (e.g., Dengue, West Nile Virus) are primarily mosquito-borne and cause serious diseases. o Negative-Sense Single-Stranded RNA (ssRNA-) Viruses: The viral RNA is complementary to mRNA and must be transcribed into a positive-sense RNA by RNA-dependent RNA polymerase before translation. ▪ Examples: ▪ Orthomyxoviruses (e.g., Influenza virus) cause flu. They have segmented genomes, leading to rapid antigenic shifts. ▪ Filoviruses (e.g., Ebola virus) cause hemorrhagic fever with high mortality rates. ▪ Rhabdoviruses (e.g., Rabies virus) have a bullet shape and affect the nervous system.  o Double-Stranded RNA (dsRNA) Viruses: These viruses have double-stranded RNA and replicate within a protective capsid to evade the host's immune response. ▪ Examples: ▪ Reoviruses (e.g., Rotavirus) cause gastroenteritis, especially in children. Retroviruses (RNA viruses with reverse transcription): Retroviruses contain ssRNA, which is reverse-transcribed into DNA, allowing them to integrate into the host genome. o Examples: ▪ HIV (Human Immunodeficiency Virus) causes AIDS by integrating into host T-cells and slowly weakening the immune system. ▪ HTLV (Human T-cell Leukemia Virus) can cause adult T-cell leukemia/lymphoma. 2. Based on Replication Strategy (Baltimore Classification): This system groups viruses into seven classes based on how they produce mRNA, which is essential for protein synthesis. Class I: Double-Stranded DNA Viruses o Examples: Herpesviruses and Adenoviruses. Class II: Single-Stranded DNA Viruses o Examples: Parvoviruses. Class III: Double-Stranded RNA Viruses o Examples: Reoviruses (like Rotavirus). Class IV: Positive-Sense Single-Stranded RNA Viruses o Examples: Flaviviruses (e.g., Dengue), Coronaviruses (e.g., SARS-CoV-2). Class V: Negative-Sense Single-Stranded RNA Viruses  oExamples: Influenza, Rabies virus. Class VI: Positive-Sense Single-Stranded RNA Viruses with Reverse Transcriptase o Examples: HIV (retroviruses). Class VII: Double-Stranded DNA Viruses with Reverse Transcriptase o Examples: Hepatitis B Virus (HBV). 3. Based on Shape and Structure: The physical shape and structure of viruses influence how they interact with host cells. Helical Viruses: These viruses have rod-like or filamentous shapes where the capsid proteins wind around the genome. o Examples: ▪ Tobacco Mosaic Virus infects plants and has a rigid, rod-shaped structure. ▪ Rabies Virus has a bullet shape and infects nerve cells. Icosahedral Viruses: These are roughly spherical and have a capsid made of 20 triangular faces, forming an icosahedron. o Examples: ▪ Adenovirus causes respiratory diseases and conjunctivitis. ▪ Poliovirus affects the nervous system and can lead to paralysis. Complex Viruses: These have more intricate structures, often with additional features like protein tails. o Examples: ▪ Bacteriophages have a head-tail structure, where the head contains the genetic material, and the  tail helps infect bacteria. ▪ Poxviruses have complex, multilayered outer walls and cause diseases like smallpox. 4. Based on Host Range: Viruses can also be classified by the type of host they infect. Animal Viruses: Infect various animal species, including humans. They can be DNA or RNA viruses and cause a range of diseases. o Examples: Influenza (affects many mammals), Rabies (affects mammals), and HIV (human-specific). Plant Viruses: These viruses specifically infect plants and are often transmitted by insect vectors. o Examples: ▪ Tobacco Mosaic Virus affects tobacco plants and other plants in the Solanaceae family. ▪ Potato Virus Y causes serious diseases in potato crops. Bacteriophages: Also known as phages, these viruses infect bacteria. They are commonly used in research and can be potential therapeutic tools. o Examples: ▪ T4 Bacteriophage infects E. coli and has a complex structure with a head-tail arrangement. ▪ Lambda Phage also infects E. coli and integrates its genome into the bacterial DNA. Archaea Viruses: Infect archaea, which are microorganisms distinct from bacteria. o Examples: The Sulfolobus turreted icosahedral virus (STIV) infects archaea in high-temperature  environments. 5. Based on Mode of Transmission: Airborne Viruses: Spread through respiratory droplets or aerosols. o Examples: Influenza virus, SARS-CoV-2, and Measles virus. Vector-Borne Viruses: Transmitted by insects or other vectors. o Examples: Dengue (spread by mosquitoes), West Nile Virus, and Zika virus. Food and Water-Borne Viruses: Transmitted via contaminated food or water. o Examples: Norovirus (common cause of gastroenteritis), Hepatitis A virus. Blood-Borne Viruses: Spread through blood and bodily fluids. o Examples: HIV, Hepatitis B, and Hepatitis C. Zoonotic Viruses: Transmitted from animals to humans, often resulting from close contact with animal hosts. o Examples: Ebola (from fruit bats or other wildlife), Rabies (from infected animals), and certain strains of Influenza. General Virology (Recap) 1. Viral Genome: Viral genomes vary in structure and are a primary basis for virus classification. They may consist of either DNA or RNA, which can be single-stranded (ss) or double-stranded (ds): o DNA Viruses include both double-stranded (dsDNA) and single-stranded (ssDNA) forms. For instance, Herpesviruses (dsDNA) have genomes that  can integrate with host mechanisms and establish latency. Adenoviruses (dsDNA) cause respiratory illnesses, while Parvoviruses (ssDNA) can infect animals and humans, causing diseases like Fifth disease. o RNA Viruses include positive-sense ssRNA, negative- sense ssRNA, and dsRNA viruses. Positive-sense ssRNA viruses (e.g., SARS-CoV-2) can translate directly into proteins. Negative-sense ssRNA viruses (e.g., Influenza) require transcription to a positive strand before translation. Retroviruses like HIV, unique among RNA viruses, reverse-transcribe RNA into DNA, allowing integration into the host genome. 2. Viral Morphology: Viruses have distinct shapes that play a role in their interactions with host cells: o Helical Viruses: These have a rod-like structure where the viral genome is coiled within a capsid cylinder, as seen in the Tobacco Mosaic Virus and Rabies virus. o Icosahedral Viruses: These have a spherical appearance due to their 20-sided capsid structure. Examples include Poliovirus and Adenovirus, known for their stability. o Complex Viruses: This category includes more complex shapes and additional structural components, such as the head-tail structure of bacteriophages and the multilayered structure of Poxviruses. 3. Enveloped vs. Naked Capsid Viruses: o Enveloped Viruses have a lipid membrane surrounding their capsid, derived from the host cell, which contains embedded viral proteins. This envelope is essential for binding to host receptors. Enveloped viruses include HIV and SARS-CoV-2.  o Naked Capsid Viruses lack this lipid envelope and consist of only a protein capsid protecting the genome, making them more resilient in external environments (e.g., Poliovirus and Adenovirus). 4. Viral Attachment Proteins: Viral attac