Presentation made by Dr. Jerriton Brewin on the bacteriology and immunology of Leprosy.

1. BACTERIOLOGY &
IMMUNOLOGY
of M. leprae
Dr. Jerriton, DVL PG,
SVMCH, Pondy
18.5.20

2. BACTERIOLOGY

4. TAXONOMY CLASSIFICATION
KINGDOM - Bacteria
PHYLUM - Actinobacteria
ORDER - Actinomycetales
SUBORDER - Corynebacterineae
FAMILY - Mycobacteriaceae
GENUS - Mycobacterium
SPECIES - Leprae

5. ORGANISM DESCRIPTION
• Obligate Intracellular
• Acid Fast – Mycolic Acid
• Aerobic
• Microaerophilic
• Non-motile
• Non-spore forming
• Pyridine extractable
• Pathogenic

6. MEASUREMENTS & SPECIAL FEATURES
• Length – 1.8 μm
• Diameter – 0.3 μm
• Length = 5 x Breath
• Parallel sides
• Rounded ends
• Bright Pink on ZN stain
• Bundle of cigars appearance

7. • Non - Cultivable
• Generation Time: 11-13 days
• Temperature: 27 - 30 ° C
• Can remain dormant in humidity for 5 months
• Undergone reductive evolution – Catabolic pathways ↓
• Unique host: Schwann cells
• Principle host: Macrophages

8. MODES OF TRANSMISSION
• Exact method unknown
• By direct contact
• Respiratory droplet spread
• Insects
NOT SPREAD BY…
• Shaking hands or hugging
• Sitting next to each other on the bus
• Sitting together at a meal
• Mother to child during pregnancy
• Sexual contact
Source: WHO & CDC

9. ANIMAL MODELS
MOUSE ARMADILLO
Immunocompetent mice
Immunodeficient mice
Inoculum size: upto 104
Bacilli Obtained: 106
Inoculum size: > 105
Bacilli Obtained: > 106
Inoculum size: 108
Bacilli Obtained: 1010

10. CELLULAR MORPHOLOGY

11. • M. leprae smears on treating with pyridine, lose the
ability to stain subsequently with carbon fuchsin and
thereby appear non-acid fast.
• It is unique to M. leprae.
PYRIDINE EXTRACTION

12. VIRULENCE FACTORS
• Phenolic Glycolipid I (PGL – I)
• Lipoarabinomannan (LAM)

13. PATHOGENESIS OF INFECTION
SCHWANN CELL (SC)
• Initial Target: Laminin α2 –> PGL 1 of M. leprae binds to it
• Laminin α2 seen in Schwann cell, Striated muscle, Placenta
• H1p/LBP21 –> potentiates interaction of M. leprae with SC
• SC processes antigen & presents it through MHC – II
• CD4+ T Cells then get activated & releases Ils –> leads to
Macrophage activation –> kills bacteria
• Concurrent nerve demyelination occurs due to inflammatory events

15. • SC membrane has laminin 2 and a laminin 2 receptor (α-
dystroglycan)
• Laminin 2 has a G domain on the α2 chain
• PGL-1 of M. leprae binds to this domain.
• This PGL-Laminin-2 complex interacts with α-dystroglycan,
leading to uptake of M. leprae.
• Laminin binding protein 21 (LBP21) of M. leprae also binds
to α-DG od SC membrane, leading to its entry.
MECHANISM OF ENTRY INTO NERVE

16. • It is a major glycolipid antigen of M. leprae.
• Is unique to M. leprae.
• It is part of lipid capsule.
• Accounts for 2% of mass of bacilli.
PGL-1

17. • Has an antigenically distinct trisaccharide linked to
phenol, which is linked to 29C phthiocerol, which are
attached 2 mycoserosic acids.
• Specific IgM antibodies develop to it, more at
lepromatous spectrum.
• Antigen specificity resides in terminal sugars, which
has been exploited for serodiagnosis.
PGL-1 (contd.)

18. • Helps in entry and colonization within phagocytes.
• Once inside phagocytes, it can scavenge ROS and
helps the bacteria survive intracellularly.
PGL-1 (contd.)

19. STAINS
Haematoxylin – Eosin stain Job-Chacko Modification of
Fite Faraco stain
Gomori – Grocott
Methenamine Silver
• Immunochemical stain
• S100 stain
• Fluorescent microscopy
• FDA – EB stain
Other stains

20. AFB STAIN

21. INDICES
BACTERIAL INDEX
- Determines bacterial load (live + dead)
- Score:
+6 = Over 1,000 bacilli + globi on an average field
+5 = Over 100 bacilli but less than 1,000 in an average field
+4 = Over 10 bacilli but less than 100 in an average field
+3 = 1 – 10 bacilli in an average field
+2 = 1 – 10 bacilli in 10 fields
+1 = 1 – 10 bacilli in 100 fields
0 = No bacilli in 100 fields

22. INDICES
BACTERIAL INDEX
Sum of values from all sites
Total number of sites
ZN stain with Acid Fast bacilli

23. INDICES
MORPHOLOGICAL INDEX
• Percentage of live bacilli from 200 bacilli
• Solid staining = Live bacilli
• Solid stain:
- Entire organism must be
uniformly stained
- Longitudinal sides are
parallel
- Both ends are rounded
- Length is 5 times width

24. • Although M. leprae has been extensively studied, and
complete genome sequencing has been done, there are
still grey areas to be explored, including the inability to
grow in vitro.
• The knowledge of M. leprae, when integrated with the
knowledge of immunological responses in the host, will
provide a better understanding for diagnosis and
treatment.
CONCLUSION

25. IMMUNOLOGY

26. CYTOKINE PROFILES

27. INNATE IMMUNITY

28. INNATE IMMUNITY

29. Entry of pathogen
Internalization
INNATE IMMUNITY

30. Recognition
Induction of cytokines
INNATE IMMUNITY

31. TT Leprosy
Induces CMI
INNATE IMMUNITY

32. Tuberculoid
Induces CMI
INNATE IMMUNITY

33. LL Leprosy
Induces humoral immunity - IL 4
(Antibody production)
Suppresses CMI - IL 10
Inhibits TLR activation
INNATE IMMUNITY

34. Antigen presentation
to lymphocytes
(Acquired immunity)
INNATE IMMUNITY

35. ACQUIRED IMMUNITY

36. TT LL
Cytokine Profile Th 1 Th 2
Immunity CMI Humoral
CD4 cells
Cytolytic cells,
more
Helper cells, less
CD8 cells Cytotoxic, more Suppressor, less
Macrophage Absent Present
NON-REACTIONAL LEPROSY STATES

37. • In lepromatous subjects, there is characteristic lymphopenia.
A. Hence macrophages contain large no. of M. leprae, but
cannot mount CMI.
B. There is B cell activation leading to antibody production,
which cannot attack intracellular M. leprae, but forms
immune complexes with tissue / circulating M. leprae
antigens. Antibodies are also exploited for diagnosis / clinical
complications (eg. IgM antibodies against PGL-1).
C. CD8 suppressor cells suppress CMI

38. Why is CMI characteristically suppressed in LL?
IMMUNOLOGICAL UNRESPONSIVENESS

39. 1. Antibody mediated suppression
2. Presence of CD8 suppressor T cells
3. Macrophages suppress T cell proliferation
4. Macrophage factors: PGE2, thromboxane, leukotrienes, IL10.
5. PGL of M. leprae
6. FOXP3 T-regs - secrete TGF-β and IL10 - leads to
suppression
7. Th phenotype paradigm - presence of Th0 phenotype in 50%
tuberculoid and 40% LL.
8. TLR2 mutation
REDUCED T CELLS IN LL IS DUE TO:

40. IMMUNOLOGY OF REACTIONS

41. • Occurs in borderline cases
• Type 4 DHT
• T cell activation (lymphocyte responsiveness)
• Reflects a switch from a Th2 toward a Th1 response
• Immunological marker: CXCL10
• Infiltration of IFN-γ and TNF-secreting CD4+ T cells in
skin lesions and nerves, resulting in edema and painful
inflammation.
• Increased Treg activity
TYPE 1 REACTION

42. • Occurs in LL > BL
• Type 3 immune complex reaction
• T cell activation (lymphocyte responsiveness)
• Increased CD4 cells
• Switch from Th2 to Th1 profile
• Massive neutrophil infiltration - TNF production - tissue
damage
• Immune complex deposition in blood vessels (vasculitis),
adipocytes (panniculitis) and eye (uveitis)
TYPE 2 REACTION

43. NERVE DAMAGE IN LEPROSY

44. • Nerve damage is due to immunological and non-
immunological events
• But not due to direct effect of bacteria.

45. 1. Intraneural macrophages produce TNFα - demyelination
2. TLR-2 activation on SC - apoptosis
3. In T1R - antigens released from SC induce DTH reaction
4. In T2R - Immune complex deposition, granulocyte
attraction and compliment activation
5. SC process and present M. leprae antigens to CD4 cells -
which gets activated, leading to SC lysis.
IMMUNOLOGICAL MECHANISMS OF
NERVE DAMAGE

46. 1. Contact dependent demyelination
2. NO producing demyelination
3. Protective role of myelin
NON-IMMUNOLOGICAL MECHANISM OF
NERVE DAMAGE

47. 1. Chromosome 6q25 - linked to PARK2/PCRG gene
regulatory region
2. Chromosome 6p21 - linked to lymphotoxin-α gene
3. Chromosome 10p13 - candidate gene not identified,
susceptible to PB (Tuberculoid)
IMMUNOGENETICS

48. • TLR 1, 2, 4 polymorphisms
TLR 2 mutation increases IL-10, an anti-inflammatory cytokine
that suppresses Th1 response
• Mutations of Mannose Receptor 1, a phagocytic
receptor that mannose capped LAM of M. leprae
• Vitamin D polymorphism (Vitamin D enhances Th2 T-
cell responses at the expense of Th1 responses)
• Laminin 2 polymorphism

49. • IL-10 polymorphism leads to increased production (IL-
10, an anti-inflammatory cytokine, inhibits Th1
response)
• IL-12 polymorphism (susceptibility to LL)
• TNF-α mutation (TNF is important for resistance to
leprosy)
• IFN-γ polymorphism

50. • Both innate and adaptive immune responses play a role in leprosy
• Lymphopenia is a characteristic feature of LL. Others include
absent CMI, presence of humoral immunity with a Th2 cytokine
profile.
• Several genetic polymorphisms make a person susceptible to
specific spectrums in leprosy, which gets reflected in their
immunological profiles.
• More understanding is needed for diagnostics, vaccine
development and therapy for a given population.
CONCLUSION

51. 1. IAL Textbook of Leprosy
2. Jopling’s Hnadbook of Leprosy
3. Bhat RM, Prakash C. Leprosy: an overview of
pathophysiology. Interdiscip Perspect Infect Dis.
2012;2012:181089.
REFERENCES