Expression and Porin Activity of P28 and OMP-1F during Intracellular Ehrlichia chaffeensis Development

Abstract

Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gramnegative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel ␤-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a ␤-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37°C and the late-exponential growth phase at 28°C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid-and late-exponential growth phases at 28°C and 37°C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.

Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen

“…OMP-1 homologous proteins are major surface antigens in Ehrlichia species (24,46,57,62,66), and OMP-1X may function similarly in the A. platys infection cycle. A. platys OMP-1X is predicted to have a ␤-barrel structure similar to those of E. chaffeensis P28 and OMP-1F (37) and is thus probably a porin.…”

“…This mechanism is thought to facilitate P44/Msp2 antigenic variation during acute and persistent infection and to facilitate adaptation to new environments, such as during transmission between tick and mammalian hosts (7,11,38,40,65,71). Purified native P44 from A. phagocytophilum and purified native OMP-1s (P28 and OMP-1F) of Ehrlichia chaffeensis have porin activity (30,37).…”

Proteomic Analysis of Neorickettsia sennetsu Surface-Exposed Proteins and Porin Activity of the Major Surface Protein P51

“…N. sennetsu was purified from P388D 1 cells by sonication at setting 2 for 32 s and by 5-m filtration. A 0.1% (wt/vol) Sarkosyl-insoluble outer membrane fraction was prepared, and outer membrane proteins were solubilized with 2% (wt/vol) octyl-␤-glucoside (OGC; Pierce) (22,26). For antibody neutralization, 25 g of pelleted outer membrane was treated for 1 h with 20 l of anti-rP51 or control rabbit serum and washed with 10 mM Tris-HCl (pH 8.0) before solubilization with 2% OGC.…”

“…Therefore, porins are an absolute necessity for the survival of this bacterium. To date, the only porins defined for the order Rickettsiales are major outer membrane proteins of Anaplasma phagocytophilum named P44s (22) and OMP-1F and P28 in Ehrlichia chaffeensis (26). These porins contain 16 (P44s) or 12 (OMP-1F and P28) transmembrane passes, and some are large enough to allow the slow diffusion of tetrasaccharides.…”

Tyrosine-Phosphorylated Ehrlichia chaffeensis and Ehrlichia canis Tandem Repeat Orthologs Contain a Major Continuous Cross-Reactive Antibody Epitope in Lysine-Rich Repeats

“…In dogs experimentally infected with E. canis, TRP19 and TRP36 (TRP47 ortholog) are among the first ehrlichial proteins to elicit antibodies (2,17). Moreover, antibodies directed at the major TR antibody epitope of the E. chaffeensis TRP120 as well as the hypervariable region of the OMP-1g outer membrane protein have been shown to reduce the bacterial burden in E. chaffeensis-infected mice, demonstrating that they are targets of protective immunity and have important functional roles that can be blocked by antibody (10,12), possibly including, but not limited to inhibiting porin activity or molecular interactions facilitating binding and internalization (8,9,24).…”