ABSTRACT
Porphyromonas gingivalis
produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the Δ
PG0027
mutant strains. Inactivation of
PG0027
caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of
P. gingivalis
W50 and the Δ
PG0027
mutant strains were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from Δ
PG0027
mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (
m
/
z
1,688) and mono-P-tetraacyl (
m
/
z
1,448) lipid A from Δ
PG0027
showed that both contained lipid A 1-phosphate, suggesting that the Δ
PG0027
mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the Δ
PG0027
mutant strains were similar, whereas the phosphatase activity in the periplasm of the Δ
PG0027
mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the Δ
PG0027
mutant (OMVs and cells) contained similar species. Thus, OMVs in
P. gingivalis
are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism.
IMPORTANCE
Gram-negative bacteria produce outer membrane vesicles (OMVs) by “blebbing” of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe
Porphyromonas gingivalis
as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in
P. gingivalis
involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.
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