A protein fragment of Rv3194c located on mycobacterial cell surface efficiently prevents adhesion of recombinant Mycobacterium smegmatis, and promises a new anti-adhesive drug

2020 ◽  
Vol 149 ◽  
pp. 104498
Author(s):  
Dongyue Zhao ◽  
Danfeng Lin ◽  
Chen Xu
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Protein Fragment ◽  
Mycobacterial Cell ◽  
Recombinant Mycobacterium Smegmatis

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

scholarly journals Rapid Memory CD8+ T-Lymphocyte Induction through Priming with Recombinant Mycobacterium smegmatis

2006 ◽  
Vol 81 (1) ◽  
pp. 74-83 ◽  
Author(s):  
Avi-Hai Hovav ◽  
Mark J. Cayabyab ◽  
Michael W. Panas ◽  
Sampa Santra ◽  
John Greenland ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
T Lymphocyte ◽  
Plasmid Dna ◽  
Mycobacterium Smegmatis ◽  
Recombinant Adenovirus ◽  
Cytotoxic T Lymphocyte ◽  
Specific Memory ◽  
Cd8 T Lymphocyte ◽  
Recombinant Mycobacterium Smegmatis ◽  
Hiv 1

ABSTRACT The most promising vaccine strategies for the induction of cytotoxic-T-lymphocyte responses have been heterologous prime/boost regimens employing a plasmid DNA prime and a live recombinant-vector boost. The priming immunogen in these regimens must elicit antigen-specific memory CD8+ T lymphocytes that will expand following the boosting immunization. Because plasmid DNA immunogens are expensive and their immunogenicity has proven disappointing in human clinical trials, we have been exploring novel priming immunogens that might be used in heterologous immunization regimens. Here we show that priming with a prototype recombinant Mycobacterium smegmatis strain expressing human immunodeficiency virus type 1 (HIV-1) gp120-elicited CD4+ T lymphocytes with a functional profile of helper cells as well as a CD8+ T-lymphocyte population. These CD8+ T lymphocytes rapidly differentiated to memory cells, defined on the basis of their cytokine profile and expression of CD62L and CD27. Moreover, these recombinant-mycobacterium-induced T lymphocytes rapidly expanded following boosting with a recombinant adenovirus expressing HIV-1 Env to gp120-specific CD8+ T lymphocytes. This work demonstrates a remarkable skewing of recombinant-mycobacterium-induced T lymphocytes to durable antigen-specific memory CD8+ T cells and suggests that such immunogens might be used as priming vectors in prime/boost vaccination regimens for the induction of cellular immune responses.

scholarly journals A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

2011 ◽  
Vol 17 (10) ◽  
pp. 1261-1268 ◽  
Author(s):  
Kari A Sweeney ◽  
Dee N Dao ◽  
Michael F Goldberg ◽  
Tsungda Hsu ◽  
Manjunatha M Venkataswamy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Recombinant Mycobacterium Smegmatis

IL-17A Autoantibody Induced by Recombinant Mycobacterium smegmatis Expressing Ag85A-IL-17A Fusion Protein

2015 ◽  
Vol 176 (7) ◽  
pp. 2018-2026 ◽  
Author(s):  
Ling Chen ◽  
Sheng Guo ◽  
Liangxia Wu ◽  
Xiaoyong Fan ◽  
Hui Ma ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Mycobacterium Smegmatis ◽  
Recombinant Mycobacterium Smegmatis

scholarly journals Conditional Depletion of KasA, a Key Enzyme of Mycolic Acid Biosynthesis, Leads to Mycobacterial Cell Lysis

2005 ◽  
Vol 187 (22) ◽  
pp. 7596-7606 ◽  
Author(s):  
Apoorva Bhatt ◽  
Laurent Kremer ◽  
Annie Z. Dai ◽  
James C. Sacchettini ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Smegmatis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Cell Lysis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Scanning Electron Micrographs ◽  
Conditional Gene Expression ◽  
Mycobacterial Cell ◽  
Key Enzyme

ABSTRACT Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two β-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of α and epoxy mycolate biosynthesis and to accumulation of α′-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a “crumpled” appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.

scholarly journals The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope

2020 ◽  
Vol 295 (32) ◽  
pp. 11184-11194 ◽  
Author(s):  
Laurie Thouvenel ◽  
Gautier Prevot ◽  
Laura Chiaradia ◽  
Julien Parra ◽  
Emmanuelle Mouton-Barbosa ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Cell Envelope ◽  
Basic Structure ◽  
Fatty Acyl ◽  
Fatty Acid Residue ◽  
Final Assembly ◽  
Straight Chain Fatty Acid ◽  
Mycobacterial Cell

Trehalose polyphleates (TPP) are high-molecular-weight, surface-exposed glycolipids present in a broad range of nontuberculous mycobacteria. These compounds consist of a trehalose core bearing polyunsaturated fatty acyl substituents (called phleic acids) and a straight-chain fatty acid residue and share a common basic structure with trehalose-based glycolipids produced by Mycobacterium tuberculosis. TPP production starts in the cytosol with the formation of a diacyltrehalose intermediate. An acyltransferase, called PE, subsequently catalyzes the transfer of phleic acids onto diacyltrehalose to form TPP, and an MmpL transporter promotes the export of TPP or its precursor across the plasma membrane. PE is predicted to be an anchored membrane protein, but its topological organization is unknown, raising questions about the subcellular localization of the final stage of TPP biosynthesis and the chemical nature of the substrates that are translocated by the MmpL transporter. Here, using genetic, biochemical, and proteomic approaches, we established that PE of Mycobacterium smegmatis is exported to the cell envelope following cleavage of its signal peptide and that this process is required for TPP biosynthesis, indicating that the last step of TPP formation occurs in the outer layers of the mycobacterial cell envelope. These results provide detailed insights into the molecular mechanisms controlling TPP formation and transport to the cell surface, enabling us to propose an updated model of the TPP biosynthetic pathway. Because the molecular mechanisms of glycolipid production are conserved among mycobacteria, these findings obtained with PE from M. smegmatis may offer clues to glycolipid formation in M. tuberculosis.

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