Whole microbe arrays accurately predict interactions and overall antimicrobial activity of galectin-8 toward distinct strains of Streptococcus pneumoniae

Abstract

Microbial glycan microarrays (MGMs) populated with purified microbial glycans have been used to define the specificity of host immune factors toward microbes in a high throughput manner. However, a limitation of such arrays is that glycan presentation may not fully recapitulate the natural presentation that exists on microbes. This raises the possibility that interactions observed on the array, while often helpful in predicting actual interactions with intact microbes, may not always accurately ascertain the overall affinity of a host immune factor for a given microbe. Using galectin-8 (Gal-8) as a probe, we compared the specificity and overall affinity observed using a MGM populated with glycans harvested from various strains of Streptococcus pneumoniae to an intact microbe microarray (MMA). Our results demonstrate that while similarities in binding specificity between the MGM and MMA are apparent, Gal-8 binding toward the MMA more accurately predicted interactions with strains of S. pneumoniae, including the overall specificity of Gal-8 antimicrobial activity. Taken together, these results not only demonstrate that Gal-8 possesses antimicrobial activity against distinct strains of S. pneumoniae that utilize molecular mimicry, but that microarray platforms populated with intact microbes present an advantageous strategy when exploring host interactions with microbes.

Figure 1

Gal-8 recognize S. pneumoniae that express self-like antigens on the microbial glycan microarray (MGM). (a) MGM array was incubated with Gal-8, Gal-8N and Gal-8C at the concentrations indicated. Error bars represent means ± standard deviation (SD). RFU, relative fluorescence units. S. pneumoniae Danish type 11A; Sp 11A, S. pneumoniae Danish type 14; Sp 14, S. pneumoniae Danish type 33F; Sp 33F. (b) Selected microbial glycans represented on the MGM were shown with binding isotherms. Error bars represent means ± SD.

Figure 2

Gal-8 recognizes specific serotypes of S. pneumoniae on the flow cytometry analysis. (a, b, and c) Flow cytometric analysis of Gal-8 (a), Gal-8N (b), and Gal-8C (c) binding to Sp 14, 33F, 2 and 11A with or without inclusion of final concentration 50 mM TDG as indicated. TDG is labeled in orange. Unstained control is labeled in gray. Each respective galectin is labeled in blue.

Figure 3

Measuring the impact of Gal-8 on the microbial viability (a, b, and c) Colony forming units (CFUs) remaining after incubation with the indicated concentrations of Gal-8 (a), Gal-8N (b), and Gal-8C (c). (d) Quantification of viable bacteria after incubation with PBS control, 5 μM Gal-8 or each of its domains with or without 50 mM TDG as indicated. Data are represented as mean values ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s test. P values less than 0.05, 0.001, and 0.0001 are summarized with one, three, and four asterisks, respectively.

Figure 4

Validation of MMA using defined monoclonal antibodies. (a) Schematic overview for the MMA. Bacteria labeled with SYTO 13 were printed to the nitrocellulose glass slide, and serotype specific monoclonal antibodies were then added to the microarray following Alexa Fluor 647 conjugated secondary antibody detection. (b) Printing layout of MMA. Isolated S. pneumoniae without a capsule was defined as serologically nontypeable (NT). Serotypes were shown and strain name is labeled in the parentheses. (c) Left panel of each figure indicate images of fluorescent signals observed on the microarray. Their corresponding targeted serotype of each antibody was labeled in red. All fluorescent intensities were normalized by the highest intensity value and shown as Normalized fluorescent intensity (NFI). Data shown correspond to the mean of triplicate spots and error bars represent means ± SD.

Figure 5

Gal-8 recognize specific strain of S. pneumoniae that express self-like antigens on the MMA. (a) MMA was incubated with Gal-8, Gal-8 N and Gal-8C at the concentrations indicated. HI: Haemophilus influenzae. Error bars represent means ± standard deviation (SD). RFU, relative fluorescence units. (b) Selected serotypes of S. pneumoniae represented on the MMA were shown with binding isotherms. Microbial glycan structures for each respective microbe are shown. Error bars represent means ± SD.