Background

Legionella pneumophila (Lpn), a Gram-negative bacterium, is the causative agent of Legionnaires’ disease, a severe form of pneumonia. Lpn is an intracellular pathogen that produces over 300 protein effectors that it secretes through either a type II secretion system (T2SS), or through a type IV secretion system (T4SS) (Hubber and Roy, 2010; White and Cianciotto, 2019). ChiA is one such T2SS protein effector. ChiA is an 81-kDa endochitinase that has a role in Lpn virulence during lung infection (Rehman et al., 2020). Lpn carrying a deletion of the ChiA gene (ΔchiA) shows decreased survival in the lungs of mice, compared to WT Lpn (DebRoy et al., 2006). While humans do not produce chitin, they do produce analogous glycoproteins, mucins, that have known properties in interacting with and blocking infection of other pathogens. In our study, we showed, for the first time, that ChiA is able to both bind to and degrade mucins (Rehman et al., 2020). To determine if live bacteria were able to bind to mucins, we utilized our previously published protocol to manipulate wild-type Lpn strain 130b and its chiA mutant to express a GFP-producing plasmid (DebRoy et al., 2006; Rondelet and Condemine, 2013). Furthermore, earlier studies showed that wheat germ agglutinin (WGA) binds to mucins (Bhavanandan and Katlic, 1979; Valdizan et al., 1992). Therefore, we utilized porcine stomach mucins which we labeled with TexasRed-conjugated WGA (Model et al., 2009). To determine whether ChiA directly binds to these mucins, we also used an ELISA based-assay with purified recombinant (N-terminally His-tagged) ChiA and detected binding using anti-His₆ antibodies.

Although mucin binding to bacteria has been studied previously (Naughton et al., 2014), and fluorescently labeled WGA has been studied in the context of Gram-positive bacteria (Fife et al., 2000) this is the first protocol to directly label mucins and bacteria and then utilize flow cytometry to measure mucin binding to a Gram-negative bacterium. Furthermore, by analyzing both bacterial binding to mucins and the binding of purified proteins to mucins, this protocol provides insight into synergistic binding of different surface exposed bacterial proteins to mucins. This protocol can be further applied to study mucin binding to other Gram-negative bacteria.

Part I: Bacterial Mucin Binding with Flow Cytometry