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Quantifying the two populations of dual oriented P6 in nontypable Haemophilus influenzae and Pal in Escherichia coli (948.6)
Author(s) -
Shaw Juliana,
Schmidt Rachel,
MacPherson Victoria,
Pichichero Michael,
Michel Lea
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.948.6
Subject(s) - periplasmic space , haemophilus influenzae , escherichia coli , bacterial outer membrane , microbiology and biotechnology , biology , chemistry , biochemistry , gene , antibiotics
Nontypable Haemophilus influenzae (NTHi) causes otitis media and other respiratory diseases in people of all ages, conferring the need for a vaccine against this bacterium. P6 is a lipoprotein and a vaccine candidate for protection against NTHi. Recent studies show that P6 is inserted into the outer membrane of NTHi in two distinct orientations‐ facing out toward the extracellular space and in toward the periplasm (Michel et al., J of Bacteriology 195: 3252). To further investigate the two orientations of P6, we utilized a cell‐impermeable biotinylating reagent (NHS‐LC‐LC‐biotin) and streptavidin‐agarose beads to separate the outward and inward facing P6. We then used standard protein detection methods to quantify the two populations. Results showed that the great majority of P6 faces in toward the periplasmic space of NTHi. Similar methods were used to quantify the two populations of Pal, P6’s homologue in Escherichia coli . Our results showed that, like P6, Pal is mostly oriented facing in toward the periplasmic space of the bacterial cell. We propose that, in light of our results, there may be more dual oriented lipoproteins in biology. Grant Funding Source : This study was supported by NIH NIDCD RO1 08671 (to MEP) and the Rochester Institute of Technology.