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.

Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain

“…To accomplish this, we employed a recently developed MMA ( Fig. 2 ), a microarray platform populated with intact microbes, which has been shown to more accurately predict the antimicrobial activity of galectins and various microbes in a high throughput manner ( 42 ). Gal-4 selectively engaged gram-positive S. pneumoniae serotypes 33F (Sp 33F) and 14 (Sp 14), as well as gram-negative E. coli O86 ( E. coli O86), K. pneumoniae O1 (KPO1), and P. alcalifaciens O5 (PAO5) ( 73 , 74 ) ( Supplemental Table S2 ).…”

“…In contrast to Gal-4, Gal-8 dimerization occurs through its N-terminal as opposed to C-terminal domain ( 41 ). Gal-8 also differs from Gal-4 in that the binding specificity of each domain is very distinct, with the C-terminal domain engaging blood group antigens, while blood group modifications actually inhibit the ability of the N-terminal domain to recognize the underlying lactosamine structure ( 38 , 41 , 42 , 66 , 81 ). In contrast to Gal-8, Gal-4N and C-terminal domains display similar binding profiles, where both domains can engage blood group antigens.…”

“…Protein was concentrated and buffer exchanged to PBS using Amicon Ultra-15 10 kDa cutoff Centrifugal Filter Units (Millipore). The protein was aliquoted and stored at −80 °C prior to use as outlined previously ( 42 , 55 , 56 ).…”

“…The MMAs were created as described earlier ( 42 ). Bacterial cells were fixed with 1% paraformaldehyde for 24 h at 4 °C and subsequently washed with PBS.…”

“…Not all galectins exhibit blood group–binding preference or possess the ability to alter microbial viability, suggesting that this activity may not be a uniform property of galectin family members ( 37 , 38 , 39 , 40 , 41 ). Prior studies also suggest that galectin quaternary structure may be important for galectin-mediated antimicrobial activity ( 38 , 41 , 42 ). For example, the C-terminal domain of Gal-3, which exists as a monomer, fails to exhibit antimicrobial activity despite its ability to bind to blood group–expressing microbes, while the full-length protein, which forms oligomers, exhibits antimicrobial activity ( 37 ).…”