Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis

https://doi.org/10.1016/j.bbapap.2004.09.021Get rights and content

Abstract

Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-l-alanyl-d-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for 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.

References (42)

  • L. Deng et al.

    Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

    J. Biol. Chem.

    (2000)
  • T. Shida et al.

    Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-l-alanine amidases CwlC and CwlV

    J. Biol. Chem.

    (2001)
  • P.J. Brennan et al.

    The envelope of mycobacteria

    Annu. Rev. Biochem.

    (1995)
  • J. Coyette et al.

    Some properties of the autolytic N-acetylmuramidase of Lactobacillus acidophilus

    J. Bacteriol.

    (1973)
  • J. Allignet et al.

    Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation

    Infect. Immun.

    (2001)
  • A.M. Berry et al.

    Contribution of autolysin to virulence of Streptococcus pneumoniae

    Infect. Immun.

    (1989)
  • E. Milohanic et al.

    The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor

    Mol. Microbiol.

    (2001)
  • M.E. Rupp et al.

    Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model

    J. Infect. Dis.

    (2001)
  • M.J. Loessner et al.

    C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates

    Mol. Microbiol.

    (2002)
  • C. Beliveau et al.

    Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin

    J. Bacteriol.

    (1991)
  • C.P. Chu et al.

    Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae

    J. Bacteriol.

    (1992)
  • Cited by (33)

    • Mycobacterium tuberculosis Raf kinase inhibitor protein (RKIP) Rv2140c is involved in cell wall arabinogalactan biosynthesis via phosphorylation

      2021, Microbiological Research
      Citation 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 Microbiology
      Citation 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 Reports
      Citation 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.

    View all citing articles on Scopus
    1

    Present address: Paratek Pharmaceuticals Inc., 75 Kneeland St., Boston, MA 02111, United States.

    2

    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.

    View full text