Project description:Proteomic investigation on the glycosylation substrates and proteome effects of altering neisserial OTases within the proteome of N. gonorrhoeae MS11
Project description:Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyze the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria but to date its role in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. Pipecolic acid derived compounds, SF235 and AN296 demonstrate inhibitory activities against CbMip and these compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain RSA439 in axenic culture with comparative proteomic demonstrating selective alterations in response to these agents. This study also showed that exposure of C. burnetii to Mip inhibitors resulted in increased sensitivity to oxidative stress. Furthermore, compounds SF235 and AN296 demonstrated protective activity in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for growth and replication and that inhibitors against CbMip offer potential as novel therapeutics against C. burnetii.
Project description:Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling these diarrheal diseases, which are a significant cause of infant morbidity in the developing world. The only approved vaccine against an apicomplexan parasite targets conserved adhesins possessing a thrombospondin repeat (TSR) domains. Orthologous TSR domain-containing proteins are commonplace in the apicomplexa and C. parvum possess 12 such proteins. Here, we explore the molecular evolution and conservation of these proteins and examine their abundance in C. parvum oocysts to assess the likelihood that they may be useful as vaccine candidates. We go onto examine the glycosylation states of these proteins using antibody-enabled and ZIC-HILIC enrichment techniques, which revealed that these proteins are modified with C-linked Hex and N-linked Hex5-6HexNAc2 glycans.
Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within pathogenic members of the Neisseria genus O-linked protein glycosylation is associated with virulence yet the depth of the glycoproteome, or if glycosylation plays additional roles in Neisserial physiology are largely unknown. Recently it was identified that even closely related members of the Neisseria genus can possess O-Oligosaccharyltransferases, pglOs, that possess distinct targeting activities suggesting extensive glycoproteome diversity in terms of the substrates capable of being glycosylated across Neisserial species. Within this work we explore this concept using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae enabling the characterization of the glycoproteomes of wild type N. gonorrhoeae MS11 as well as a recently reported panel of strains expressing different pglO allelic chimeras (15 PglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy independent of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and the impact of glycosylation on bacterial species.