Unique structural features of the Mycobacterium ribosome.

Protein synthesis in all the living cells is mediated by a large protein-RNA complex called the ribosome. These macromolecular complexes can range from 2.5 (prokaryotes) to 4.2 MDa. (eukaryotes) in size and undergo various conformational transitions during protein synthesis to translate the genetic code into the nascent polypeptide chains. Recent advances in cryo-electron microscopy (cryo-EM) and image processing methods have provided numerous detailed structures of ribosomes from diverse sources and in different conformational states resolved to near-atomic resolutions. These structures have not only helped in better understanding of the translational mechanism but also revealed species-specific variations or adaptations in the ribosome structures. Structural investigations of the ribosomes from Mycobacterium smegmatis (Msm) and its closely related pathogenic Mycobacterium tuberculosis (Mtb) lead to the identification of two additional ribosomal proteins named as bS22 and bL37 and several unique extensions in ribosomal-protein and ribosomal-RNA. Hibernation Promoting Factor (HPF) bound structure of Msm ribosome, termed as the hibernating ribosome, possibly indicates a new mechanism of ribosome protection during dormancy. These studies enabled the identification of the mycobacteria-specific ribosomal features and provides an opportunity to understand their function and target them for further drug-discovery purposes. Here we review the unique structural features identified in Msm ribosome and their possible implications in comparison to a well-studied Escherichia coli (Ec) ribosome.

SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions.

SIRT3 (sirtuin 3), a mitochondrial protein deacetylase, maintains respiratory function, but its role in the regulation of innate immune defense is largely unknown. Herein, we show that SIRT3 coordinates mitochondrial function and macroautophagy/autophagy activation to promote anti-mycobacterial responses through PPARA (peroxisome proliferator activated receptor alpha). SIRT3 deficiency enhanced inflammatory responses and mitochondrial dysfunction, leading to defective host defense and pathological inflammation during mycobacterial infection. Antibody-mediated depletion of polymorphonuclear neutrophils significantly increased protection against mycobacterial infection in sirt3-/- mice. In addition, mitochondrial oxidative stress promoted excessive inflammation induced by Mycobacterium tuberculosis infection in sirt3-/- macrophages. Notably, SIRT3 was essential for the enhancement of PPARA, a key regulator of mitochondrial homeostasis and autophagy activation in the context of infection. Importantly, overexpression of either PPARA or TFEB (transcription factor EB) in sirt3-/- macrophages recovered antimicrobial activity through autophagy activation. Furthermore, pharmacological activation of SIRT3 enhanced antibacterial autophagy and functional mitochondrial pools during mycobacterial infection. Finally, the levels of SIRT3 and PPARA were downregulated and inversely correlated with TNF (tumor necrosis factor) levels in peripheral blood mononuclear cells from tuberculosis patients. Collectively, these data demonstrate a previously unappreciated function of SIRT3 in orchestrating mitochondrial and autophagic functions to promote antimycobacterial responses. Abbreviations: Ab: antibody; BCG: M. bovis Bacillus Calmette-Guérin; Baf-A1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; CFU: colony forming unit; CXCL5: C-X-C motif chemokine ligand 5; EGFP: enhanced green fluorescent protein; ERFP: enhanced red fluorescent protein; FOXO3: forkhead box O3; HC: healthy controls; H&E: haematoxylin and eosin; HKL: honokiol; IHC: immunohistochemistry; IL1B: interleukin 1 beta; IL6: interleukin 6; IL12B: interleukin 12B; MDMs: monocyte-derived macrophages; MMP: mitochondrial membrane potential; Mtb: Mycobacterium tuberculosis; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PMN: polymorphonuclear neutrophil; PPARA: peroxisome proliferator activated receptor alpha; ROS: reactive oxygen species; SIRT3: sirtuin 3; TB: tuberculosis; TEM: transmission electron microscopy; TFEB: transcription factor EB; TNF: tumor necrosis factor.

Efferocytosis and extrusion of leukocytes determine the progression of early mycobacterial pathogenesis.

Macrophages and neutrophils are the first responders to invading pathogens and contribute strongly to the host defense against intracellular pathogens. The collective interplay and dynamic interactions between these leukocytes are to a large extent not understood. In the present study, we have investigated their role using a combination of confocal laser-scanning and electron microscopy in a zebrafish model for tuberculosis, a local Mycobacterium marinum infection in the tissue of the larval tail fin. Our results show that neutrophils are efficient in phagocytosis of mycobacteria and that they contribute largely to their dissemination. Macrophages appear to play a major role in efferocytosis, phagocytosis of dead cells that contain bacterial content. Phagocytic cells with large bacterial aggregates are formed that can be extruded out of the tissue after cell death. Alternatively, these excessively infected cells can undergo necrosis leading to immediate recruitment of surrounding leukocytes and subsequent phagocytosis of released bacteria. Our data show that these necrotic burst events result in progression of the infection, whereas extrusion abates the infection.

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp Polybia dimorpha.

Mycobacterium abscessus subsp. massiliense, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from Polybia dimorpha Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different M. abscessus subsp. massiliense strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with M. abscessus subsp. massiliense induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the M. abscessus subsp. massiliense cell wall and reduce 40-90% of the bacterial load both in vitro and in vivo. The presented results encourage further studies on the use of Polydim-I as one of the components for M. abscessus subsp. massiliense treatment.

Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins.

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

Microscopic cords, a virulence-related characteristic of Mycobacterium tuberculosis, are also present in nonpathogenic mycobacteria.

The aggregation of mycobacterial cells in a definite order, forming microscopic structures that resemble cords, is known as cord formation, or cording, and is considered a virulence factor in the Mycobacterium tuberculosis complex and the species Mycobacterium marinum. In the 1950s, cording was related to a trehalose dimycolate lipid that, consequently, was named the cord factor. However, modern techniques of microbial genetics have revealed that cording can be affected by mutations in genes not directly involved in trehalose dimycolate biosynthesis. Therefore, questions such as "How does mycobacterial cord formation occur?" and "Which molecular factors play a role in cord formation?" remain unanswered. At present, one of the problems in cording studies is the correct interpretation of cording morphology. Using optical microscopy, it is sometimes difficult to distinguish between cording and clumping, which is a general property of mycobacteria due to their hydrophobic surfaces. In this work, we provide a new way to visualize cords in great detail using scanning electron microscopy, and we show the first scanning electron microscopy images of the ultrastructure of mycobacterial cords, making this technique the ideal tool for cording studies. This technique has enabled us to affirm that nonpathogenic mycobacteria also form microscopic cords. Finally, we demonstrate that a strong correlation exists between microscopic cords, rough colonial morphology, and increased persistence of mycobacteria inside macrophages.

Inhibition of mycobacterial arylamine N-acetyltransferase contributes to anti-mycobacterial activity of Warburgia salutaris.

In this study, we show that extracts and a purified compound of Warburgia salutaris exhibit anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG Pasteur. The extracts did not inhibit growth of Escherichia coli and were not toxic to cultured mammalian macrophage cells at the concentrations at which anti-mycobacterial activity was observed. The extract and pure compound inhibited pure recombinant arylamine N-acetyltransferase (NAT), an enzyme involved in mycobacterial cell wall lipid synthesis. Moreover, neither extract nor pure compound inhibited growth of a strain of M. bovis BCG in which nat has been deleted suggesting that NAT may indeed be a target within the mycobacterial cell. The purified compound is a novel drimane sesquiterpenoid lactone, 11alpha-hydroxycinnamosmolide. These studies show that W. salutaris is a useful source of anti-tubercular compounds for further analysis and supports the hypothesis of a link between NAT inhibition and anti-mycobacterial activity.

Isolation and characterization of polycyclic aromatic hydrocarbon-degrading Mycobacterium isolates from soil.

Bioremediation of soils contaminated with wood preservatives containing polycyclic aromatic hydrocarbons (PAHs) is desired because of their toxic, mutagenic, and carcinogenic properties. Creosote wood preservative-contaminated soils at the Champion International Superfund Site in Libby, Montana currently undergo bioremediation in a prepared-bed land treatment unit (LTU) process. Microbes isolated from these LTU soils rapidly mineralized the (14)C-labeled PAH pyrene in the LTU soil. Gram staining, electron microscopy, and 16S rDNA-sequencing revealed that three of these bacteria, JLS, KMS, and MCS, were Mycobacterium strains. The phylogeny of the 16S rDNA showed that they were distinct from other Mycobacterium isolates with PAH-degrading activities. Catalase and superoxide dismutase (SOD) isozyme profiles confirmed that each isolate was distinct from each other and from the PAH-degrading mycobacterium, Mycobacterium vanbaalenii sp. nov, isolated from a petroleum-contaminated soil. We find that dioxygenase genes nidA and nidB are present in each of the Libby Mycobacterium isolates and are adjacent to each other in the sequence nidB-nidA, an order that is unique to the PAH-degrading mycobacteria.

Mycobacterium-inducible Nramp in striped bass (Morone saxatilis).

In mammals, the natural resistance-associated macrophage protein 1 gene, Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized an Nramp gene in this species and obtained evidence that there is induction following Mycobacterium exposure. The striped bass Nramp gene (MsNramp) and a 554-amino-acid sequence contain all the signal features of the Nramp family, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely grouped MsNramp with other teleost Nramp genes and revealed high sequence similarity with mammalian Nramp2. MsNramp expression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection of Mycobacterium marinum, MsNramp expression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels of MsNramp were three- and sixfold higher on days 3 and 15, respectively. Injection of Mycobacterium shottsii resulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of an Nramp gene by mycobacteria in a poikilothermic vertebrate.

Mycobacterium and the coat of many lipids.

Pathogenic Mycobacterium reside inside vacuoles in their host macrophages. These vacuoles fail to fuse with lysosomes yet interact with early endosomes. Glycoconjugates released by the intracellular bacilli traffic through the host cell and are released through exocytosis. These molecules represent both antigens for immune recognition and modulators of immune function. The molecules play key roles in the induction and maintenance of the granuloma, a tissue response that limits bacterial spread yet ensures persistence of the infection.

Virulence and drug susceptibility of Mycobacterium celatum.

The virulence and drug susceptibility of a clinical isolate of Mycobacterium celatum which showed smooth transparent (ST) and smooth opaque (SO) colonies were studied. While ST cells multiplied intracellularly and maintained their coccobacillary form in a human macrophage model of infection, SO cells formed long filaments and completely destroyed the phagocytes. In BALB/c mice, the ST variant, but not the SO variant, grew efficiently in the spleen, liver and lung. The ST variant was usually more resistant in vitro than the SO variant to drugs, with MIC values for clarithromycin (CLA), azithromycin (AZI), ciprofloxacin, sparfloxacin, amikacin, clofazimine, ethambutol and isoniazid being higher than those of the SO variant. In beige mice infected with the more highly virulent variant ST, CLA and AZI were the most active drugs in terms of viable count reduction in organs and mutant selection. Together, these observations indicate that the ST variant of M. celatum is a virulent form that can be efficiently inhibited in vivo by CLA and AZI.

Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog.

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or alpha-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Acción del Agua del Volcán Copahue (Neuquén, Argentina) sobre la morfología de las microbacterias / Action of the Copahue Volcano (Neuquén, Argentine) water of mycobateria morphology

Se efectuó un estudio de la acción del Agua del Volcán Copahue (AVC), Neuquén, Argentina, sobre 15 cepas de microbacterias Mycobacterium tuberculosis: M. bovis y los mycobacterum no tuberculosos ("Atipicos"), poniendo especial interés en los que forman "cuerdas". Se utilizó AVC con su pH l,3 y se la llevó a pH 6,5. En los bacilos que resistieron la acción del agua hasta el momento de la coloración, se advirtieron elementos más o menos alterados. Al aumentar el tiempo de contacto se llegó a la destrucción total, observándose en algunos casos muy pocos bacilos aislados, material deteriorado y formas granulares. Estas alteraciones fueron mucho más marcadas en las suspensiones que en el líquido del sedimento con el AVC; en las primeras directamente no se llegó a reparar la formación de cuerdas en ningun momento aun en bacilos que deberían formarla. Su acción no estaría asociada a una reacción bioquímica responsable en la síntesis de la pared celular, como la transpeptidación. El AVC actuaría sobre la síntesis del ácido micólico y se trataría de un agente bacteriolítico. Además se realizaron estudios en el "Laboratorio de tratamiento de imágenes", INEUCI (Instituto de Neurociencia), CONICET

Mycobacteria glycolipids as potential pathogenicity effectors: alteration of model and natural membranes.

Four mycobacterial wall glycolipids were tested for their effects on phospholipidic liposome organization and passive permeability and on oxidative phosphorylation of isolated mitochondria. From fluorescence polarization of diphenylhexatriene performed on liposomes it was concluded that the two trehalose derivatives (dimycoloyltrehalose and polyphthienoyltrehalose) rigidified the fluid state of liposomes, the triglycosyl phenolphthiocerol slightly fluidized the gel state, while the peptidoglycolipid ("apolar" mycoside C) just shifted the phase transition temperature upward. Dimycoloyltrehalose was without effect on liposome passive permeability, as estimated from dicarboxyfluorescein leak rates, and polyphthienoyltrehalose and triglycosyl phenolphthiocerol slightly decreased leaks, while mycoside C dramatically increased leaks. Activity of these lipids on mitochondrial oxidative phosphorylation was examined. The two trehalose derivatives have been tested previously: both had the same type of inhibitory activity, dimycoloyltrehalose being the most active. Triglycosyl phenolphthiocerol was inactive. Mycoside C was very active, with effects resembling those of classical uncouplers: this suggested that its activity on mitochondria was related to its effect on permeability. All these membrane alterations were called nonspecific because it is likely that they result from nonspecific lipid-lipid interactions, and not from recognition between specific molecular structures. Such nonspecific interactions could be at the origin of some of the effects of mycobacteria glycolipids on cells of the immune system observed in the last few years.

[Change in the structural organization of Mycobacterium rubrum cells upon the action of ethidium bromide]. / Izmenenie strukturnoi organizatsii kletok Mycobacterium rubrum pri deistvii étidiuma bromida.

The action of ethidium bromide on Mycobacterium rubrum cells was studied. The culture growth was found to depend on ethidium bromide (EB) concentration in the medium. The reaction of EB with nucleoid DNA was shown to be specific and changes in the nucleoid structure were detected. Low EB doses (ca. 2 micrograms/ml) caused DNA despiralization in many cells. The process was reversible, which accounted for the elevated ability of reactivation at low EB doses. A higher EB dose (ca. 5-10 micrograms/ml and more) made the nucleoid structure coarser and denser in most cells and the nucleoid broke down to small fragments. As a result, due to the pool of enzymes present in the cells prior to EB addition, secondary changes developed. They involved all the cellular structures as well as the metabolism of lipids, polyphosphates, and glycogen. As a rule, these changes were incompatible with the cell viability.

Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves.

Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.

In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.

Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.

Two strains of an unclassified Mycobacterium species were isolated after 18 and 30 months of incubation of media inoculated with resected intestinal tissues from patients with Crohn's disease. These strains represented the third and fourth isolates of this organism from Crohn's disease patients. Ultrastructural examination of this strain and two previously isolated strains revealed the presence of spheroplasts which eventually transformed into the bacillary form of a previously unrecognized Mycobacterium species. These cell wall-deficient forms did not stain with conventional dyes and failed to grow on hypertonic media. Restriction polymorphism of the ribosomal DNA genes was used to determine the relationship between the cell wall-deficient and bacillary forms. Identical restriction patterns of the ribosomal DNA genes were found between the spheroplasts and Mycobacterium sp. isolates with EcoRI, BamHI, and XhoI restriction endonucleases, thus providing definitive evidence of their origin. Unidentified spheroplasts were isolated from an additional 12 patients with Crohn's disease, of which 7 of 10 seroagglutinated with antiserum prepared against the Mycobacterium sp. Spheroplasts were isolated from 16 of 26 (61%) patients with Crohn's disease but not from tissues of 13 patients with ulcerative colitis or 13 patients with other diseases of the bowel. These findings support the role of mycobacteria as etiologic agents in some cases of Crohn's disease.