Coxiella burnetii acid phosphatase inhibits the release of reactive oxygen intermediates in polymorphonuclear leukocytes - PubMed (original) (raw)

Coxiella burnetii acid phosphatase inhibits the release of reactive oxygen intermediates in polymorphonuclear leukocytes

J Hill et al. Infect Immun. 2011 Jan.

Abstract

Coxiella burnetii, the etiological agent of Q fever, is a small, Gram-negative, obligate intracellular bacterium. Replication of C. burnetii during infection has been shown to be increased by decreasing oxidative stress using p47(phox -/-) and iNOS(-/-) mice in vivo and by pharmacologic inhibitors in vitro. Building upon this model, we investigated the role polymorphonuclear leukocytes (PMN) play in the control of infection, since NADPH oxidase-mediated release of reactive oxygen intermediates (ROI) is a primary bactericidal mechanism for these cells that is critical for early innate clearance. Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN through expression of an unidentified acid phosphatase (ACP). Recent genomic annotations identified one open reading frame (CBU0335) which may encode a Sec- and type II-dependent secreted ACP. To test this model, viable C. burnetii propagated in tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP) was combined with human PMN induced with 4-phorbol 12-myristate 13-acetate (PMA). The release of ROI was inhibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were challenged with electron beam-inactivated C. burnetii. C. burnetii extracts and rACP were also able to inhibit PMA-induced formation of NADPH oxidase complex on PMN membranes, suggesting a molecular mechanism responsible for this inhibition. These data support a model in which C. burnetii eludes the primary ROI killing mechanism of activated PMN by secreting at least one acid phosphatase.

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Figures

FIG. 1.

FIG. 1.

Coxiella burnetii does not stimulate ROI release from human PMN. (A) Human PMN (106) were challenged with live or killed NMI or NMII or live E. coli at an MOI of 10 bacteria to 1 PMN. (B) At 30 min postchallenge, 50 ng/ml PMA was added. Cells were incubated at 37°C and 5% CO2 for the duration of the experiment. Data represent mean ± SD of triplicate values from one experiment representative of a total of three. *, P < 0.05; **, P < 0.005.

FIG. 2.

FIG. 2.

Amino acid alignment of CBU0335 with ClustalW and predicted cleavage site. (A) The cleavage site was determined for ORF CBU0335 using the Gram-negative secretion prediction group from SignalP. n-region, h-region, and c-region, N terminus, middle, and C terminus, respectively, of the predicted signal sequence. (B) Sequences of L. pneumophilia strain Paris ORF lpp2674 (Lp_Paris), C. burnetii RSA493 ORF CBU0335 (Cb_RSA493), and F. tularensis subsp. Schu S4 ORF FTT0156 (Ft_SCHU_S4). Asterisks indicate identical amino acids; colons indicate conserved substitutions; periods indicate semiconserved substitutions.

FIG. 3.

FIG. 3.

Detection of ACP. (A) Western blot using anti-rACP primary antibody of rACP (lane 1) and C. burnetii extract (lane 2). (B) Silver stain of SDS-PAGE marker (lane 1), rACP (lane 2), and C. burnetii extract (lane 3).

FIG. 4.

FIG. 4.

Acid phosphatase activity of C. burnetii extract and rACP. C. burnetii extract (A) and rACP (B) (0.01 U and 0.00001 U, respectively) were added to 100 μl of either 1 M sodium acetate buffer (pH 3 to 6) or TBS (pH 7 to 8). DiFMUP (100 μM) was added, and the increase in fluorescence was monitored. Data represent mean ± SD of triplicate values from one experiment representative of a total of three. *, P < 0.05; **, P < 0.005.

FIG. 5.

FIG. 5.

Inhibition of acid phosphatase activity. 0.0001 U rACP or 0.08 U C. burnetii extract were combined with 10 μM sodium molybdate, 10 μM sodium orthovanadate, or 10 mM sodium tartrate in 100 μl reaction buffer. Acid phosphatase activity was detected by an increase in fluorescence as described in Materials and Methods. Percent inhibition is relative to that of the untreated control. Data represent the mean of triplicate values from one experiment representative of a total of three.

FIG. 6.

FIG. 6.

Inhibition of ROI release by rACP and ACE. Human PMN (106) were incubated with 0.0001 U rACP or 0.08 U ACE for 10 min. Cells were then treated with 50 ng/ml PMA or not treated and were incubated for 30 min at 37°C. Data represent mean ± SD of triplicate values from one experiment representative of a total of three. *, P ≤ 0.005; **, P ≤ 0.001.

FIG. 7.

FIG. 7.

ACE and rACP inhibit the movement of p47phox to the membrane. E. coli (30 min), NMII (30 min), ACE (10 min), or rACP (10 min) was incubated with PMN and stimulated or not stimulated with 50 ng/ml PMA (10 min). Membranes were run on SDS-polyacrylamide gels, blotted to polyvinylidene difluoride (PVDF), and probed with anti-p47phox or anti-actin. Fold induction = (average pixel density of α-p47phox/average pixel density of α-actin)/(average pixel density of α-p47phox control/average pixel density of α-actin control). Data represent means ± SD of values from three experiments.

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