Intracellular Pathogens – Listeria monocytogenes & Mycobacterium tuberculosis

Intracellular‐versus‐Extracellular Pathogenesis

• Extracellular pathogens
  • Kill host cells from the outside and replicate outside host cells.
  • Example: enterohemorrhagic E. coli (EHEC) secretes toxins that damage intestinal epithelium but stays in the intestinal lumen.
• Intracellular pathogens
  • Invade, survive, and replicate within host cells.
  • The intracellular lifestyle is often essential for virulence.
  • Two medically important models explored in this lecture:
  • Listeria monocytogenes
  • Mycobacterium tuberculosis

Listeria monocytogenes – Food-borne Intracellular Pathogen

Outbreak Example (Blue Bell Creameries, USA)

• Full product recall on $April~20^{th},~2015$ due to potential Listeria contamination.
• $10$ confirmed listeriosis cases; $3$ deaths.
• Investigation timeline
  • Routine screening in South Carolina found L. monocytogenes in ice-cream products.
  • Production plants in Texas & Oklahoma subsequently tested positive.
  • Bacterial isolates were uploaded to PulseNet; identical genome profiles linked factory strains to hospitalized patients across $4$ states (Arizona, Kansas, Oklahoma, Texas) over a $5$-year window.

Basic Biology & Ecology

• Gram-positive bacillus.
• Natural saprophyte in soil; feeds on decaying organic matter.
• Broad host range (protists, arthropods, animals, humans).
• Named for Joseph Lister (pioneer of surgical sterility).
• Species epithet "monocytogenes" reflects marked monocytosis during infection.
• Transmission to humans: ingestion of contaminated processed meats, dairy, other refrigerated foods.
• Physical/physiological tolerances confer food-borne fitness:
  • High salt, low moisture, and cold growth (can multiply in refrigerators).

Pathogenesis in Humans

• Colonizes gastrointestinal tract after ingestion.
• Healthy individuals: asymptomatic carriage or self-limiting diarrhea; bacteria shed.
• Immunocompromised, elderly, pregnant, or neonates:
  • Crosses intestinal epithelium ➔ lymphatic spread ➔ systemic dissemination.
  • Possible courses (incubation $3$–$4$ weeks):
  • Septicemia (bloodstream invasion).
  • Central nervous system disease (meningitis, encephalitis) after traversing blood-brain barrier.
  • Trans-placental infection in pregnancy ➔ miscarriage / neonatal sepsis.

Cellular Invasion – Internalins (InlA & InlB)

• Listeria expresses surface adhesins Internalin A (InlA) & Internalin B (InlB).
• Bind host epithelial receptors, initiating signaling cascades that remodel the actin cytoskeleton.
• Membrane ruffling induces a forced phagocytosis (epithelial cells are normally non-phagocytic).
• Bacterium is enclosed in a phagosome instead of a digestive lysosome.

Phagosomal Escape – Listeriolysin O (LLO)

• LLO = cholesterol-dependent pore-forming cytolysin.
  • Synthesized as soluble monomers, active at acidic pH (phagosome environment).
  • Monomers bind cholesterol-rich phagosomal membrane ➔ oligomerize ➔ insert β-barrel pores.
  • Ionic imbalance & membrane rupture release bacterium into cytoplasm.
• Cholesterol-dependent cytolysins are a common virulence mechanism among Gram-positives.

Intracellular Motility & Cell-to-Cell Spread

• Cytoplasm provides rich nutrients & shields bacterium from complement, antibodies, professional phagocytes.
• Replication limited by host nutrient pool of a single cell; dissemination requires active movement.
• ActA protein displayed at one pole recruits host Arp2/3 complex ➔ nucleates actin comet tails.
  • Continuous polymerization propels bacterium through cytoplasm.
  • Rapid Brownian jostling ensures filaments fill any gap, generating forward thrust.
  • Listeria can form protrusions that are engulfed by neighboring cells, entering them within a double membrane.
  • LLO mediates escape from both membranes, restarting the cycle.
• Outcome: direct cell-to-cell spread through tissues without extracellular exposure.

Concept-Check Summaries (Lecture Polls)

• “Why is Listeria so good at causing food‐borne illness?” – Answer: All of the above (resistant to freezing, high salt, drying).
• “How does LLO bind specifically to host cells?” – Answer: It binds cholesterol-rich membranes.
• “How does Listeria move inside the host?” – Answer: Induces polymerization of host actin.

Mycobacterium tuberculosis – Ancient & Chronic Intracellular Pathogen

Historical Perspective

• Acid-fast bacterium responsible for tuberculosis (TB).
• Co-migrated with early humans out of Africa ~$70{,}000$ years ago.
• Western Europe’s “White Plague” ($17^{th}$–$18^{th}$ centuries): virtually universal infection; TB caused $25\%$ of all deaths.
• Present day: rare in developed nations yet second leading infectious killer worldwide.

U.S. Epidemiology

• TB incidence declined steadily with improved sanitation, nutrition, housing.
• Marked spike late $1980$s – early $1990$s due to:
  • HIV/AIDS epidemic (immunosuppression).
  • Rising homelessness (crowding + limited healthcare).

Transmission Dynamics

• Spread via inhalation of aerosolized respiratory droplets/dust; risk escalates in crowded settings.
• Requires relatively high infectious dose.
• Two clinical stages:
  1. Primary TB
  • Initial exposure; mild, self-limiting “cold-like” illness.
  • Not contagious at this stage.
  1. Secondary (reactivation) TB
  • Emerges when cellular immunity wanes (months→years later).
  • Symptoms: persistent, productive cough, fever, weight loss/emaciation (“consumption”).
  • Highly infectious owing to continuous cough.
• The asymptomatic interval reflects successful immune containment, not bacterial clearance.

Cellular & Molecular Determinants of Persistence

1. Extremely slow growth
   • Doubling time ≈ $14$ h.
   • Evades rapid immune detection & undermines antibiotics that target division.
2. Acid-fast cell envelope
   • Rich in long-chain mycolic acids; yields waxy colonies.
   • Confers formidable resistance to:
     • Chemical disinfectants
     • Desiccation (survives in airborne droplets & dust)
3. Intracellular survival in macrophages
   • After inhalation, alveolar macrophages phagocytose bacilli.
   • Mtb secretes cell-wall lipids that block phagosome–lysosome fusion, avoiding acid hydrolases.
   • Bacteria replicate slowly inside the unacidified phagosome (they do not escape to cytoplasm).
4. Granuloma formation
   • Lipids released by Mtb act as MAMPs (microbe-associated molecular patterns) ➔ stimulate cytokine networks.
   • Host response walls off infection within granulomas composed of concentric layers of:
     • Infected macrophages
     • T lymphocytes & other immune cells
     • Fibroblasts depositing collagen.
   • Function: contain, not eradicate, bacilli; oxygen & nutrient limitation slows but does not kill Mtb.
   • Bacilli may persist viable decades.
   • Immune compromise (HIV, aging, malnutrition, immunosuppressive therapy) ➔ granuloma breakdown ➔ reactivation & contagious secondary TB.

Concept-Check Summaries (Lecture Polls)

• “Which stage of TB is most contagious?” – Answer: Secondary TB.
• “What is the function of granulomas?” – Answer: To contain Mtb.

Comparative Themes & Key Takeaways

• Both pathogens establish intracellular niches to avoid humoral immunity.
• Listeria: rapid cytosolic growth, actin-based motility, short incubation, acute dissemination.
• M. tuberculosis: slow intraphagosomal growth, granuloma formation, long latency, chronic disease.
• Understanding distinct intracellular survival strategies guides targeted therapeutics and public‐health interventions.

Numerical & Statistical References (for quick review)

• $3303$ – Course number (BIOL $3303$, Lecture $22$).
• $April~20^{th},~2015$ – Blue Bell recall date.
• $10$ cases, $3$ deaths – Blue Bell outbreak toll.
• $4$ states, $5$ years – Geographic & temporal spread linked by PulseNet.
• $70{,}000$ years – Approximate age of Mtb–human association.
• $25\%$ – Deaths caused by TB during Europe’s White Plague.
• $14$ h – Mtb doubling time.
• Late $1980$s/early $1990$s – U.S. TB spike (HIV + homelessness).