Mycobacterium tuberculosis Mycobacterium tuberculosis Rv2224c - - PowerPoint PPT Presentation

Mycobacterium tuberculosis Mycobacterium tuberculosis Rv2224c modulates innate Rv2224c modulates innate immune responses immune responses

Jyothi Rengarajan, Elissa Murphy, Arnold Park, Arnold Park, Cassandra L. Krone, Erik C. Hett, Barry R. Bloom, Laurie H. Glimcher, and Eric J.Rubin. The Proceedings of the National Academy of Sciences 105:264-269, 2008.

Speaker Speaker： ：Ming Ming-

• Cheng Chen

Cheng Chen Advisor Advisor： ：Chii Chii-

• Shen

Shen Yang, Ph.D. Yang, Ph.D. Date Date： ：2008/12/16 2008/12/16

1

Mycobacterium Mycobacterium tuberculosis(Mtb tuberculosis(Mtb) )

Tuberculosis Mycolic acid Primary intracellular niche

is within macrophages

Chronic infection

2

Tuberculosis in the world Tuberculosis in the world

• range = 200-300

yellow = 100-200 green = 50-100 blue = <50

For every year For every year

8 million people become ill with tuberculosis 2 million people die from the disease worldwide

The leading c The leading cause of death ause of death among people with AIDS among people with AIDS

3

In the history In the history… …

Simon Bolivar (Venezuelan revolutionary leader) King Tutankhamen (Egyptian pharaoh)

4

The infection pathway of The infection pathway of Mtb Mtb in in human human

spread through the air form granulomas in the lung

5

The infection pathway of The infection pathway of Mtb Mtb in in human human

6

tubercle tubercle

• ld age
• ld age

malnutritio n malnutritio n HIV-coinfection HIV-coinfection

Tissue section in TB patients Tissue section in TB patients

mtb mtb

Caseous necrosis Caseous necrosis Tissue staining of granulomas Tissue staining of granulomas

7

The possible outcomes of The possible outcomes of Mtb Mtb exposure exposure

High innate immunity High innate immunity High innate immunity Innate immunity Innate immunity Innate immunity Innate immunity Innate immunity Innate immunity Mtb clearance Mtb Mtb clearance clearance Defective adaptive immunity Defective adaptive immunity Defective adaptive immunity Adaptive immunity Adaptive immunity Adaptive immunity

No infection No infection No infection Disease <10 % Disease <10 % Disease <10 % Latent infection Latent infection Latent infection Containment >90 % Containment >90 % Containment >90 % Disease reactivation Disease reactivation Disease reactivation Immunosuppression Immunosuppression Immunosuppression

8

Mtb Mtb is a successful human pathogen is a successful human pathogen

9

Mtb Mtb The bacterial factors that mediate these processes are poorly understood ! The bacterial factors that mediate these processes are poorly understood !

fusion of phagosomes with lysosome

antigen presentations

IFN-γ-mediated signaling pathways

transcriptional responses

In the previous study In the previous study… …

Constructing transposon mutant library 126/2863 gene is essentially important Rv2224c is critically important for Mtb

survival in macrophages

Gene Unactivated

pre-infection IFNg post-infection IFNg

Rv2224c 0.0873 0.0395 0.0395 Rv2282c 0.373 0.729 0.313 Rv1332 0.476 0.379 0.478 rimJ 4.419 2.901 2.94

………….

10

The construction of the The construction of the transposon transposon mutant library mutant library

11 + gene X transposon transposon insertion gene X loss function

Mtb growth in different conditions Different fluorescent strength

B is essential for Mtb surviving in macrophage

Experimental strategy Experimental strategy

12

The location of Rv2224c Influence of Rv2224c for Mtb growth in vivo

The relationship between Rv2224c and the innate immune response

The regulation target for Rv2224c The importance of Rv2224c for Mtb virulence

Rv2224c is predicted as a cell Rv2224c is predicted as a cell envelope envelope-

• associated

associated hydrolase hydrolase

Whole cell Cell wall CytoplasmCell menbrane

Catalytic triad Catalytic triad

Lipoprotein attachment site Lipoprotein attachment site

Hydrolase activity Hydrolase activity

13

cytoplasmic heat-shock-protein

cell wall-associated protein

Mice infected with wild Mice infected with wild-

• type and

type and Rv2224c Rv2224c mutant strain mutant strain

wt:Rv2224c::tn wt:Rv2224c::tn wt:Rv2224c::tn 2224c wt:Rv2224c::tn 2224c wt:Rv2224c::tn S228A wt:Rv2224c::tn S228A

mixed infected

The Rv2224c enzymatic activity is required for optimal Mtb growth in vivo. The Rv2224c enzymatic activity is required for optimal

• ptimal Mtb

Mtb growth growth in vivo. in vivo.

14

Lung

Disruption of Disruption of Rv2224c Rv2224c prolongs prolongs survival and reduces lung survival and reduces lung immunopathology immunopathology

• f infected mice
• f infected mice

Median survival time = 195 day Median survival time = 195 day Median survival time = 336 day Median survival time = 336 day

Mice infected with mutant live longer Mice infected with mutant live longer

15

The cell wall structure of The cell wall structure of Mtb Mtb

waxy coating on the cell surface —mycolic acids (1) auramine-rhodamine stain (2) H&E stain

16

hematoxylin basophilic structures blue-purple hue Nucleic acid

ribosomes chromatin-rich cell nucleus cytoplasmatic regions rich in RNA

eosin Y eosinophilic structures bright pink protein cytoplasm red blood cell

H&E H&E staining staining

H&E H&E-

• stained lung tissue sections from

stained lung tissue sections from wild wild-

• type

type-

• r mutant
• r mutant-
• infected mice

infected mice

The tissue was stained 20 weeks after infection

Wild type Rv2224c::tn 80–90% of the tissue containing cellular infiltrates 80–90% of the tissue containing cellular infiltrates retained 80% of alveolar space retained 80% of alveolar space

17

Conclusion(1/4)

Rv2224c is a cell membrane lipoprotein with

hydrolase activity.

What’s the role of Rv2224c in innate immune response ?

18

Absence of Rv2224c attenuates the

virulence of Mtb in vivo.

Rv2224c Rv2224c modulates innate modulates innate immune responses immune responses

Median survival time = 64 day Median survival time = 64 day Median survival time = 37 day Median survival time = 37 day Median survival time = 50 day Median survival time = 50 day Median survival time = 84 day Median survival time = 84 day intravenous aerosol

RAG-/- mice infected with mutant survive longer RAG-/- mice infected with mutant survive longer

RAG-/- mice = lack T and B cells, RAG-/- mice = lack T and B cells,

19 80–90% of the tissue containing cellular infiltrates 80–90% of the tissue containing cellular infiltrates retained 80% of alveolar space retained 80% of alveolar space

Lung tissue were stained 35 days after aerosol infection

Day 35 400x Time of death 400x Time of death 100x

Wild type Rv2224c::tn

Auramine O Auramine O mocylic acid mocylic acid reddish-yellow fluorescence reddish-yellow fluorescence

the cfus become the same the cfus become the same

Auramine/rhodamine Auramine/rhodamine staining staining

• f lung tissue
• f lung tissue

Aerosol-infected RAG-/- mice on day 35

20

Conclusion(2/4)

Rv2224c mutant strains are not inherently

defective in their ability to replicate but fail to do so in the face of early innate immune responses.

What’s the difference after Rv2224c in Mtb is mutated?

21

Rv2224c can influence the Rv2224c can influence the inflammatory response inflammatory response

2 days after infection

proinflammatory cytokines：TNF, IL1, and IL6 neutrophil and lymphocyte recruitment： IL8, MIP1, and RANTES

22

Dampened inflammatory milieu may be generated by Rv2224c::tn-infected macrophages in vivo Dampened inflammatory milieu may be generated by Rv2224c::tn-infected macrophages in vivo

Deficient in Rv2224c increases Deficient in Rv2224c increases susceptibility to susceptibility to lysozyme lysozyme

When losts Rv2224c function The resistance to lysozome is decreased When losts Rv2224c function The resistance to lysozome is decreased

23

Mtb can resistant the hydrolysis of lysozyme Mtb can resistant the hydrolysis of lysozyme

Each strain in the presence/absence of lysozyme Each strain in the presence/absence of lysozyme

Conclusion(3/4)

Rv2224c::tn mutant was markedly more

susceptible to lysozyme

What’s the physiological target for Rv2224c in Mtb?

24

Influence innate immune response by

secreting cytokines and chemokines

Rv2224c promotes processing of Rv2224c promotes processing of GroEL2 GroEL2

Rv2224c cell envelope-associated hydrolytic activity Rv2224c cell envelope-associated hydrolytic activity

Small protein

(active form)

Small protein

(active form)

Full-length protein Full-length protein

Whole cell Cell wall Cytoplasm Cell menbrane Whole cell pellets Culture supernatant

25

smaller version is produced by cleavage of full-length GroEL2 smaller version is produced by cleavage of full-length GroEL2

GroEL GroEL-

• heat shock protein in

heat shock protein in Mtb Mtb

GroEL GroEL+GroES

Two kinds of function

(1) Helps the folding of naïve protein (1) Helps the folding of naïve protein (2)Regulates the immune response (2)Regulates the immune response

GroEL2 secreted by Mtb can induce chronic inflammation response GroEL2 secreted by Mtb can induce chronic inflammation response

26

Rv2224c promotes processing of Rv2224c promotes processing of GroEL2 GroEL2

Rv2223c+Rv2224c Rv2223c+Rv2224c catalytic triad in S228-H490-D463

Whole cell pellets Culture supernatant

27

Conclusion(4/4)

The extracellular releasing of GroEL2

depends on Rv2224c function.

28

Releasing of GroEL2 to cell can modulate

inflammation response.

Summary Summary

Rv2224c locates on the cell membrane and with hydrolase activity. Rv2224c locates on the cell membrane and with hydrolase activity.

29

It’s critical of Mtb virulence in vivo, and influence the Mtb resistance to innate immunoresponse. It’s critical of Mtb virulence in vivo, and influence the Mtb resistance to innate immunoresponse. It helps the release of GroEL2. Thus, it can modulate inflammation response. It helps the release of GroEL2. Thus, it can modulate inflammation response. In the long run, it causes the formation of granulomas and makes patient in latent infection. In the long run, it causes the formation of granulomas and makes patient in latent infection.

Rv2224c

New therapy for tuberculosis!!!

GroEL2

granulomas

Thank you for your Attention!! Thank you for your Attention!!

30

RAG

(recombination activating genes )

The recombination activating genes encode

enzymes that play an important role in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules during the process of VDJ recombination.

RAG-1 and RAG-2 are essential to the generation of

mature B and T lymphocytes, two cell types that are crucial components of the adaptive immune system.

The RAG proteins initiate V(D)J recombination,

which is essential for the maturation of pre-B and pre-T cells.

31

Chronic inflammation

• Chronic inflammation is a pathological condition characterised by

concurrent active inflammation, tissue destruction, and attempts at

• repair. Chronic inflammation is not characterised by the classic signs
• f acute inflammation listed above. Instead, chronically inflamed

tissue is characterised by the infiltration of mononuclear immune cells, tissue destruction, and attempts at healing, which include angiogenesis and fibrosis.

• Endogenous causes include persistent acute inflammation.

Exogenous causes are varied and include bacterial infection, prolonged exposure to chemical agents such as silica, tobacco smoke, or autoimmune reactions such as rheumatoid arthritis.

• In chronically inflamed tissue the stimulus is persistent, and

therefore recruitment of monocytes is maintained, existing macrophages are tethered in place, and proliferation of macrophages is stimulated.

32

Cellular immune response to Cellular immune response to Mtb Mtb

33

Acid-fast organisms are difficult to characterize

using standard microbiological techniques (e.g. Gram staining. Once stained, these organisms resist the dilute acid and/or ethanol-based de- colorization procedures common in many staining protocols—hence the name acid-fast.

34

35

Summary of the infectious Summary of the infectious processes of processes of Mtb Mtb

36

The red acid-fast Mtb is in haled

MHC II Antigen presentation CD4

IL-12 IL-1

T Fever TH 1 TH 1 CD4+ TH 1 cells Activated Macrophage Caseous Necrosis

IL-2 INF-γ

Epithelioid macrophage (activated) Alveolus macrophage

(1) (2) (3) (4) (5) (6)

Alveolus macrophage Mycobacterium tuberculosis Alveolus macrophage

phagocytosis

37

Auramine O can be used to stain acid-fast bacteria (e.g. Mycobacterium), where it binds to the mycolic acid in its cell wall in a way similar to Ziehl-Neelsen stain.