Mucinases as Emerging Players in Legionella pneumophila Pathogenesis

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PROJECT SUMMARY / ABSTRACT Legionella pneumophila (Lp) is the agent of Legionnaires disease pneumonia, which is increasing in incidence. Previously, we showed that a chitinase (ChiA) secreted by the type II secretion system (T2SS) is needed for optimal survival of Lp in lungs. Since chiA mutants were not impaired for intracellular infection of macrophages or epithelia, we posited that ChiA mediates an overlooked extracellular event(s). Since chitin is not made by mammals, we further surmised that ChiA is bi-functional and acts on a chitin-like factor in infected lungs. Thus, we recently determined ChiA's structure, and functional analyses revealed a C-terminal domain (CTD) with the chitinase active site. Yet, as we had hypothesized, the CTD also had a unique Zn-dependent, peptidase active site that mediates the cleavage of mucins, including MUC5AC, a mucus gel-forming glycoprotein expressed in lungs. Most significantly, we found that i) chiA mutant supernatants are deficient for cleavage of mucins and ii) the chiA mutant is impaired for the ability to penetrate an in vitro mucin layer. Thus, we hypothesize that ChiA promotes Lp dissemination through mucin layers in lungs as a step toward accessing its coveted intracellular niches. Though mucinases have been studied in various bacteria, our work was the first documentation of a Lp mucinase and one of the few defined for a lung pathogen or an intracellular parasite of macrophages and epithelia. Extending our inquiry, we wondered if ChiA might also degrade mucin-like glycoproteins and found that purified ChiA-CTD cleaves human C1-INH. Since C1-INH is a component of the complement system, we hypothesize that ChiA may have yet another role in infection, i.e., promoting Lp's long-known but still poorly understood resistance to complement-mediated killing. To our knowledge, ChiA currently stands alone as an exoenzyme that degrades i) chitin, ii) gel-forming mucins of the lung, and iii) C1-INH of the human complement system, besides aiding Lp in the lungs. Thus, this proposal will discern if ChiA-mediated degradation of mucin promotes Lp access into human cells, if ChiA-mediated cleavage of C1-INH confers Lp resistance to killing by human complement, and if the mucinase-peptidase domain vs the chitinase domain of ChiA fosters Lp growth in lungs. Despite the link between ChiA and mucin, the chiA mutant did retain some mucin-degradation activity, suggesting that Lp secretes additional proteins that act on gel-forming mucins. Thus, this proposal will also seek to identify which of the other T2SS-dependent peptidases that we previously identified degrades mucin and perhaps also complement components. Besides generating much-needed data on Lp mucinase- peptidases and complement-resistance, this work will both improve our broad understanding of mucinases during lung infection and intracellular parasitism and potentially reveal new targets for disease intervention.