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<p>In <i>Mycobacterium leprae (M. leprae)</i>, the small heat shock protein Hsp18 has been reported to function as a chaperone <i>in vitro</i>. The stability and activity of Hsp18 protein was increased by stress induced post-translational modification. The significant identity and similarity with a conserved sequence GVDP and proteolytic active site between <i>M. leprae</i> Hsp18 and <i>E. coli</i> and <i>M. smegmatis</i> Lon proteases reveals that, Hsp18 will have additional role <i>in vivo</i> that remains unexplored. In this study, we characterized the protease activity of <i>M. leprae</i> Hsp18 by complementing the Lon protease of <i>Escherichia coli (E. coli)</i>. The MG1655Δ<i>lon</i> mutant of <i>E. coli</i>, which lack the Lon protease overexpress capsular polysaccharide and form mucoid colonies. Similarly, <i>E. coli</i> SG20780 is the <i>cps</i>B10::<i>lac</i> fusion that is <i>lac</i>+ and expresses <i>lac</i>Z, and both the strains are sensitive to methyl methane sulphonate (MMS) induced DNA damage. Upon induced expression of <i>M. leprae</i> Hsp18 in MG1655Δ<i>lon</i> strain the mucoid phenotype was reversed by downregulating <i>rcsA</i> and in SG20780 mutant, <i>lac</i>Z expression was inhibited. In these <i>E. coli</i> mutants <i>M. leprae</i> Hsp18 expression conferred resistance to MMS in a concentration-dependent manner. In patients with leprosy, the polymorphic variants Hsp18S and Hsp18P have been identified, where the codon 52 encodes serine and proline respectively. Among these variants, Hsp18S showed higher protease activity and MMS resistance than the Hsp18P variant. Induced expression of N- and C-terminal domains of <i>M. leprae</i> Hsp18 in MG1655Δ<i>lon</i> and SG20780, restored the Δ<i>lon</i> phenotype suggesting that both the terminals act in synergy for the proteolytic activity. Thus, our study demonstrates that <i>M. leprae</i> Hsp18 is a protease which has similar function as that of Lon protease in <i>E. coli</i>. The chaperone activity and the proteolytic activity of Hsp18 is pivotal for the survival of <i>M. leprae in vivo</i> within the macrophages as chaperone by refolding the misfolded proteins and proteolytic cleavage of misfolded and damaged proteins during pathogenesis.</p>

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Mycobacterium leprae small heat shock protein-18 complements Lon protease in Escherichia coli

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  • 发表日期：

    2026年4月9日

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  • Elengikal A. Rehna,
  • Kuppamuthu Dharmalingam,
  • Sankar Natesan

摘要

In Mycobacterium leprae (M. leprae), the small heat shock protein Hsp18 has been reported to function as a chaperone in vitro. The stability and activity of Hsp18 protein was increased by stress induced post-translational modification. The significant identity and similarity with a conserved sequence GVDP and proteolytic active site between M. leprae Hsp18 and E. coli and M. smegmatis Lon proteases reveals that, Hsp18 will have additional role in vivo that remains unexplored. In this study, we characterized the protease activity of M. leprae Hsp18 by complementing the Lon protease of Escherichia coli (E. coli). The MG1655Δlon mutant of E. coli, which lack the Lon protease overexpress capsular polysaccharide and form mucoid colonies. Similarly, E. coli SG20780 is the cpsB10::lac fusion that is lac+ and expresses lacZ, and both the strains are sensitive to methyl methane sulphonate (MMS) induced DNA damage. Upon induced expression of M. leprae Hsp18 in MG1655Δlon strain the mucoid phenotype was reversed by downregulating rcsA and in SG20780 mutant, lacZ expression was inhibited. In these E. coli mutants M. leprae Hsp18 expression conferred resistance to MMS in a concentration-dependent manner. In patients with leprosy, the polymorphic variants Hsp18S and Hsp18P have been identified, where the codon 52 encodes serine and proline respectively. Among these variants, Hsp18S showed higher protease activity and MMS resistance than the Hsp18P variant. Induced expression of N- and C-terminal domains of M. leprae Hsp18 in MG1655Δlon and SG20780, restored the Δlon phenotype suggesting that both the terminals act in synergy for the proteolytic activity. Thus, our study demonstrates that M. leprae Hsp18 is a protease which has similar function as that of Lon protease in E. coli. The chaperone activity and the proteolytic activity of Hsp18 is pivotal for the survival of M. leprae in vivo within the macrophages as chaperone by refolding the misfolded proteins and proteolytic cleavage of misfolded and damaged proteins during pathogenesis.