🧱 Bacterial Cell Envelope
Layers common to all bacteria
- Cytoplasmic (inner) membrane
- Carries cell-wall-building enzymes
- Transpeptidases and carboxypeptidases = penicillin-binding proteins (PBPs)
- PBPs are the targets of β-lactam drugs
- Peptidoglycan
- Rigid mesh that resists osmotic lysis
- Thickness explains Gram stain behavior
Gram-positive envelope
- Very thick peptidoglycan
- Retains crystal violet, stains purple
- No outer membrane
- Teichoic acids (in wall)
- Lipoteichoic acid (anchored in membrane)
- Both help adhesion to host surfaces
- Surface proteins vary by genus
- Example: M protein of Streptococcus pyogenes
- Large peptidoglycan–teichoic acid fragments
- Act as structural toxins
- Attract neutrophils
Gram-negative envelope
- Thin peptidoglycan in the periplasmic space
- Loses crystal violet, counterstains pink
- Outer membrane covers the wall
- Porins let small molecules through
- Lipoprotein anchors outer membrane to peptidoglycan
- Outer leaflet is lipopolysaccharide (LPS)
- Lipid A: embedded, the toxic part
- Core polysaccharide
- O antigen: variable chain, used for serotyping
- Periplasm holds enzymes such as β-lactamases
What LPS does in the host
Lipid A binds TLR-4→Macrophage activation→TNF-α, IL-1, IL-6 release→Fever, vasodilation, shock risk
- Also activates B cells without T-cell help (T-independent)
Comparison
| Feature | Gram-positive | Gram-negative |
|---|---|---|
| Peptidoglycan | Thick | Thin |
| Outer membrane | Absent | Present (LPS) |
| Endotoxin | No | Yes (lipid A) |
| Teichoic acids | Present | Absent |
| Periplasmic space | Minimal | Prominent |
| Main adhesion tool | Teichoic acids | Pili (fimbriae) |
Concept
- Carriers harbor a potential pathogen without overt disease.
🛠️ Peptidoglycan Synthesis and Antibiotic Targets
Polymer structure
- Backbone: alternating NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid)
- Linked by β-1,4 bonds; lysozyme cuts this bond
- Peptide stem hangs from each NAM
- Begins as a pentapeptide
- Ends in D-Ala–D-Ala
- Third residue carries a free amine (L-lysine or diaminopimelic acid)
- Cross-link formed between
- Amine on residue 3 of one stem
- D-Ala at position 4 of a neighboring stem
- Terminal D-Ala is released during cross-linking
Synthesis steps
1 Cytoplasm: make NAG, NAM, add pentapeptide
2 Membrane: unit attached to bactoprenol carrier
3 NAG added to complete the disaccharide-peptide unit
4 Flipped across the membrane
5 Transglycosylase adds unit to growing chain
6 Transpeptidase cross-links stems (PBP)
7 Carrier recycled: pyrophosphate-carrier to phosphate-carrier
- Carboxypeptidases trim terminal D-Ala residues
Why it is a good drug target
- Peptidoglycan exists only in bacteria, so human cells are largely unaffected.
Cell-wall inhibitors
| Drug | Step blocked | Resistance |
|---|---|---|
| Bacitracin | Carrier recycling (dephosphorylation of bactoprenol pyrophosphate) | Mostly used topically |
| β-lactams | Bind PBPs, block transpeptidation |
|
| Vancomycin | Binds D-Ala–D-Ala, blocks transglycosylation and transpeptidation | VanA and VanB change the terminus (D-Ala–D-Lac), so drug cannot bind |
Antibiotic targets across the cell
| Target | Drugs |
|---|---|
| Cell wall synthesis | β-lactams, vancomycin, bacitracin, polymyxin, daptomycin, isoniazid, ethambutol, ethionamide, cycloserine |
| 30S ribosome | Aminoglycosides, tetracyclines, tigecycline |
| 50S ribosome | Macrolides, clindamycin, chloramphenicol, linezolid, quinupristin-dalfopristin, telithromycin |
| DNA replication or integrity | Fluoroquinolones, metronidazole, clofazimine |
| RNA synthesis | Rifampin, rifabutin |
| Folate pathway (antimetabolites) | Sulfonamides, trimethoprim, dapsone, para-aminosalicylic acid |
Exam trap
- Tetracyclines bind the 30S subunit, whereas macrolides bind the 50S subunit.
- Antibiotics work best on rapidly dividing cells in log phase.
📈 Endospores, Growth and Culture
Endospores
- Formed by Bacillus and Clostridium (gram-positive rods)
- Purpose: survival, not reproduction
- One cell makes one spore
- Trigger: nutrient depletion
- Germination: warmth, moisture, nutrients
- Withstand heat, drying, radiation, freezing, chemicals
- Resistance features
- Dehydrated core rich in calcium dipicolinate
- Protective keratin-like protein coat
- Thick, less cross-linked cortex of peptidoglycan
- Heat-resistant enzymes
- Core holds DNA, ribosomes, glycolytic enzymes
- Autoclaving (steam under pressure) is needed to kill them
Vegetative cell→Starvation→Spore→Favorable conditions→Vegetative cell
- Fungal spores differ: they serve reproduction
Growth curve phases
| Phase | What happens |
|---|---|
| Lag |
|
| Log (exponential) |
|
| Stationary |
|
| Death |
|
- Binary fission: 1 → 2 → 4 → 8 cells
Culture principles
- Obligate intracellular organisms cannot grow on artificial media
- Viruses, rickettsiae, chlamydiae
- Need cell culture, eggs or animals
- Some organisms cannot be cultured at all, e.g., syphilis spirochete
- Selective medium: favors certain organisms with nutrients or antibiotics
- Differential medium: separates organisms by colony color or appearance
Special media
| Organism | Medium | Type |
|---|---|---|
| Anaerobes | Thioglycolate broth | Oxygen-depleting |
| Corynebacterium | Löffler serum; tellurite agar | Selective; differential |
| Enteric gram-negatives | MacConkey; eosin methylene blue | Selective and differential; differential |
| Enteric pathogens (Salmonella, Shigella) | Hektoen; xylose-lysine-deoxycholate | Differential |
| Vibrio cholerae | TCBS (alkaline) | Selective |
| Legionella | Charcoal yeast extract with cysteine | Selective |
| Mycobacterium | Löwenstein-Jensen | Selective |
| Neisseria (sterile sites), Haemophilus | Chocolate agar | Enriched |
| Neisseria (nonsterile sites) | Thayer-Martin: chocolate agar plus vancomycin, colistin, nystatin | Selective |
Special growth requirements
| Requirement | Organism |
|---|---|
| Cholesterol, purines, pyrimidines | Mycoplasma |
| Cysteine | Francisella, Brucella, Legionella, Pasteurella |
| X factor (hemin) and V factor (NAD) | Haemophilus influenzae and H. aegyptius need both |
🎯 Virulence Strategies
1. Colonization
- Adhesion to host surfaces
- Pili or fimbriae: main tool in gram-negatives
- Teichoic acids: main tool in gram-positives
- Adhesins: colonization factors, pertussis toxin, hemagglutinins
- Biofilm formation on inert surfaces
- Staphylococcus epidermidis (catheters, prostheses)
- Streptococcus mutans (teeth)
- Pseudomonas aeruginosa (lungs, catheters)
2. Avoiding phagocytosis and mucosal defense
- Polysaccharide capsules
- Streptococcus pneumoniae
- Klebsiella pneumoniae
- Haemophilus influenzae type b
- Pseudomonas aeruginosa
- Neisseria meningitidis
- Cryptococcus neoformans (yeast)
- Surface proteins that block uptake
- S. pyogenes: M protein
- S. aureus: protein A (binds Fc of IgG)
- N. gonorrhoeae: pili
- IgA protease cleaves secretory IgA
- Neisseria, Haemophilus, S. pneumoniae
3. Acquiring nutrients
- Siderophores chelate and import host iron
4. Antigenic variation
- N. gonorrhoeae: pili and outer membrane proteins
- Trypanosoma brucei: surface antigen switching
- Enterobacteriaceae: capsular and flagellar antigens switched on or off
- Influenza and HIV: mutation-driven change in surface proteins
5. Surviving inside cells
- Block phagosome–lysosome fusion
- Mycobacterium tuberculosis
- Escape the phagosome into cytoplasm
- Listeria monocytogenes
- Invasins drive entry into non-phagocytic cells
- Best characterized in Yersinia pseudotuberculosis
- Viral injury comes from intracellular replication
- Cell death, transformation or silent latency
6. Type III secretion
- Needle-like channel injects effector proteins into host cells
- Seen in E. coli, Salmonella, Yersinia, Pseudomonas aeruginosa, Chlamydia
7. Immune-mediated injury
| Mechanism | Example |
|---|---|
| Antibody cross-reactivity with host tissue | Rheumatic fever after group A strep |
| Immune complex deposition | Post-streptococcal glomerulonephritis |
| Delayed hypersensitivity and granulomas |
|
| Wall fragments driving inflammation | Gram-positive peptidoglycan–teichoic acid |
8. Physical damage
- Swelling in a closed space
- Meningitis, neurocysticercosis
- Bulk of the organism
- Ascaris blocking the bile duct
High-yield
- Intracellular pathogens trigger different immune responses, need different antibiotics and different culture methods.
☠️ Endotoxin and Exotoxin Principles
Endotoxin (LPS)
- Structural component of the gram-negative outer membrane
- Toxic part: lipid A
- Released mainly when bacteria die or lyse
- Neisseria meningitidis sheds outer membrane blebs while alive
- Heat stable
- Weakly immunogenic, so cannot be made into a toxoid
- Main driver of gram-negative sepsis
Pathway to septic shock
LPS→TLR-4 on macrophages→TNF-α, IL-1, IL-6→Endothelial injury, vasodilation→Hypotension, shock
- IL-1 is the major fever mediator
- Bradykinin-mediated vasodilation worsens shock
- Hageman factor (XII) activation triggers clotting
- Leads to DIC
Exotoxins
- Secreted protein toxins
- Produced by both gram-positive and gram-negative species
- Highly potent in tiny amounts
- Can be inactivated by heat or chemicals into a toxoid
- Keeps immunogenicity, loses toxicity
- Basis of tetanus and diphtheria vaccines
- Types by action
- Enterotoxins: act on gut
- Neurotoxins: act on nerves
- Cytotoxins: kill cells
A–B toxin design
- B subunit: binds a host receptor and delivers A into the cell
- A subunit: enzymatic, often ADP-ribosyltransferase
Cytolysins
- Damage membranes directly from outside
- Clostridium perfringens alpha toxin: lecithinase (phospholipase C)
- Staphylococcus aureus alpha toxin: forms membrane pores
Superantigens
- Bind MHC II and T-cell receptor outside the antigen groove
- Massive polyclonal T-cell activation with cytokine surge
Exam trap
- Endotoxin cannot be toxoided, but exotoxins can.
💉 Major Exotoxins by Mechanism
Protein synthesis inhibitors
| Organism | Toxin | Mechanism | Effect |
|---|---|---|---|
| Corynebacterium diphtheriae (gram +) | Diphtheria toxin | ADP-ribosylates elongation factor 2 (eEF-2) | Epithelial necrosis with pseudomembrane; cardiac and nerve injury |
| Pseudomonas aeruginosa (gram −) | Exotoxin A | ADP-ribosylates eEF-2 | Hepatocyte injury, tissue necrosis |
| Shigella dysenteriae (gram −) | Shiga toxin | Inactivates 60S ribosome (rRNA cleavage) | Enterotoxic, cytotoxic and neurotoxic; bloody diarrhea |
| Enterohemorrhagic E. coli (gram −) | Shiga-like toxin (verotoxin) | Inactivates 60S ribosome | Hemorrhagic colitis; hemolytic uremic syndrome |
Neurotoxins
| Organism | Toxin | Mechanism | Effect |
|---|---|---|---|
| Clostridium tetani (+) | Tetanospasmin | Blocks release of inhibitory glycine and GABA | Spastic paralysis, lockjaw |
| Clostridium botulinum (+) | Botulinum toxin | Blocks acetylcholine release at cholinergic junctions | Flaccid paralysis |
Superantigens
| Organism | Toxin | Effect |
|---|---|---|
| Staphylococcus aureus (+) | TSST-1 | Fever, rash, capillary leak, shock; heightened sensitivity to LPS |
| Streptococcus pyogenes (+) | Exotoxin A (pyrogenic or erythrogenic) | Similar illness, scarlet fever rash, cardiotoxicity |
Toxins that raise cAMP
| Organism | Toxin | Mechanism | Effect |
|---|---|---|---|
| Enterotoxigenic E. coli (−) | Heat-labile toxin (LT) | ADP-ribosylates Gs, locking adenylate cyclase on | Secretory diarrhea (heat-stable toxin ST acts through cGMP) |
| Vibrio cholerae (−) | Cholera toxin | Same as LT | Profuse watery diarrhea |
| Bacillus anthracis (+) | Anthrax toxins (three proteins) |
| Edema, impaired phagocytosis, cell death |
| Bordetella pertussis (−) | Pertussis toxin | ADP-ribosylates Gi, removing inhibition of adenylate cyclase | Lymphocytosis, increased histamine sensitivity, insulin release |
Cytolysins
| Organism | Toxin | Mechanism | Effect |
|---|---|---|---|
| Clostridium perfringens (+) | Alpha toxin | Lecithinase | Membrane breakdown, myonecrosis (gas gangrene) |
| Staphylococcus aureus (+) | Alpha toxin | Inserts to form pores | Leaky, lysed cells |
High-yield
- Diphtheria toxin and Pseudomonas exotoxin A share the same target, eEF-2.
- Cholera, ETEC LT and pertussis toxin all alter G-protein signaling to change cAMP.
- Tetanus blocks inhibitory transmitters, so muscles contract; botulism blocks excitatory transmitter, so muscles relax.