J Bacteriol Virol.  2008 Sep;38(3):109-118. 10.4167/jbv.2008.38.3.109.

Cytokine Profiles of Macrophages by Mycobacterium abscessus Conditional Morphotype Variants and Comparison of Their Extracellular-Protein Expressions

• Lee KS
• Sohn H
• Lee SS
• Lee BS
• Kim HJ
• Shin SJ

• KMID: 1483969
• DOI: http://doi.org/10.4167/jbv.2008.38.3.109

Abstract

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Previously, it was documented that a spontaneously formed rough variant of M. abscessus causes persistent and invasive infection in mice, while a smooth isogenic variant does not. However, little is known for immune responses elicited by M. abscessus variants artificially induced by culture conditions and their culture filtrate antigens. Thus, morphological variants of M. abscessus type strain (ATCC19977T) were generated by an acidic and low oxygen culture conditions. Overall comparison between the variant and its original smooth strain showed that the rough variant was less virulent than original smooth strain in murine bone-marrow derived macrophage. To understand the basis for the difference, the protein expression pattern in the culture filtrates of each strain was analyzed by 1-dimensional electrophoresis. Generally, the protein expressions were more influenced by pH conditions than oxygen pressures. Interestingly, several proteins, mainly lower than 30 kDa molecular weight, were uniquely expressed in normal culture conditions. In contrast, several high molecular weight proteins (>55 kDa) were induced by acidic and low oxygen culture conditions. These findings not only provide new insights of association between morphological change and the virulence, but may also be useful in the design of immunological diagnosis and vaccines for M. abscessus infection.

Keyword

Mycobacterium abscessus; Morphological variants; Culture filtrate; Protein expression

MeSH Terms

Animals
Electrophoresis
Humans
Hydrogen-Ion Concentration
Immunologic Tests
Macrophages
Mice
Molecular Weight
Mycobacterium
Oxygen
Proteins
Sprains and Strains
Vaccines
Oxygen
Proteins
Vaccines

Figure

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  Figure 1. Colony morphology of M. abscessus type strain under variable culture conditions. (A) Normal oxygen tension (21%) and pH 6.0, (B) pH 7.5, (D) pH 5.0 and (E) low oxygen tension (13%), pH 5.0 culture conditions affected the morphology of M. abscessus. The smooth (C) and rough (F) variants were obtained from modified culture conditions after 7 days of incubation on 7H10 agar plate at 37°C. The arrows indicate rough variants of M. abscessus.

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  Figure 2. The frequency of morphotype changes under acidic and low oxygen tension culture conditions. In vitro conditional culture system affects the colony morphology of M. abscessus. Rough colonies of M. abscessus have mutated from smooth type strain.

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  Figure 3. Growth of M. abscessus conditional variants strains in BMDM. M. abscessus # 1 (•) and # 4 (Δ) survive and more actively multiply in BMDMs than M. abscessus # 2 (○) and # 3 (▾). BMDMs were infected with 2.5×105 M. abscessus strains for 4 h. Bacterial cfu in cell lysates was determined at the indicated intervals. Data are the mean of duplicated determinations of two separate experiments. Data are presented as the mean ± SD of two experiments. Significant differences (∗p<0.05) compared with conditional morphotype variants are indicated. M. abscessus strains were grown under each indicated condition as # 1 (•); 21% O2, pH 6.0, # 2 (○); 21% O2, pH 5.0, # 3 (▾); 13% O2, pH 5.0, and # 4 (Δ); 21% O2, pH 7.5.

• 

  Figure 4. TNF-α and IL-12p40 production in BMDMs stimulated with M. abscessus conditional variants and treated with culture filtrate (CF). BMDMs were infected for 4 hr with M. abscessus # 1 and # 3 variants at a MOI of 1:1, washed three times, and further incubated for 72 hr (A, B). The CF was used at a concentration of 1 μg/ml (C, D). TNF-α and IL-12p40 were measured in culture supernatants at 4, 24 and 72 hr post-infection. Data are presented as the mean ± SD of three experiments. Significant differences (∗p<0.05; ∗∗p<0.01) compared with strain or CF of smooth variant are indicated.

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  Figure 5. Comparison of the culture filtrate (CF) protein profiles from M. abscessus variants grown under 21% O2, pH 6.0 (2), 21% O2, pH 7.5 (3), 21% O2, pH 5.0 (4), 13% O2, pH 5.0 (5) conditions. The first lane (1) was loaded with protein molecular weight marker. The CF proteins (30 μg of total proteins for each lane) were separated by 12% SDS-PAGE. The gel was stained with 0.25% Coomassie brilliant blue R250.

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