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. ： Ming Speaker ： Ming- -Cheng Chen Cheng Chen Speaker ： Chii Advisor ： Chii- -Shen Shen Yang, Ph.D. Yang, Ph.D. Advisor ： 2008/12/16 Date ： 2008/12/16 Date 1
Mycobacterium tuberculosis(Mtb tuberculosis(Mtb) ) Mycobacterium � Tuberculosis � Mycolic acid � Primary intracellular niche is within macrophages � Chronic infection 2
Tuberculosis in the world Tuberculosis in the world orange = 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 cause of death The leading c ause of death among people with AIDS among people with AIDS 3
In the history … In the history … King Tutankhamen Simon Bolivar (Egyptian pharaoh) (Venezuelan revolutionary leader) 4
The infection pathway of Mtb Mtb in in The infection pathway of human human � spread through the air � form granulomas in the lung 5
The infection pathway of Mtb Mtb in in The infection pathway of human human tubercle tubercle old age old age malnutritio malnutritio n n HIV-coinfection HIV-coinfection 6
Tissue section in TB patients Tissue section in TB patients Tissue staining of granulomas Tissue staining of granulomas Caseous necrosis Caseous necrosis mtb mtb 7
The possible outcomes of Mtb Mtb exposure exposure The possible outcomes of High innate immunity Mtb clearance clearance High innate immunity Mtb No infection No infection High innate immunity Mtb clearance No infection Innate immunity Defective adaptive immunity Disease <10 % Innate immunity Defective adaptive immunity Disease <10 % Innate immunity Defective adaptive immunity Disease <10 % Innate immunity Adaptive immunity Innate immunity Adaptive immunity Latent infection Latent infection Innate immunity Adaptive immunity Latent infection Immunosuppression Immunosuppression Immunosuppression Disease reactivation Disease reactivation Containment >90 % Containment >90 % Disease reactivation Containment >90 % 8
Mtb is a successful human pathogen is a successful human pathogen Mtb fusion of phagosomes with lysosome antigen presentations IFN- γ -mediated signaling pathways Mtb Mtb transcriptional responses The bacterial factors that mediate these The bacterial factors that mediate these processes are poorly understood ! processes are poorly understood ! 9
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 0.373 0.729 0.313 Rv2282c 0.476 0.379 0.478 Rv1332 4.419 2.901 2.94 rimJ …………. 10
The construction of the The construction of the transposon mutant library transposon mutant library + gene X transposon transposon insertion gene X loss function B is essential for Mtb surviving in macrophage � Mtb growth in different conditions � Different fluorescent strength 11
Experimental strategy Experimental strategy 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 12
Rv2224c is predicted as a cell Rv2224c is predicted as a cell envelope- -associated associated hydrolase hydrolase envelope Catalytic triad Catalytic triad Hydrolase activity Hydrolase activity Lipoprotein attachment site Lipoprotein attachment site cytoplasmic heat-shock-protein cell wall-associated protein Whole cell Cell wall CytoplasmCell menbrane 13
Mice infected with wild- -type and type and Mice infected with wild Rv2224c mutant strain mutant strain Rv2224c Lung 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 The Rv2224c enzymatic activity is required for optimal optimal Mtb Mtb growth growth in vivo. in vivo. for optimal Mtb growth in vivo. 14
Disruption of Rv2224c Rv2224c prolongs prolongs Disruption of survival and reduces lung survival and reduces lung immunopathology of infected mice immunopathology of infected mice Median survival time = 336 day Median survival time = 336 day Median survival time = 195 day Median survival time = 195 day Mice infected with mutant live longer Mice infected with mutant live longer 15
The cell wall structure of Mtb Mtb The cell wall structure of H&E staining staining H&E 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 � waxy coating on the cell surface —mycolic acids � (1) auramine-rhodamine stain � (2) H&E stain 16
H&E- -stained lung tissue sections from stained lung tissue sections from H&E wild- -type type- - or mutant- -infected mice infected mice wild or mutant Wild type Rv2224c::tn 80–90% of the tissue containing retained 80% of alveolar space 80–90% of the tissue containing retained 80% of alveolar space cellular infiltrates cellular infiltrates 17 The tissue was stained 20 weeks after infection
Conclusion(1/4) � Rv2224c is a cell membrane lipoprotein with hydrolase activity. � Absence of Rv2224c attenuates the virulence of Mtb in vivo . What’s the role of Rv2224c in innate immune response ? 18
Rv2224c modulates innate modulates innate Rv2224c immune responses immune responses intravenous aerosol Median survival time = 50 day Median survival time = 50 day Median survival time = 84 day Median survival time = 84 day RAG -/- mice = lack T and B cells, RAG -/- mice = lack T and B cells, Median survival time = 64 day Median survival time = 64 day Median survival time = 37 day Median survival time = 37 day RAG -/- mice infected RAG -/- mice infected with mutant survive longer with mutant survive longer 80–90% of the tissue 80–90% of the tissue retained 80% of alveolar space retained 80% of alveolar space containing containing 19 cellular infiltrates cellular infiltrates Lung tissue were stained 35 days after aerosol infection
Auramine/rhodamine staining Auramine/rhodamine staining of lung tissue of lung tissue Wild type Rv2224c::tn Auramine O Auramine O Day 35 mocylic acid mocylic acid 400x reddish-yellow fluorescence reddish-yellow fluorescence the cfus become the cfus become Time of the same the same death 400x Time of death 100x 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 Dampened inflammatory milieu may be generated by Dampened inflammatory milieu may be generated by Rv2224c :: tn -infected macrophages in vivo Rv2224c :: tn -infected macrophages in vivo 22
Deficient in Rv2224c increases Deficient in Rv2224c increases susceptibility to lysozyme lysozyme susceptibility to When losts Rv2224c function When losts Rv2224c function The resistance to The resistance to lysozome is decreased lysozome is decreased Mtb can resistant the Mtb can resistant the hydrolysis of lysozyme hydrolysis of lysozyme Each strain in the Each strain in the presence/absence of lysozyme presence/absence of lysozyme 23
Conclusion(3/4) � Rv2224c :: tn mutant was markedly more susceptible to lysozyme � Influence innate immune response by secreting cytokines and chemokines What’s the physiological target for Rv2224c in Mtb ? 24
Rv2224c promotes processing of Rv2224c promotes processing of GroEL2 GroEL2 Small protein Small protein Full-length protein Full-length protein (active form) (active form) Rv2224c Rv2224c � cell envelope-associated � cell envelope-associated � hydrolytic activity � hydrolytic activity Culture supernatant smaller version is produced by Whole cell pellets smaller version is produced by cleavage of full-length GroEL2 cleavage of full-length GroEL2 Whole cell Cell wall Cytoplasm Cell menbrane 25
GroEL- -heat shock protein in heat shock protein in Mtb Mtb GroEL (1) Helps the (1) Helps the folding of naïve folding of naïve protein protein GroEL GroEL+GroES Two kinds of function GroEL2 secreted by Mtb can induce GroEL2 secreted by Mtb can induce (2)Regulates the immune response (2)Regulates the immune response chronic inflammation response chronic inflammation response 26
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