Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

Buruli disease, caused by Mycobacterium ulcerans, is the third most important mycobacterial disease in humans besides tuberculosis and leprosy. We have compared systemic and intralesional cytokine production in patients presenting with a nodular form and a necrotizing, ulcerative form of the disease. Gamma interferon (IFN-␥) levels in response to whole M. ulcerans and Mycobacterium bovis BCG bacilli and in response to purified Ag85 protein from BCG were lower in peripheral blood mononuclear cells (PBMC) cultures from Buruli disease patients than in PBMC from healthy purified protein derivative-positive contacts. Interleukin-4 (IL-4) and IL-13 content was below the detection threshold in these PBMC cultures. IFN-␥ production after stimulation with M. ulcerans was significantly lower (P < 0.05) in PBMC cultures from patients with ulcers than in those from patients with nodules. On the other hand, PBMC from Buruli disease patients produced significant levels of IL-10 in response to M. ulcerans (but not to M. bovis BCG) and production was highest in patients with the ulcerative form. Third, semiquantitative reverse transcription-PCR analysis demonstrated a similar difference in the local, intralesional cytokine profile for the two forms of the disease: high IFN-␥ but low IL-10 mRNA levels in nodular lesions and high IL-10 but low IFN-␥ mRNA levels in ulcerative lesions. Intralesional IL-4 and IL-13 mRNA levels were low and only detected in patients with the ulcerative form. Our results indicate, although they do not formally prove, that production of IL-10 rather than production of IL-4 or IL-13 by Th2-type T cells may be involved in the low M. ulcerans-specific IFN-␥ response in Buruli disease patients.

show abstract

“…At least two other enzymes, a phospholipase C and a hemolysin, may also exert toxic effects in M. ulcerans infection (11,12).…”

Section: Discussionmentioning

confidence: 99%

Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease

et al. 2004

View full text Add to dashboard Cite

show abstract

“…This notion will have to be reassessed by larger epidemiological studies. Further investigation is necessary to elucidate the virulence factors of the M. kansasii subtypes 1 and 2, such as hemolytic activity and the production of phospholipase C or other similar enzymes, possibly related to pathogenicity (13).…”

Section: Discussionmentioning

confidence: 99%

Clinical Implications of Mycobacterium kansasii Species Heterogeneity: Swiss National Survey

Taillard¹,

Greub²,

Weber³

et al. 2003

J Clin Microbiol

122

102

View full text Add to dashboard Cite

Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n ‫؍‬ 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n ‫؍‬ 128), while subtypes 2 and 3 comprised 21% (n ‫؍‬ 40) and 8% (n ‫؍‬ 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n ‫؍‬ 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.

show abstract

“…Others have identified cytotoxic properties of the MUCFs in protein-free media (14) and have defined phospholipase activities (7,8) and cytolysins (A. Mve-Obiang, A. Gomez, J. Bujnicki, R. Kotlowski, L. Rychlewski, J. Remacle, F. Portaels, and P. A. Fonteyne, unpublished data) from the M. ulcerans exudate that may additionally contribute to the cytotoxicity seen in this cell model. Future studies will focus on the utility of this cell model in the delineation of the factors from the MUCF directing SW872 cellular apoptosis and their role in the pathogenesis of BU.…”

mentioning

confidence: 99%

“…The high protein content of such media impeded the characterization of the secreted proteins of M. ulcerans, including their role, if any, in cytotoxicity. Recent developments in the culture of M. ulcerans in protein-free media have led to the discovery of serodiagnostic proteins (1) and phospholipases (7,8) in the MUCF and facilitated this study, in which a human adipose cell model of M. ulcerans infection and MUCF cytotoxicity was established.M. ulcerans infection of human adipose cells.…”

mentioning

confidence: 99%

Mycobacterium ulceransCytotoxicity in an Adipose Cell Model

et al. 2001

View full text Add to dashboard Cite

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis upon Mycobacterium ulcerans infection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors in M. ulcerans may be involved in Buruli ulcer pathogenesis.Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans infection, is characterized by chronic necrotizing skin ulcers, with tissue destruction often observed in the adipose tissue and at sites distal to acid-fast bacilli. Based on these observations, the pathogenesis of BU has been hypothesized to be due to one or more secreted mycobacterial toxins. Early studies on the elucidation of a toxin led to the description of a heat-stable and pronase-and lipase-labile factor from the culture filtrate of M. ulcerans (10). Further studies revealed that M. ulcerans filtrates possessed immunosuppressive properties (16). The advent of more sensitive biochemical methods led to the identification and characterization of a unique polyketide in M. ulcerans, named mycolactone, which possesses distinct cytotoxic and immunosuppressive characteristics (4-6, 15).A perusal of the literature demonstrates that previous studies of the cytotoxicity of M. ulcerans culture filtrate (MUCF) relied upon albumin-or serum-supplemented media. The high protein content of such media impeded the characterization of the secreted proteins of M. ulcerans, including their role, if any, in cytotoxicity. Recent developments in the culture of M. ulcerans in protein-free media have led to the discovery of serodiagnostic proteins (1) and phospholipases (7,8) in the MUCF and facilitated this study, in which a human adipose cell model of M. ulcerans infection and MUCF cytotoxicity was established.M. ulcerans infection of human adipose cells. M. ulcerans strain 94-816 (kindly provided by Francoise Portaels) was grown by serial 10-fold passage to 100 ml in Middlebrook 7H9 oleic acid-albumin-dextrose-catalase broth until the optical density (600-nm ) was 0.5 to 0.7. Single-cell bacterial suspensions were made by pulse sonication at 4°C using a Branson 450 cell sonicator fitted with a cup horn attachment (Branson Ultrasonics, Danbury, Conn.).Human adipose cells (SW872) were obtained from the American Type Culture Collection (ATCC HTB 92; Manassas, Va.). Cells were maintained in L-15S (Leibovitz-15 medium with L-glutamine [Gibco/BRL, Grand Island, N.Y.], supplemented with 10% heat-inactivated fetal bovine serum [HyClone Laboratories Inc., Logan, Utah] and sodium bicarbonate [1.5 g/liter; Gibco]) and incubated at 37°C and 5% CO 2 . Prior to use in experimental assays, the cells were released from the culture flask with 0.25% trypsin (Gibco), washed twice with fresh medium, and seeded onto 6-or 24-well microculture plates as needed. Viable cell counts were confirmed prior to each experiment using trypan exclusion (13).For electron microscopic (EM) analysis, fresh SW872 cells were trypsinized (0.25.%), washe...

show abstract

Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

Cited by 24 publications

References 43 publications

Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease

Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease

Clinical Implications of Mycobacterium kansasii Species Heterogeneity: Swiss National Survey

Mycobacterium ulceransCytotoxicity in an Adipose Cell Model

Contact Info

Product

Resources

About