Genomic, Metabolic, and Immunological Characterization of GMP-Grade Mycobacterium phlei.

Mycobacterium phlei (M. phlei) is an understudied microbe with medical values as an immunomodulating agent. Here, we establish an industrial strain of M. phlei, CUD, and characterize its genomic, metabolic, and immunological profiles. The established strain has been stably passed for more than a decade, indicated by next-generation sequencing of its 5.3 Mb genome. We show that the intramuscular inoculation of heat-inactivated CUD in immunocompetent mice is well tolerated, and can mount immunological responses. Immunophenotyping demonstrates induced innate and adaptive immune responses in peripheral blood, spleen, and inguinal lymph nodes of CUD-treated mice. Using GC-TOF-MS, we find that the metabolomic profiles of different batches are highly concordant. These results demonstrate a highly reproducible production of M. phlei under GMP conditions. IMPORTANCE Heat-inactivated M. phlei demonstrates promising efficacy to treat BCG-unresponsive non-muscle-invasive bladder cancer patients in clinical trials. However, lack of GMP-grade heat-inactivated M. phlei hampers further clinical investigations. Here, we described a GMP-grade, heat-inactivated M. phlei product, and presented initial characterization of its safety and immunomodulating properties. This product will serve as a starting point for further preclinical studies as well as clinical trials such as in combination with immune checkpoint inhibitors to treat bladder cancer.

Effects of dietary addition of heat-killed Mycobacterium phlei on growth performance, immune status and anti-oxidative capacity in early weaned piglets.

The contradiction between high susceptibility of early weaned piglets to enteric pathogens and rigid restriction of antibiotic use in the diet is still prominent in the livestock production industry. To address this issue, the study was designed to replace dietary antibiotics partly or completely by an immunostimulant, namely heat-killed Mycobacterium phlei (M. phlei). Piglets (n = 192) were randomly assigned to one of the four groups: (1) basal diet (Group A), (2) basal diet + a mixture of antibiotics (80 mg/kg diet, Group B), (3) basal diet + a mixture of antibiotics (same as in Group B, but 40 mg/kg diet) + heat-killed M. phlei (1.5 g/kg diet) (Group C) and (4) basal diet + heat-killed M. phlei (3 g/kg diet) (Group D). All piglets received the respective diets from days 21 to 51 of age and were weaned at the age of 28 d. Compared with the Control (Group A), in all other groups the average daily gain, average daily feed intake, small intestinal villus height:crypt depth ratio and protein levels of occludin and ZO-1 in the jejunal mucosa were increased. A decreased incidence of diarrhoea in conjunction with an increased sIgA concentration in the intestinal mucosa and serum IL-12 and IFN-γ concentrations was found in groups supplemented with heat-killed M. phlei (Groups C and D), but not in Group B. Groups C and D also showed decreased IL-2 concentrations in the intestinal mucosa with lower TLR4 and phosphor-IκB protein levels. The antioxidant capacity was reinforced in Groups C and D, as evidenced by the reduction in malondialdehyde and enhanced activities of antioxidant enzymes in serum. These data indicate that heat-killed M. phlei is a promising alternative to antibiotic use for early weaned piglets via induction of protective immune responses.

The application of metabolite profiling to Mycobacterium spp.: determination of metabolite changes associated with growth.

In order to decipher the complex biological networks underlying biochemical and physiological processes, cellular regulation at all levels must be studied. The metabolites determined by metabolomics represent the end-point of cellular regulation and thus vital components of any integrative network. In the case of pathogenic agents such as Mycobacterium tuberculosis metabolomics offers an ideal opportunity to gain a better understanding of how this species adapts to environmental conditions and antimicrobial treatments. In the present study a metabolite profiling protocol for Mycobacterium including optimised quenching, extraction and analysis has been devised. These methods have been applied to three different Mycobacterium spp. demonstrating potential translation across the genus. Steady-state levels of metabolites during growth have been determined for Mycobacterium smegmatis, Mycobacterium phlei and Mycobacterium bovis BCG (Bacillus Calmette-Guérin). The changes of designated biomarkers emphasised phenotypical differences (e.g. nitrogen metabolism) and similarities (e.g. cysteine biosynthesis) between the bacteria. Each time point showed distinguishable metabolic characteristics from early lag to late stationary phase/beginning of non-replicating phase. The combination of the metabolic results with published "omics" data indicated that transcription appeared to be the most predominant mode of cellular regulation utilised by these bacteria studied.

A method to extract intact and pure RNA from mycobacteria.

We describe a high-yielding, simple, and aerosol-free protocol for the isolation of RNA from mycobacteria that does not require sophisticated instruments. The method yielded 50 µg of RNA from 10(7) cells, 50 times more than a recently reported method. Our method can extract total RNA from aerobically grown bacteria and from in vitro hypoxia-induced dormant bacilli and mycobacteria residing within infected macrophages.

Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Direct molecular mass determination of trehalose monomycolate from 11 species of mycobacteria by MALDI-TOF mass spectrometry.

Direct estimation of the molecular mass of single molecular species of trehalose 6-monomycolate (TMM), a ubiquitous cell-wall component of mycobacteria, was performed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. When less than 1 microg TMM was analysed by MALDI-TOF mass spectrometry, quasimolecular ions [M+Na]+ of each molecular species were demonstrated and the numbers of carbons and double bonds (or cyclopropane rings) were determined. Since the introduction of oxygen atoms such as carbonyl, methoxy and ester groups yielded the appropriate shift of mass ions, the major subclasses of mycolic acid (alpha, methoxy, keto and wax ester) were identified without resorting to hydrolytic procedures. The results showed a marked difference in the molecular species composition of TMM among mycobacterial species. Unexpectedly, differing from other mycoloyl glycolipids, TMM from Mycobacterium tuberculosis showed a distinctive mass pattern, with abundant odd-carbon-numbered monocyclopropanoic (or monoenoic) alpha-mycolates besides dicyclopropanoic mycolate, ranging from C75 to C85, odd- and even-carbon-numbered methoxymycolates ranging from C83 to C94 and even- and odd-carbon-numbered ketomycolates ranging from C83 to C90. In contrast, TMM from Mycobacterium bovis (wild strain and BCG substrains) possessed even-carbon-numbered dicyclopropanoic alpha-mycolates. BCG Connaught strain lacked methoxymycolates almost completely. These results were confirmed by MALDI-TOF mass analysis of mycolic acid methyl esters liberated by alkaline hydrolysis and methylation of the original TMM. Wax ester-mycoloyl TMM molecular species were demonstrated for the first time as an intact form in the Mycobacterium avium-intracellulare group, M. phlei and M. flavescens. The M. avium-intracellulare group possessed predominantly C85 and C87 wax ester-mycoloyl TMM, while M. phlei and the rapid growers tested contained C80, C81, C82 and C83 wax ester-mycoloyl TMM. This technique has marked advantages in the rapid analysis of not only intact glycolipid TMM, but also the mycolic acid composition of each mycobacterial species, since it does not require any degradation process.

Pro-apoptotic and immunomodulatory activity of a mycobacterial cell wall-DNA complex towards LNCaP prostate cancer cells.

BACKGROUND: We have isolated a mycobacterial cell wall-DNA complex (MCC) possessing anti-cancer activity against bladder cancer cells. The anti-cancer activity of MCC appears to be due to two effects: a direct interaction with bladder cancer cells resulting in the induction of apoptosis and an indirect effect via the stimulation of monocytes and macrophages cytokine synthesis. In this study, the direct effect of MCC towards LNCaP cancer cells was evaluated. METHODS: Inhibition of proliferation, cell cycle arrest and induction of apoptosis were evaluated in vitro using LNCaP cells treated with MCC. The synthesis of IL-12, GM-CSF, and TNF-alpha by LNCaP cells in response to MCC was also determined. Experiments were performed to gain insight into the mechanism of action of MCC towards LNCaP cells. RESULTS: MCC caused a dose-dependent inhibition of the proliferation of LNCaP cells that was associated with cell cycle arrest at the G0/G1 phase. MCC-induced apoptosis of LNCaP cells was consistent with a mitochondrial pathway involving mitochondrial disruption, release of cytochrome c, and an increase in Bax protein levels leading to caspase-3 and -7 activation and cleavage of poly (ADP-ribose) polymerase and nuclear mitotic apparatus protein. Surprisingly, MCC also directly induced the synthesis of IL-12 and GM-CSF, but not TNF-alpha, by LNCaP cells. CONCLUSIONS: MCC possesses the ability to directly induce apoptosis of LNCaP cells and to trigger the synthesis of IL-12 and GM-CSF by these cells, suggesting a potential role of MCC for the treatment of prostate cancer.

Mycobacterial cell wall extract for treatment of carcinoma in situ of the bladder.

PURPOSE: Bacillus Calmette-Guerin (BCG) established immunotherapy as an effective modality for carcinoma in situ of the bladder and remains the most effective agent for treatment. However, as a live organism it has the potential for undesirable side effects and toxicity. This result has led to the search for other active and safer biological response modifiers. We investigated the efficacy of a mycobacterial cell wall extract (MCWE) from Mycobacterium phlei, which does not contain live bacteria, for management of carcinoma in situ of the bladder in humans. MATERIALS AND METHODS: The requirement for an emulsified preparation was investigated with photon correlation spectroscopy to determine the stability of the bacterial fragments. A total of 61 patients with histologically documented carcinoma in situ completed the study. Cell wall extract from M. phlei suspended in oil droplets to form an emulsion were instilled into the bladder at a dose of 4 mg. once weekly for 6 weeks and then monthly for 1 year. Response assessment was performed at 3-month intervals. Complete response to treatment indicated the absence of endoscopic and histological evidence of carcinoma in situ. Partial responders were those cases in which cystoscopy and biopsies were negative but cytology was suspicious for malignant cells. All other cases were considered failures. RESULTS: The need for an emulsified suspension of the cell wall extract was confirmed by the demonstration that the cell wall extract alone in urine aggregated, whereas the MCWE emulsion had remained stable. Kaplan-Meier estimates showed negative cystoscopy and biopsies in 62.5% at 12, 49.3% at 24 and 41.1% of patients at 60 weeks after therapy. After this point the number of responders had remained stable. Excellent tolerance with minimal toxicity was observed. CONCLUSIONS: Our study demonstrates clinical activity of low doses of MCWE against human bladder cancer. The results observed at the dosage used in our trial are less than those observed with live BCG. However, MCWE has a better toxicity profile and can be instilled in the presence of a disrupted urothelium. It also appears to exhibit activity in patients in whom BCG has failed.

Anticancer activity of mycobacterial DNA: effect of formulation as chitosan nanoparticles.

Mycobacterium phlei (M. phlei) DNA inhibits cancer cell division but is susceptible to degradation by DNase. Chitosan forms nanoparticulate polyelectrolyte complexes with DNA, and may thus reduce nuclease degradation. We have characterized chitosan-DNA nanoparticle formation, determined DNase susceptibility, and evaluated their antiproliferative activity. Nanoparticle diameter initially decreased with increasing phosphate charge density. However nanoparticle diameter increased above 6 micromol of phosphate. Particle aggregation occurred at 16.2 micromol phosphate and was related to reduced surface charge. Incorporation of DNA within chitosan nanoparticles significantly decreased degradation by DNase. The ability of M. phlei DNA-chitosan nanoparticles to inhibit melanoma cell division was determined relative to M. phlei DNA and a cationic liposomal M. phlei DNA formulation. M. phlei DNA had antiproliferative activity (MTT reduction, IC50 = 0.9 mg/ml) without intrinsic cytotoxicity (LDH release, ED50 > 50 microg/ml). Cationic polyphosphate chitosan nanoparticles were inert (antiproliferative IC50 > 1 mg/ml, ED50 > 1 mg/ml). M. phlei DNA-chitosan nanoparticles were 20-fold more potent than M. phlei DNA. Cationic DOTAP/DOPE liposomes were cytostatic (IC50 = 49 microg/ml) and cytotoxic (ED50 = 87 microg/ml), and complexation of M. phlei DNA resulted in a significant reduction of antiproliferative activity. Chitosan nanoparticles may therefore be appropriate delivery vehicles for M. phlei DNA.

Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells.

Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phlei mycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms--a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte-macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12.

Isolation, purification and characterization of intracellular calmodulin like protein (CALP) from Mycobacterium phlei.

A monomeric acidic protein of 14,000 Da with an isoelectric point of 4.5 was isolated from Mycobacterium phlei, which stained poorly with Coomassie brilliant blue. This protein showed retardation in mobility in SDS-PAGE upon treatment with calcium, similar to eukaryotic calmodulin proteins. Activation of cAMP phosphodiesterase and NAD kinase by this protein was observed. The CD spectral analysis indicated that the CALP has 52% of beta-conformation. The regular beta-conformation of the calmodulin like protein was shifted to 46% alpha-helical structure when calcium ions reacted with the protein, however, 42% of the CALP still retained its original beta-conformation. These observations indicated homology of this calcium binding protein with that of eukaryotic calmodulins in few structural and functional properties.

Effect of growth temperature on the fatty acid composition of Mycobacterium phlei.

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35 degrees C to 26-20 degrees C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10 degrees C. Thus, down to 26-20 degrees C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10 degrees C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that the mechanism behind the responses might be more complex than generally believed.