Concept: Mycobacterium smegmatis
Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organizations of these molecules remain unclear. Here we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate (TDM) in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified TDM and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensible structural role of trehalose mycolates in architectural design of the exposed surface of mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.
The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) has been shown to be active against Mycobacterium tuberculosis (Mtb) in clinical tuberculosis (TB) treatment. However, the mechanism of action of TMP-SMX against Mtb is still unknown. To unravel this, we have studied the effect of TMP and SMX by deleting folP2 gene in Mycobacterium smegmatis (Msm), and overexpressing the Mtb and Msm folP1/2 genes in Msm. Knocking out of the folP2 gene in Msm reduced the minimum inhibitory concentration (MIC) of SMX 8-fold compared with wild type. Overexpression of the folP1 genes from Mtb and Msm increased the MICs by 4 and 2-fold in Msm for SMX and TMP respectively. We show a strong correlation between the expression of folP1 and folP2 genes and TMP-SMX resistance in mycobacteria. This suggests that a combination of FolP2 inhibitor and SMX could be used for TB treatment with a better outcome.
Exposure of an isogenic bacterial population to a cidal antibiotic typically fails to eliminate a small fraction of refractory cells. Historically, fractional killing has been attributed to infrequently dividing or nondividing “persisters.” Using microfluidic cultures and time-lapse microscopy, we found that Mycobacterium smegmatis persists by dividing in the presence of the drug isoniazid (INH). Although persistence in these studies was characterized by stable numbers of cells, this apparent stability was actually a dynamic state of balanced division and death. Single cells expressed catalase-peroxidase (KatG), which activates INH, in stochastic pulses that were negatively correlated with cell survival. These behaviors may reflect epigenetic effects, because KatG pulsing and death were correlated between sibling cells. Selection of lineages characterized by infrequent KatG pulsing could allow nonresponsive adaptation during prolonged drug exposure.
Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants with substantial economic impacts to the cattle industry. Johne’s disease is known for its long latency period and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as MAP can survive for extended periods within the environment, resulting in new infections in naïve animals (3). This study explored the use of a biosecure, static composting structure to inactivate MAP. Mycobacterium smegmatis was also assessed as a surrogate for MAP. Two structures were constructed to hold three cattle carcasses each. Naturally-infected tissues and ground beef inoculated with laboratory cultured MAP and M. smegmatis were placed in nylon and plastic bags to determine effect of temperature and compost environment on viability over 250 (days) d. After removal, samples were cultured and growth of both organisms assessed after 12 weeks (wks). After 250 d, MAP was still detectable by PCR, while M. smegmatis was not detected after 67 d of composting. Furthermore, MAP remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophillic (55-65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of MAP after exposure to 80°C for 90 d. Naturally-infected lymph tissues were mixed with and without compost. After 90 d, MAP remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating MAP associated with cattle mortalities.
The integrity of the distinguishing, multilaminate cell envelope surrounding mycobacteria is critical to its survival and pathogenesis. The prevalence of phosphatidylinositol mannosides in the cell envelope suggests an important role in the mycobacterial life cycle. Indeed, deletion of the pimE gene (ΔpimE) encoding the first committed step in phosphatidylinositol hexamannoside biosynthesis in Mycobacterium smegmatis results in the formation of smaller colonies than wildtype colonies on Middlebrook 7H10 agar. To further investigate potential contributors to cell-envelope mannan biosynthesis while taking advantage of this colony morphology defect, we isolated spontaneous suppressor mutants of ΔpimE that reverted to wildtype colony size. Of 22 suppressor mutants, 6 accumulated significantly shorter lipomannan or lipoarabinomannan. Genome sequencing of these mutants revealed mutations in genes involved in the lipomannan/lipoarabinomannan biosynthesis, such as those encoding the arabinosyltransferase EmbC and the mannosyltransferase MptA. Furthermore, we identified three mutants carrying a mutation in a previously uncharacterized gene, MSMEG_5785, that we designated lmeA Complementation of these suppressor mutants with lmeA restored the original ΔpimE phenotypes, and deletion of lmeA in wildtype M. smegmatis resulted in smaller lipomannan as observed in the suppressor mutants. LmeA carries a predicted N-terminal signal peptide, and density gradient fractionation and detergent extractability experiments indicated that LmeA localizes to the cell envelope. Using a lipid ELISA assay, we found that LmeA binds to plasma membrane phospholipids such as phosphatidylethanolamine and phosphatidylinositol. LmeA is widespread throughout the Corynebacteriales; therefore, we concluded that LmeA is an evolutionarily conserved cell-envelope protein critical for controlling the mannan chain length of lipomannan/lipoarabinomannan.
Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process.IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)-the synthesis of which is an attractive drug target-combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis FDAA incorporation predominates at one of the two poles in M. smegmatis In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 4 years ago
Mycobacteria grow and divide asymmetrically, creating variability in growth pole age, growth properties, and antibiotic susceptibilities. Here, we investigate the importance of growth pole age and other growth properties in determining the spectrum of responses of Mycobacterium smegmatis to challenge with rifampicin. We used a combination of live-cell microscopy and modeling to prospectively identify subpopulations with altered rifampicin susceptibility. We found two subpopulations that had increased susceptibility. At the initiation of treatment, susceptible cells were either small and at early stages of the cell cycle, or large and in later stages of their cell cycle. In contrast to this temporal window of susceptibility, tolerance was associated with factors inherited at division: long birth length and mature growth poles. Thus, rifampicin response is complex and due to a combination of differences established from both asymmetric division and the timing of treatment relative to cell birth.
A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo.
Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc(2)155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.
Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multi-drug resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2- a]pyridine derivatives, is believed to target the pathogen’s homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome b-cc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a “supercomplex” with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis which has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding, and provide structural insight into a third class of the bc-complex superfamily.