scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type ◽  
Mycobacterial Cell Wall

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival for mycobacteria. However, the mechanisms underlying the biosynthesis of mycobacterial cell wall remain poorly understood. aceE encodes the E1 component of pyruvate dehydrogenase (PDH)complex. This study aimed to know the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.Results: We observed that the colony morphology of aceE-deficient mutants(aceE-mut)was quite different from the wild-type(WT) strain during the transposon library screening of M.smegmatis, smaller and smoother on the solid culture medium. Notably, the aceE-mut lost its ability of growing aggregately and biofilm forming, which are two very important features of mycobacteria.The morphological changes of the aceE-mut strain were further confirmed by electron microscopy that presented shorter, smoother and thinner images in contrast withWT strain.Additionally, the analysis of mycolic acid(MA)using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceEgene restored the composition of MA. Conclusions: Overall, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology and biofilm formation of M.smegmatis.

scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant ( aceE -mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis , basonym Mycobacterium smegmatis ( M. smegmatis ). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis. Results: We observed that the colony morphology of aceE -mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis . Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE -mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE -mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE -mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE -mut strain whereas complementation of the aceE -mut with a wild-type aceE gene restored the composition of MA. Conclusions: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

scholarly journals The aceE involves in mycolic acid synthesis and biofilm formation in Mycobacterium smegmatis

2020 ◽  
Author(s):  
Suting Chen ◽  
Tianlu Teng ◽  
Shuan Wen ◽  
Tingting Zhang ◽  
Hairong Huang
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Morphological Changes ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Wall Structure ◽  
Wild Type

Abstract Background: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis.Results: We observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA. Conclusions: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.

scholarly journals TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ei’ichi Iizasa ◽  
Yasushi Chuma ◽  
Takayuki Uematsu ◽  
Mio Kubota ◽  
Hiroaki Kawaguchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Macrophage Activation ◽  
Mycolic Acid ◽  
Dependent Manner ◽  
Independent Manner ◽  
Lectin Receptors ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

AbstractMycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

scholarly journals FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis

2005 ◽  
Vol 187 (19) ◽  
pp. 6603-6611 ◽  
Author(s):  
Liem Nguyen ◽  
Satheesh Chinnapapagari ◽  
Charles J. Thompson
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Single Mutation ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Colonial Morphology ◽  
Mycobacterial Cell Wall

ABSTRACT Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating α,α′-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.

Contrasting roles of the innate receptors TREM2 versus Mincle in the recognition and response of macrophages to mycolic acid-containing lipids in mycobacterial cell walls

2020 ◽  
Author(s):  
Ei'ichi Iizasa ◽  
Yasushi Chuma ◽  
Takayuki Uematsu ◽  
Mio Kubota ◽  
Hiroaki Kawaguchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterial Infection ◽  
Mycolic Acid ◽  
Dependent Manner ◽  
Independent Manner ◽  
Lectin Receptors ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

Abstract Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, their mechanism of action remains unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages responded to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages responded to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhanced Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerated the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

scholarly journals Synthesis of a homologous series of galactofuranose-containing mycobacterial arabinogalactan fragments

2016 ◽  
Vol 94 (11) ◽  
pp. 976-988 ◽  
Author(s):  
Maju Joe ◽  
Todd L. Lowary
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Chemical Synthesis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Complex Cell ◽  
Human Pathogen ◽  
Structural Element ◽  
Target Molecules ◽  
Mycobacterial Cell Wall

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, produce a complex cell wall structure made of carbohydrates and lipids. The major structural element of the mycobacterial cell wall is a glycoconjugate called the mycolic acid – arabinogalactan – peptidoglycan (mAGP) complex. Inhibition of mAGP biosynthesis is a proven strategy for developing anti-mycobacterial drugs, and thus, understanding the pathways and enzymes involved in the assembly of this molecule is of interest. In this paper, we describe the chemical synthesis of a panel of nine oligosaccharide fragments (4–12) of the galactan domain of the mAGP complex designed as biosynthetic probes. These structures, ranging in size from a hexasaccharide to a tetradecasaccharide, are potential substrates for two biosynthetic enzymes, GlfT2 and AftA, and represent the largest mycobacterial galactan fragments synthesized to date. The route developed was iterative and provided multimilligram quantities of the target molecules 4–12 in good overall yield.

scholarly journals Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance and Persistence

2021 ◽  
Author(s):  
Kirsi Savijoki ◽  
Henna Myllymäki ◽  
Hanna Luukinen ◽  
Lauri Paulamäki ◽  
Leena-Maija Vanha-aho ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Secretion ◽  
Biofilm Formation ◽  
Asymmetric Cell Division ◽  
Glyoxylate Cycle ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Mycobacterium Marinum ◽  
Surface Proteins ◽  
Label Free

ABSTRACTThe complex cell wall and biofilm matrix (ECM) act as key barriers to antibiotics in mycobacteria. Here, the ECM-proteins of Mycobacterium marinum ATCC927, a non-tuberculous mycobacterial model, was monitored over three months by label-free proteomics and compared with cell-surface proteins on planktonic cells to uncover pathways leading to virulence, tolerance, and persistence. We show that ATCC927 forms pellicle-type (PBFs) and submerged-type (SBFs) biofilms after two weeks and two days of growth, respectively, and that the increased CelA1 synthesis in this strain prevents biofilm formation and leads to reduced rifampicin tolerance. The proteomic data suggests that specific changes in mycolic acid synthesis (cord factor), Esx1-secretion, and cell-wall adhesins explain the appearance of PBFs as ribbon-like cords and SBFs as lichen-like structures. A subpopulation of cells resisting the 64 × MIC rifampicin (persisters) were detected in both biofilm subtypes, and already in one-week-old SBFs. The key forces boosting their development could include subtype-dependent changes in asymmetric cell division, cell wall biogenesis, tricarboxylic acid/glyoxylate cycle activities, and energy/redox/iron metabolisms. The effect of varying ambient oxygen tensions on each cell type and non-classical protein secretion are likely factors explaining majority of the subtype-specific changes. The proteomic findings also imply that Esx1-type protein secretion is more efficient in PL and PBF cells, while SBF may prefer both the Esx5- and non-classical pathways to control virulence and prolonged viability/persistence. In conclusion, this study reports a first proteomic insight into aging mycobacterial biofilm-ECMs and indicates biofilm subtype-dependent mechanisms conferring increased adaptive potential and virulence on non-tuberculous mycobacteria.IMPORTANCEMycobacteria are naturally resilient and mycobacterial infections are notoriously difficult to treat with antibiotics, with biofilm formation being the main factor complicating the successful treatment of TB. The present study shows that non-tuberculous Mycobacterium marinum ATCC927 forms submerged- and pellicle-type biofilms with lichen- and ribbon-like structures, respectively, as well as persister cells under the same conditions. We show that both biofilm subtypes differ in terms of virulence-, tolerance- and persistence-conferring activities, highlighting the fact that both subtypes should be targeted to maximize the power of antimycobacterial treatment therapies.

scholarly journals An essential mycolate remodeling program for mycobacterial adaptation in host cells

2018 ◽  
Author(s):  
Eliza J.R. Peterson ◽  
Rebeca Bailo ◽  
Alissa C. Rothchild ◽  
Mario Arrieta-Ortiz ◽  
Amardeep Kaur ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
New Technology ◽  
Mycolic Acid ◽  
Host Cells ◽  
Macrophage Infection ◽  
Cell Wall Remodeling ◽  
Mycobacterial Cell Wall

AbstractThe success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA. Using Path-seq we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/A2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.One Sentence SummaryNovel technology (Path-seq) discovers cell wall remodeling program during Mycobacterium tuberculosis infection of macrophages

scholarly journals Roles of Lsr2 in Colony Morphology and Biofilm Formation of Mycobacterium smegmatis

2006 ◽  
Vol 188 (2) ◽  
pp. 633-641 ◽  
Author(s):  
Jeffrey M. Chen ◽  
Greg J. German ◽  
David C. Alexander ◽  
Huiping Ren ◽  
Tracy Tan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Mycolic Acid ◽  
Colony Morphology ◽  
Insertion Mutant ◽  
Transposon Insertion ◽  
Layer Chromatography ◽  
Wall Components

ABSTRACT The lipid-rich cell wall is a defining feature of Mycobacterium species. Individual cell wall components affect diverse mycobacterial phenotypes including colony morphology, biofilm formation, antibiotic resistance, and virulence. In this study, we describe a transposon insertion mutant of Mycobacterium smegmatis mc2155 that exhibits altered colony morphology and defects in biofilm formation. The mutation was localized to the lsr2 gene. First identified as an immunodominant T-cell antigen of Mycobacterium leprae, lsr2 orthologs have been identified in all sequenced mycobacterial genomes, and homologs are found in many actinomycetes. Although its precise function remains unknown, localization experiments indicate that Lsr2 is a cytosolic protein, and cross-linking experiments demonstrate that it exists as a dimer. Characterization of cell wall lipid components reveals that the M. smegmatis lsr2 mutant lacks two previously unidentified apolar lipids. Characterization by mass spectrometry and thin-layer chromatography indicate that these two apolar lipids are novel mycolate-containing compounds, called mycolyl-diacylglycerols (MDAGs), in which a mycolic acid (α- or α′-mycolate) molecule is esterified to a glycerol. Upon complementation with an intact lsr2 gene, the mutant reverts to the parental phenotypes and MDAG production is restored. This study demonstrates that due to its impact on the biosynthesis of the hydrophobic MDAGs, Lsr2 plays an important role in the colony morphology and biofilm formation of M. smegmatis.

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