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.