Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis
Introduction
Mycobacterium tuberculosis, the microbe that causes tuberculosis (TB), is the leading cause of death from infection worldwide. In recent years, multidrug-resistant strains have emerged as a new threat in both developed and developing countries, and are strongly associated with mortality in AIDS patients. The M. tuberculosis cell envelope is unusually thick, rigid, and waxy, making it impermeable to many conventional antimicrobial drugs and thereby limiting the number of agents effective against tuberculosis [1], [2], [3]. Bacterial peptidoglycan hydrolases, also called autolysins, solubilize the bacterial cell wall by hydrolyzing specific bonds in cell wall peptidoglycan (Fig. 1). These enzymes, which are classified into glycosidases (including muramidases and glucosaminidases), amidases and peptidases [4], [5], have been proposed to have roles in bacterial surface growth, cell division [6], [7] and bacterial pathogenesis, including adhesion and invasion of host cells [8], [9], [10], [11], [12].
Genes encoding cell wall autolysins have been extensively studied in many bacteria [9], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]; however, little is known about the autolysins of mycobacteria, including M. tuberculosis. In 1977, two autolysins, an amidase and an endopeptidase, were described in Mycobacterium smegmatis [26]; however, their physical, biochemical, and enzymatic properties were not defined. Li et al. [27] described the partial characterization of an autolysin from Mycobacterium phlei. We have previously reported that ethambutol, an anti-TB drug, stimulates an endogenous enzymatic activity that cleaves the cell wall of M. smegmatis. In the current study, we identified an open reading frame cwlM (Rv3915, accession number: AAK48399) in the M. tuberculosis genome [28], [29], which had 31% homology and 21% identity with the cwlB gene of Bacillus subtilis [20] (Fig. 2). The entire 1218-bp gene encoding a 44-kDa protein was cloned and expressed, and its genetic product, a 47-kDa fusion protein, was purified and characterized. To our knowledge, this represents the first identification and cloning of a peptidoglycan hydrolase gene from M. tuberculosis. This enzyme may permit the development of a novel class of permeability drugs that can be used to enhance the effectiveness of existing antibiotics against TB.
Section snippets
Bacterial strains and growth conditions
M. smegmatis mc2155 [30] was used as a source for mycobacterial cell wall extract. M. tuberculosis genomic DNA was purified from M. tuberculosis clinical isolate BMC# 2732386. Mycobacterial strains were cultured in Middlebrook 7H9 broth or on 7H10 agar, enriched in each case with OADC. Cloned cwlM was expressed in E. coli strain HMS174 (DE-3)F [31], which was routinely cultured in LB broth containing 10 μg/ml tetracycline. All bacterial cultures in this study were incubated at 37 °C, and liquid
Sequence analysis of cwlM
Searches of GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) and ExPASy (http://www.expasy.ch) revealed that the M. tuberculosis cwlM gene (Rv3915) has 31% homology and 21% identity with cwlB of B. subtilis 168S, which was cloned by Kuroda and Sekiguchi [20] and Shida et al. [39]. cwlB encodes an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), which is one of several bacterial peptidoglycan hydrolases involved in peptidoglycan catabolism. Homologous genes have also been identified in
Discussion
M. tuberculosis and other mycobacteria have a thick, rigid, and waxy cell envelope that protects them against harsh environmental conditions (including the intracellular degradative enzymes that constitute an important defense against foreign organisms) and is also impermeable to many conventional antimicrobial drugs [1], [2], [3]. Cell wall autolysins have been extensively studied in many bacteria and shown to be essential for growth. However, little is known about the autolysins of M.
Acknowledgments
This work was supported by United States Health Service Grant AI-45617 from NIAID, by a research grant RG-107-N from the American Lung Association, and by The Research Service of the Department of Veteran Affairs.
We are grateful to Margaret Condron and to Drs. Heinz Remold, Eric Rubin, John S. Anderson, Gerald Pier, Xiaoling Chen, Jeff Hall, Jean Lee, and Katie O'Riordan for technical assistance and expert advice with this project. We thank Dr. Joe Keane of the BU Pulmonary Center for M.
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Cited by (33)
Mycobacterium tuberculosis Raf kinase inhibitor protein (RKIP) Rv2140c is involved in cell wall arabinogalactan biosynthesis via phosphorylation
2021, Microbiological ResearchCitation Excerpt :Rv3915 and Rv0050, the corresponding ortholog of regulated phosphoproteins relevant to cell wall biosynthesis in M. smegmatis has been reported to affect cell wall permeability and induce morphological alteration in MTB. ( Deng et al., 2005; Gao et al., 2019). Intriguingly, a large spectrum of phosphoproteins regulated by Rv2140c, were involved in amino acids and carbohydrate transport.
Effects of CwlM on autolysis and biofilm formation in Mycobacterium tuberculosis and Mycobacterium smegmatis
2019, International Journal of Medical MicrobiologyCitation Excerpt :Additionally, Bacillus cereus autolysin CwlB plays an important role in bacterial cell lysis; if the autolysin gene is deleted, bacterial fragmentation and the delayed release of virulence proteins occur (Yang et al., 2013). Previous studies showed that the CwlM protein is encoded by the Rv3915 gene in M. tuberculosis and by the MSMEG_6935 gene in M. smegmatis (Deng et al., 2005). However, the study of the Rv3915 and MSMEG_6935 genes has not been comprehensive and deep.
Two Faces of CwlM, an Essential PknB Substrate, in Mycobacterium tuberculosis
2018, Cell ReportsCitation Excerpt :CwlM is predicted to be an N-acetylmuramoyl-l-alanine amidase; however, its actual activity remains uncertain. While Deng et al. (2005) have previously demonstrated CwlM to possess peptidoglycan hydrolyzing activity, in a more recent study, no such activity was detected, presumably due to the lack of two essential catalytic residues (Boutte et al., 2016). As mentioned above, Boutte et al. (2016) have proposed that phospho-CwlM controls peptidoglycan generation by activating MurA; however, the possible functions of non-phosphorylated CwlM were not addressed.
Effect of Mycobacterium tuberculosis Rv3717 on cell division and cell adhesion
2018, Microbial PathogenesisFalse positives in using the zymogram assay for identification of peptidoglycan hydrolases
2018, Analytical Biochemistry
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Present address: Paratek Pharmaceuticals Inc., 75 Kneeland St., Boston, MA 02111, United States.
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Present address: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Mail Slot 511, 4301 W Markham Street, Little Rock, AR 72205, United States.