Comparative proteomic investigation unravels the pathobiology of Mycobacterium fortuitum biofilm.

Biofilm formation by Mycobacterium fortuitum causes serious threats to human health due to its increased contribution to nosocomial infections. In this study, the first comprehensive global proteome analysis of M. fortuitum was reported under planktonic and biofilm growth states. A label-free Q Exactive Quadrupole-Orbitrap tandem mass spectrometry analysis was performed on the protein lysates. The differentially abundant proteins were functionally characterized and re-annotated using Blast2GO and CELLO2GO. Comparative analysis of the proteins among two growth states provided insights into the phenotypic switch, and fundamental pathways associated with pathobiology of M. fortuitum biofilm, such as lipid biosynthesis and quorum-sensing. Interaction network generated by the STRING database revealed associations between proteins that endure M. fortuitum during biofilm growth state. Hypothetical proteins were also studied to determine their functional alliance with the biofilm phenotype. CARD, VFDB, and PATRIC analysis further showed that the proteins upregulated in M. fortuitum biofilm exhibited antibiotic resistance, pathogenesis, and virulence. Heatmap and correlation analysis provided the biomarkers associated with the planktonic and biofilm growth of M. fortuitum. Proteome data was validated by qPCR analysis. Overall, the study provides insights into previously unexplored biochemical pathways that can be targeted by novel inhibitors, either for shortened treatment duration or for eliminating biofilm of M. fortuitum and related nontuberculous mycobacterial pathogens. KEY POINTS: • Proteomic analyses of M. fortuitum reveals novel biofilm markers. • Acetyl-CoA acetyltransferase acts as the phenotype transition switch. • The study offers drug targets to combat M. fortuitum biofilm infections.

INNO-LiPA DNA line probe assay misidentification of M. smegmatis as Mycobacterium fortuitum complex.

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65-, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required.

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells.

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.

Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard.

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons. Mycobacterium chelonae and M. fortuitum are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome. Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M. fortuitum infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.

SDS-PAGE for identification of species belonging to the Mycobacterium fortuitum complex.

We performed a study to determine the usefulness of SDS-PAGE of whole cell proteins for the characterization of species of rapidly growing mycobacteria belonging to the Mycobacterium fortuitum complex. Strains included 37 M. fortuitum, 32 M. chelonae, 10 M. peregrinum, 5 M. abscessus, and 3 M. mucogenicum. Eight collection strains (including type strains of the five species) were also included in the study. All strains yielded between 44 and 58 bands in the electrophoretograms. Intraspecies similarity showed Dice coefficients higher than 95%, with only one strain of M. fortuitum having a six-band difference (Dice coefficient 87.75%). However, interspecies similarity was always below 75%, the similarity being higher between M. fortuitum and M. peregrinum (75.51%) and between M. chelonae and M. abscessus (54.9%). Visual examination of the electrophoretograms was sufficient for species characterization. SDS-PAGE of whole cell proteins is a useful technique for identification of isolates of the M. fortuitum complex, and is easy to perform without the need for complex or expensive equipment.

Mycobacterial di-O-acyl-trehalose inhibits mitogen- and antigen-induced proliferation of murine T cells in vitro.

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.

Use of gas chromatographic fatty acid and mycolic acid cleavage product determination to differentiate among Mycobacterium genavense, Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis.

Three Mycobacterium genavense strains and three American Type Culture Collection reference strains each of Mycobacterium fortuitum, Mycobacterium simiae, and Mycobacterium tuberculosis were subcultured onto Mycobacteria 7H11 agar (Difco Laboratories, Detroit, Mich.) supplemented with mycobactin J (Allied Laboratories, Fayette, Mo.). After 4 weeks of incubation at 37 degrees C in 10% CO2, the cultures were analyzed by gas-liquid chromatography (GLC) for their fatty acids and mycolic acid cleavage products. M. fortuitum was clearly differentiated from M. genavense by the presence of the specific marker 2-methyloctadecenoic acid in M. fortuitum and by the ratio of tetracosanoic acid to hexacosanoic acid. This ratio was <1 for M. genavense and >3 for M. fortuitum. M. fortuitum also contained docosanoic acid, which was not detected in M. genavense. M. genavense, M. simiae, and M. tuberculosis, which have similar GLC profiles, were also differentiated from each other by the presence of either cis-10-hexadecenoic acid or cis-11-hexadecenoic acid and by tetradecanoic acid content.