DIA analysis supports the expression of different pglOalleles results in widespread proteomic alterations in N. gonorrhoeae.
While FAIMS enabled glycoproteomics and western analysis supports changes in the glycoproteome of strains expressing chimeric pglOalleles, how this impacts the N. gonorrhoeae proteome as well as individual glycoproteins is unclear. To explore this, we undertook a quantitative assessment of the proteomes of N. gonorrhoeaeexpressing different pglO alleles seeking to understand changes both in identified glycoproteins as well as the broader proteome. Using DIA analysis, four biological replicates of each strain were assessed leading to the identification of 1459 proteins with > 96% of proteins observed in each replicate and all biological replicates examined demonstrating a Pearson correlation of >0.9 (Supplementary Figure 5A and B, Supplementary table 3). PCA analysis reveals modest separation of pglO alleles yet clustering of most biological groups (Figure 3A) with ANOVA analysis supporting diverse proteome changes across the panel with 481 proteins differentially altered within at least one biological group (Figure 3B and C). Clustering of these 481 differentially altered proteins reveals the separation of pglO alleles into three groups, denoted as group one, composed of pglOmeningitidis and pglOhybrid 16; group two, composed of pglOcinerea as well as hybrid 1, 2, 3, 4; and group three, pglO hybrid 5, 6, 7, 8, 9, 10, 11 and 12 (Figure 3C). Enrichment analysis of these differentially abundant proteins demonstrates a small enrichment within proteins assigned as “integral component of membrane [GO:0016021]” (Fisher exact test, enrichment value 1.6041, p-value = 7.01*10-11, Supplementary table 5) supporting the enrichment in changes within the membrane proteome in response to changes in pglO alleles. To further understand the differential changes between pglO alleles, we examined protein alterations (defined as proteins with a fold change of ± 1-fold (log2) and a -log10(p-value) >2.0 respectively, Supplementary table 3) observed betweenpglOmeningitidis and the remaining members of the panel revealing 76 differentially altered proteins (Figure 4A). Akin to protein alterations enriched within the ANOVA analysis, multiple differentially abundant proteins corresponded to membrane proteins including permeases (NGO_0196, NGO_0216, NGO_2011 and NGO_2012, highlighted in blue) and transporters (NGO_0395, NGO_1957 and NGO_1435 highlighted in red) again supporting differential changes in the membrane proteome in response to changes in pglO alleles (Figure 4B). Surprisingly, across these 76 altered proteins only a single known glycoprotein was observed to be differentially impacted (NGO_1577, highlighted in green Figure 4B) supporting that despite alterations in glycosylation in response to changes in pglOalleles, most glycoproteins appear unaffected. Combined these finding support that across N. gonorrhoeae strains expressing differentpglO alleles, notable changes in the proteome, but not known glycoproteins, are observed with these alterations being enriched for membrane components.