Killing of Mycobacterium avium subspecies paratuberculosis within macrophages
© Bannantine and Stabel; licensee BioMed Central Ltd. 2002
Received: 21 November 2001
Accepted: 30 January 2002
Published: 30 January 2002
Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host.
Serial plating of M. paratuberculosis infected macrophage lysates on Herold's egg yolk medium showed that mycobacterial replication takes place between 0 and 24 hours post-infection. This initial growth phase was followed by a steady decline in viability over the next six days. Antibodies against M. paratuberculosis were affinity purified and used in conjunction with transmission electron microscopy to track the development of intracellular bacilli. Immunogold labeling of infected macrophages with antibody against M. paratuberculosis showed degraded intracellular mycobacteria that were unrecognizable by morphology alone. Conversely, when macrophages were heavily infected with M. paratuberculosis, no degraded forms were observed and macrophages were killed.
We present a general description of M. paratuberculosis survival within cultured macrophages using transmission electron microscopy and viability counts. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years.
Johne's disease, also called paratuberculosis, is a chronic granulomatous enteritis of ruminant animals caused by Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis). While Johne's disease can end in death of the animal, the economic impact of this disease is much more significant [1, 2]. Losses are estimated to be $200/clinically infected cow/year and are a result of animal culling, reduced milk production, poor reproductive performance, and reduced carcass value [1, 3]. Research on the pathogenesis and immunology of M. paratuberculosis infections of cattle is necessary to allow design of more rational diagnostic and control procedures.
A small number of specialized microorganisms can survive inside macrophages designed specifically to kill bacteria. However, a hallmark of mycobacterial pathogenesis is their ability to survive, and even replicate, within macrophages. These include Mycobacteria, Salmonella, Listeria, Coxiella and Corynebacteria. Different mechanisms are employed as survival strategies and the mycobacteria are exceptional in the duration and persistence of this interaction. Survival of pathogenic mycobacteria is attributed to the fact that the mycobacterial phagosome does not fuse with lysosomes [4–6]. The mechanism that prevents phagosome maturation is still unknown as are any mycobacterial genes that contribute to the delayed maturation.
Several studies surrounding the interactions of M. paratuberculosis with macrophages have been published because of its importance in pathogenesis. These studies include entry into J774 macrophages [7, 8], an electron microscopic examination of goat tissue [9, 10] and an assessment of intracellular fate of M. paratuberculosis within bovine monocytes/macrophages [11, 12]. However, many assumptions regarding M. paratuberculosis interactions with macrophages are based on analogies to M. tuberculosis or M. avium. In this communication, we present an analysis of M. paratuberculosis survival within J774 macrophages using transmission electron microscopy to show temporal events early during infection.
Viability of M. paratuberculosis within resting J774 macrophages
Immunoelectron microscopy of intracellular M. paratuberculosis
Temporal events during M. paratuberculosis infection of J774 macrophages
Growth and survival of mycobacterial species within macrophages has been an area of intensive study because of its implications in pathogenicity. For example, M. bovis has been shown to grow within macrophages whereas BCG strains do not . This observation is controversial since there are reports of immunocompromised patients with disseminated BCG. Although multi-species studies are complex and multi-factorial, M. avium appears to be able to survive in secondary lysosomes better than does M. tuberculosis. In macrophages co-infected with Coxiella burnetii, an intracellular pathogen known to inhabit and replicate within secondary lysosomes , M. avium growth was not impaired . Whereas M. tuberculosis bacilli that co-localized with C. burnetii containing vacuoles, did show reduced growth . A recent report by Thomsen et al.  showed a higher percentage of degraded M. paratuberculosis in euthymic as compared to athymic mice. The in vivo study by Thomsen supports data from our in vitro study, indicating that J774 cultured macrophages are a good model for pathogenesis studies.
Many bacteria such as Staphylococcus aureus are rapidly endocytosed and digested by macrophages within a few hours postinfection . The present study showed an initial 25 hour intracellular growth period of M. paratuberculosis followed by a slow decline in the viability within macrophages over a period of 7 days. This survival curve is similar to that observed by Zhao et al.  which showed M. paratuberculosis intracellular growth in the first six days followed by killing after day six. The experiments described herein were performed using the type strain of M. paratuberculosis, which may have affected the survivability within macrophages since it is not a recent field isolate. However, M. paratuberculosis 6783  and M. paratuberculosis BO45  are field isolates that have both showed a decline in viability inside macrophages over time, although much slower than observed with the type strain. No attempt has been made to activate the macrophages in this study, although several laboratories have shown that macrophages activated with cytokines such as interferon-γ increase the ability to kill mycobacteria [26–30].
A very recent communication of M. paratuberculosis interactions with macrophages was published during the course of our experiments . This comprehensive study showed that M. paratuberculosis-containing vacuoles were mildly acidified (pH 6.3) as compared to latex beads (pH 5.2). In addition, the M. paratuberculosis phagosome was characterized by the presence of LAMP 1 and absence of LAMP 2 lysosomal membrane protein markers as well as other endocytic tracer molecules. These cell biology studies clearly add to our understanding of M. paratuberculosis interactions with macrophages.
This study has also provided a general description of early events in M. paratuberculosis infection of cultured macrophages. Several intracellular pathogens such as Chlamydia or Coxiella undergo readily distinguishable morphological changes during infection of host cells [31, 32]. This is clearly not the case for M. paratuberculosis or other mycobacteria as they remain morphologically similar at least up until 4 days postinfection. However, an increased percentage of degraded mycobacterial forms was observed over time. These degraded forms appeared by 24 hours postinfection and were hardly recognizable morphologically, but did label with immunogold particles indicating the presence of mycobacterial antigen. At low moi, the bacilli were tightly clustered into a single vacuole throughout the observed time period. This mycobacterial vacuole is most likely characteristic of a late endosome, based on previous studies with M. avium, M. tuberculosis[33, 34] and M. paratuberculosis.
All intracellular bacterial pathogens enter host cells surrounded by a membrane bound vacuole [35, 36]. Some pathogens in heavily infected cells collect into a single vacuole within the same cell . J774 macrophages infected at a moi of 5:1 showed separate M. paratuberculosis-containing vacuoles within the same macrophage even after 48 hours postinfection (Figure 2). Likewise, M. tuberculosis and M. avium appear to remain in distinct phagosomes that do not harbor more than one bacilli per vacuole (see Figures 1A and 4B in  for example). However, in M. avium-infected macrophages, one of the first phenotypic alterations following activation with cytokines is the coalescence of individual M. avium-containing vacuoles into communal vacuoles with many bacilli . The significance of separate M. paratuberculosis-containing vacuoles observed in this study is still unclear.
There appears to be a tenuous relationship to gain control between M. paratuberculosis and the macrophage with survival at stake. The macrophage can control growth and even kill M. paratuberculosis. However, the mycobacteria are cytotoxic to macrophages or induce apoptosis at high moi. The mechanism that enables M. paratuberculosis to persist within cattle for several years remains unclear as high moi are not likely observed at early stages of Johne's disease.
In vitro assays to quantify survival of bacteria in macrophages provide useful insights into host-pathogen relations. The results of this study provides further insight surrounding M. paratuberculosis-macrophage infections and have implications in the pathogenesis of M. paratuberculosis, a pathogen known to persist inside cattle for many years. Further studies need to address the tenuous relationship between mycobacteria and macrophage in relation to disease outcome.
Materials and methods
Bacterial strains and growth conditions
M. paratuberculosis ATCC19698 was grown in Middlebrook 7H9 broth (pH 6.0) supplemented with oleic acid albumin dextrose complex (Becton Dickinson Microbiology) and 0.05% Tween 80 and ferric mycobactin J (2 mg/L). For viability counts, M. paratuberculosis was cultured on Herold's egg yolk medium (HEYM) prepared as described elsewhere .
Culture of M. paratuberculosis in macrophages
The murine macrophage cell line, J774.16, was cultured in Dulbecco's modified Eagle medium (MEM; Life Technologies) supplemented with 10% defined fetal calf serum (Hyclone Laboratories) at 37°C in 5% CO2. Because phagocytosis of mycobacteria was shown to be enhanced with complement protein C3 , macrophages were infected with M. paratuberculosis in the presence of 10% serum from a clinical cow with Johne's disease. Multiplicities of infection (moi) ranged between 2:1 and 30:1 (bacteria: macrophage ratio) in PBS. The number of macrophages was 3.0 × 106/well as determined on an Angel CellTrak 3B cell counter. The input inoculum was 3.0 × 107mycobacteria/well for a 10:1 moi experiment. After 2 hours, extracellular bacteria were removed by washing the monolayers twice with fresh media. Infected macrophages were then incubated in MEM at 37°C and in 5% CO2. Medium was exchanged every third day during the course of the infection. To determine survival of M. paratuberculosis within macrophages, 24-well plates of macrophages were infected at an moi of 10:1. Infected macrophages were lysed in cold sterile distilled H2O at 0, 24, 48, 72, 96, 120, 144, and 168 hours postinfection. Following lysis, serial platings of infected macrophage lysates on HEYM slants were performed. Lysates from three separate wells were plated for each time point.
Colloidal gold-conjugated goat anti-rabbit IgG was purchased from Ted Pella, Inc., Redding, CA. Two New Zealand white rabbits were immunized with sonicated preparations of M. paratuberculosis as previously described . Antibodies directed at a whole cell sonicated extract of M. paratuberculosis were affinity purified using AminoLink columns (Pierce Chemical Company, Rockford, IL, USA). Briefly, the sonicated extract of M. paratuberculosis was coupled by reductive amination to a 4% agarose support column (Pierce Chemical Company). Sera from rabbits immunized with a killed preparation of M. paratuberculosis (1–2 ml) were passed over the column followed by three washes and elution according to the instructions of the manufacturer. Eluted fractions were evaluated by spectrophotometry and immunoblot analysis . Fractions with the highest absorbance at 280 nm and the strongest reactivity by immunoblot were neutralized in 1 M Tris-HCl (pH 9.5) buffer and stored at 4°C.
All fixation and staining procedures were conducted at room temperature. Noninfected and M. paratuberculosis-infected macrophages were cultured on membrane inserts for times indicated in each experiment described below. Cells were fixed for 2–4 h in 2.5% glutaraldehyde in 0.1 M Cacodylate buffer, pH 7.4. Fixed cells were washed in the same buffer three times and were postfixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, for 2 h. After washing in the same buffer, cells were incubated with 30% ethanol for 10 min. The cells were further dehydrated with a graded series of ethanol and embedded in epoxy resin (Embed 812). Ultrathin sections for immunoelectron microscopy were washed in buffer 15 min three times and etched with saturated sodium metaperiodate for 15 min. Cells were then blocked with 5% BSA for 30 min at room temperature. Cells were treated with affinity purified rabbit IgG against M. paratuberculosis (diluted 1:20) in the blocking solution for 2 h at room temperature. Cells were washed in Tris buffer containing 0.1% Tween 20 and 0.1% BSA four times for 10 min each and then incubated with goat anti-rabbit IgG conjugated to colloidal gold (10 nm diameter) in Tris buffer for 2 h. Immunolabeled sections were washed in Tris buffer four times and fixed with 1% glutaraldehyde in Tris for 10 min. All ultrathin sections were double stained with uranyl acetate and Reynolds lead citrate and then observed under a Philips 410 microscope.
The expert technical assistance of Janis Hansen and Trudy Bosworth is appreciated. We gratefully acknowledge Judy Stasko for the embedding and staining of samples for electron microscopy.
- Ott SL, Wells SJ, Wagner BA: Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med. 1999, 40: 179-192. 10.1016/S0167-5877(99)00037-9.View ArticlePubMedGoogle Scholar
- Johnson-Ifearulundu Y, Kaneene JB, Lloyd JW: Herd-level economic analysis of the impact of paratuberculosis on dairy herds. J Am Vet Med Assoc. 1999, 214: 822-825.PubMedGoogle Scholar
- Wells SJ, Wagner BA, Dargatz DA: Factors associated with M. a. paratuberculosis infection in U.S. dairy herds. In: Proceedings of the Sixth International Colloquium on Paratuberculosis;. 1999, Melbourne, Australia., 62-65.Google Scholar
- Armstrong JA, Hart PD: Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J Exp Med. 1975, 142: 1-16.View ArticlePubMedGoogle Scholar
- Frehel C, de Chastellier C, Lang T, Rastogi N: Evidence for inhibition of fusion of lysosomal and prelysosomal compartments with phagosomes in macrophages infected with pathogenic Mycobacterium avium. Infect Immun. 1986, 52: 252-262.PubMed CentralPubMedGoogle Scholar
- Hart PD, Young MR: Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J Exp Med. 1991, 174: 881-889.View ArticlePubMedGoogle Scholar
- Goethe R, Kuehnel MP, Darji A, Weiss S, Rohde M, Gerlach G-F, Valentin-Weigand P: Interactions of Mycobacterium avium subsp. paratuberculosis with murine macrophages: intracellular survival and modulation of macrophage functions. In: Proceedings of the Sixth International Colloquium on Paratuberculosis;. 1999, Melbourne, Australia., 635-637.Google Scholar
- Rastogi N, Blom-Potar MC, David HL: Comparative intracellular growth of difficult-to-grow and other mycobacteria in a macrophage cell line. Acta Leprol. 1989, 7: 156-159.PubMedGoogle Scholar
- Paliwal OP, Rehbinder C: Ultrastructural studies of paratuberculosis (Johne's disease) in goats. Acta Vet Scand. 1981, 22: 180-188.PubMedGoogle Scholar
- Sigur-Dardottir OG, Press CM, Evensen O: Uptake of Mycobacterium avium subsp. paratuberculosis through the distal small intestinal mucosa in goats: an ultrastructural study. Vet Pathol. 2001, 38: 184-189. 10.1354/vp.38-2-184.View ArticlePubMedGoogle Scholar
- Bendixen PH, Bloch B, Jorgensen JB: Lack of intracellular degradation of Mycobacterium paratuberculosis by bovine macrophages infected in vitro and in vivo: light microscopic and electron microscopic observations. Am J Vet Res. 1981, 42: 109-113.PubMedGoogle Scholar
- Zhao BY, Czuprynski CJ, Collins MT: Intracellular fate of Mycobacterium avium subspecies paratuberculosis in monocytes from normal and infected, interferon-responsive cows as determined by a radiometric method. Can J Vet Res. 1999, 63: 56-61.PubMed CentralPubMedGoogle Scholar
- Russell DG, Sturgill-Koszycki S, Vanheyningen T, Collins H, Schaible UE: Why intracellular parasitism need not be a degrading experience for Mycobacterium. Philos Trans R Soc Lond B Biol Sci. 1997, 352: 1303-1310. 10.1098/rstb.1997.0114.PubMed CentralView ArticlePubMedGoogle Scholar
- Mohagheghpour N, van Vollenhoven A, Goodman J, Bermudez LE: Interaction of Mycobacterium avium with human monocyte-derived dendritic cells. Infect Immun. 2000, 68: 5824-5829. 10.1128/IAI.68.10.5824-5829.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- de Chastellier C, Berche P: Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria. Infect Immun. 1994, 62: 543-553.PubMed CentralPubMedGoogle Scholar
- Read RC, Zimmerli S, Broaddus C, Sanan DA, Stephens DS, Ernst JD: The (alpha2–>8)-linked polysialic acid capsule of group B Neisseria meningitidis modifies multiple steps during interaction with human macrophages. Infect Immun. 1996, 64: 3210-3217.PubMed CentralPubMedGoogle Scholar
- Kuehnel MP, Goethe R, Habermann A, Mueller E, Rohde M, Griffiths G, Valentin-Weigand P: Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol. 2001, 3: 551-566. 10.1046/j.1462-5822.2001.00139.x.View ArticlePubMedGoogle Scholar
- Gomes MS, Paul S, Moreira AL, Appelberg R, Rabinovitch M, Kaplan G: Survival of Mycobacterium avium and Mycobacterium tuberculosis in acidified vacuoles of murine macrophages. Infect Immun. 1999, 67: 3199-3206.PubMed CentralPubMedGoogle Scholar
- Heinzen RA, Scidmore MA, Rockey DD, Hackstadt T: Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. Infect Immun. 1996, 64: 796-809.PubMed CentralPubMedGoogle Scholar
- Sebbagh M, Renvoize C, Hamelin J, Riche N, Bertoglio J, Breard J: Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol. 2001, 3: 346-352. 10.1038/35070019.View ArticlePubMedGoogle Scholar
- Gan YH, Peng SQ, Liu HY: Molecular mechanism of apoptosis induced by ricin in HeLa cells. Acta Pharmacol Sin. 2000, 21: 243-248.PubMedGoogle Scholar
- Saafi EL, Konarkowska B, Zhang S, Kistler J, Cooper GJ: Ultrastructural evidence that apoptosis is the mechanism by which human amylin evokes death in RINm5F pancreatic islet beta-cells. Cell Biol Int. 2001, 25: 339-350. 10.1006/cbir.2000.0643.View ArticlePubMedGoogle Scholar
- Aldwell FE, Wedlock DN, Slobbe LJ, Griffin JF, Buddle BM, Buchan GS: In vitro control of Mycobacterium bovis by macrophages. Tuberculosis. 2001, 81: 115-123. 10.1054/tube.2000.0280.View ArticlePubMedGoogle Scholar
- Thomsen BV, Steadham EM, Gallup JM, Ackerman MR, Brees DJ, Cheville NF: T cell-dependent inducible nitric oxide synthase productional and ultrastructural morphology in BALB/c mice infected with Mycobacterium avium subspecies paratuberculosis. Journal of Comparative Pathology. 2001, 125: 137-144. 10.1053/jcpa.2001.0491.View ArticlePubMedGoogle Scholar
- Schmitt-Slomska J, Michailova L, Ivanova E, Toshkov A: Adhesion and phagocytosis of Staphylococcus aureus L-forms. J Basic Microbiol. 1986, 26: 429-440.View ArticlePubMedGoogle Scholar
- Schaible UE, Sturgill-Koszycki S, Schlesinger PH, Russell DG: Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol. 1998, 160: 1290-1296.PubMedGoogle Scholar
- Xing Z, Zganiacz A, Santosuosso M: Role of IL-12 in macrophage activation during intracellular infection: IL-12 and mycobacteria synergistically release TNF-alpha and nitric oxide from macrophages via IFN-gamma induction. J Leukoc Biol. 2000, 68: 897-902.PubMedGoogle Scholar
- Bonay M, Bouchonnet F, Pelicic V, Lagier B, Grandsaigne M, Lecossier D, Grodet A, Vokurka M, Gicquel B, Hance AJ: Effect of stimulation of human macrophages on intracellular survival of Mycobacterium bovis Bacillus Calmette-Guerin. Evaluation with a mycobacterial reporter strain. Am J Respir Crit Care Med. 1999, 159: 1629-1637.View ArticlePubMedGoogle Scholar
- Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D, Deretic V: Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci. 1998, 111: 897-905.PubMedGoogle Scholar
- Zurbrick BG, Follett DM, Czuprynski CJ: Cytokine regulation of the intracellular growth of Mycobacterium paratuberculosis in bovine monocytes. Infect Immun. 1988, 56: 1692-1697.PubMed CentralPubMedGoogle Scholar
- Heinzen RA, Hackstadt T, Samuel JE: Developmental biology of Coxiella burnettii. Trends Microbiol. 1999, 7: 149-154. 10.1016/S0966-842X(99)01475-4.View ArticlePubMedGoogle Scholar
- Rockey DD, Matsumoto A: The chlamydial developmental cycle. In: Prokaryotic Development Washington, D.C. Edited by: Brun YV, Shimkets LJ. 2000, American Society for Microbiology, 403-425.View ArticleGoogle Scholar
- Sturgill-Koszycki S, Schaible UE, Russell DG: Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. Embo J. 1996, 15: 6960-6968.PubMed CentralPubMedGoogle Scholar
- Sturgill-Koszycki S, Schlesinger PH, Chakraborty P, Haddix PL, Collins HL, Fok AK, Allen RD, Gluck SL, Heuser J, Russell DG: Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science. 1994, 263: 678-681.View ArticlePubMedGoogle Scholar
- Small PL, Ramakrishnan L, Falkow S: Remodeling schemes of intracellular pathogens. Science. 1994, 263: 637-639.View ArticlePubMedGoogle Scholar
- Garcia-del Portillo F, Finlay BB: The varied lifestyles of intracellular pathogens within eukaryotic vacuolar compartments. Trends Microbiol. 1995, 3: 373-380. 10.1016/S0966-842X(00)88982-9.View ArticlePubMedGoogle Scholar
- Sinai AP, Joiner KA: Safe haven: the cell biology of nonfusogenic pathogen vacuoles. Annu Rev Microbiol. 1997, 51: 415-462. 10.1146/annurev.micro.51.1.415.View ArticlePubMedGoogle Scholar
- Xu S, Cooper A, Sturgill-Koszycki S, van Heyningen T, Chatterjee D, Orme I, Allen P, Russell DG: Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol. 1994, 153: 2568-2578.PubMedGoogle Scholar
- Merkal RS, Curran BJ: Growth and metabolic characteristics of Mycobacterium paratuberculosis. Appl Microbiol. 1974, 28: 276-279.PubMed CentralPubMedGoogle Scholar
- Schlesinger LS, Bellinger-Kawahara CG, Payne NR, Horwitz MA: Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol. 1990, 144: 2771-2780.PubMedGoogle Scholar
- Stabel JR, Ackermann MR, Goff JP: Comparison of polyclonal antibodies to three different preparations of Mycobacterium paratuberculosis in immunohistochemical diagnosis of Johne's disease in cattle. J Vet Diagn Invest. 1996, 8: 469-473.View ArticlePubMedGoogle Scholar
- Bannantine JP, Stabel JR: Identification of two Mycobacterium avium subspecies paratuberculosis gene products differentially recognised by sera from rabbits immunised with live mycobacteria but not heat-killed mycobacteria. J Med Microbiol. 2001, 50: 795-804.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.