Infect Immun. 2009 Oct;77(10):4446-54. Epub 2009 Aug 3.

Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa.

Van Alst NE, Wellington M, Clark VL, Haidaris CG, Iglewski BH.

Source

Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642, USA.

Abstract

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (Delta narGH) and nitrite reductase (Delta nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to Delta nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.

PMID:: 19651860; [PubMed - indexed for MEDLINE]
PMCID: PMC2747934

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FIG. 1.

Schematic of the denitrification pathway in P. aeruginosa. The representative operons are in italics.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 4.

Detection of T3SS exoproduct secretion by Western blotting. All strains were grown for 6 h in LB broth supplemented with 0.5 M EGTA to chelate calcium and induce expression of T3SS exoproducts (23). Culture supernatant proteins were precipitated and analyzed by Western blotting with a mouse MAb against either ExoU (A) or PcrV (C) or with rabbit polyclonal antiserum to ExoT (B). Representative Western blot assays are shown. T3SS exoproducts were not detected in the supernatant from either the ΔnarGH or the ΔnirS mutant. Supernatants from the ΔexoU (A), ΔexoT (B), and MAR2xT7::pcrV (C) mutant control strains were also negative for secretion of the corresponding exoproducts. c. ΔnarGH and c. ΔnirS, genetically complemented ΔnarGH and ΔnirS mutant strains, respectively.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 2.

P. aeruginosa interaction with human monocytic cell line THP-1. (A) Wild-type PA14 and membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) mutant strains were cocultured with THP-1 cells at an MOI of 0.1 for up to 6 h. The ΔnarGH and ΔnirS mutants displayed reduced viability over time compared to that of wild-type PA14, as measured by CFU per milliliter. (B) Wild-type PA14 and NO reductase (MAR2xT7::norB), nitrous oxide reductase (MAR2xT7::nosZ), and T3SS (ΔexoU) mutants were cocultured with THP-1 cells as for panel A. Only ΔexoU displayed reduced viability over time. (C) Comparison of wild-type PA14 and ΔnarGH and ΔnirS mutant strain interactions with THP-1 cells at different MOIs. At all of the MOIs used, the two mutants displayed a reduction in viability compared to that of wild-type PA14. At MOIs of 1.0 and 10, the viability of the ΔnirS mutant was significantly less than that of the ΔnarGH mutant.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 3.

Cytotoxicity of P. aeruginosa for THP-1 cells. (A) Cytotoxicity for THP-1 cells was determined by measuring LDH release following interaction with P. aeruginosa. Wild-type PA14 and denitrification pathway mutants were cocultured with THP-1 cells at an MOI of 0.1 for 6 h. T3SS (ΔexoU, ΔexoT, and MAR2xT7::pcrV) mutants were also examined. Compared to wild-type PA14, the membrane nitrate reductase (ΔnarGH) and nitrite reductase (ΔnirS) mutants induced significantly less cytotoxicity against THP-1 cells, similar to the ΔexoU and MAR2xT7::pcrV T3SS mutants. In contrast, the genetically complemented ΔnarGH and ΔnirS mutant strains (c. ΔnarGH and c. ΔnirS, respectively), as well as the ΔexoT, MAR2xT7::norB, and MAR2xT7::nosZ T3SS mutants, induced cytotoxicity comparably to that of wild-type PA14. (B) Comparison of cytotoxicity against THP-1 cells at MOIs of 0.1 and 1.0. At the higher MOI of 1.0, all of the strains induced comparable levels of cytotoxicity. There was no significant release of LDH from strain PA14 in the absence of THP-1 cells (data not shown).

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 7.

Detection of P. aeruginosa exoproduct gene expression by RT-PCR. Table 2 describes the oligonucleotides used for RT-PCR and the predicted size of each amplicon. (A) RT-PCR of mRNA extracted from axenic culture under T3SS-inducing conditions after 6 h of growth. (B) RT-PCR of mRNAs extracted from P. aeruginosa strains cocultured with the human monocytic cell line THP-1 at an MOI of 0.1. (C) RT-PCR of mRNAs extracted from P. aeruginosa strains cocultured with the human monocytic cell line THP-1 at an MOI of 1.0. In accordance with their secreted protein profiles, transcripts for proteases and T3SS exoproducts were not detected by RT-PCR in the ΔnarGH and ΔnirS mutants under any of the conditions tested. toxA transcripts were detected in wild-type PA14 and the ΔnarGH and ΔnirS mutants under T3SS-inducing culture conditions (A) and during the P. aeruginosa interaction with THP-1 cells at an MOI of 1.0 (C) but not at an MOI of 0.1 (B). The toxA expression profiles of the ΔnarGH and ΔnirS mutants correlate with the cytotoxicity data shown in Fig. 3B. −RT, control without reverse transcriptase.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 6.

Total protease, elastase, and exotoxin A secretion by denitrification pathway mutants. Culture supernatants from wild-type PA14 and denitrification pathway and T3SS mutants were compared for total protease (A) and elastase (B) activities. Only the ΔnarGH and ΔnirS mutants displayed significantly lower levels of protease and elastase secretion than wild-type PA14. Protease secretion was restored to normal in the genetically complemented ΔnarGH and ΔnirS mutant strains (c. ΔnarGH and c. ΔnirS, respectively). Supernatants from each strain were tested in triplicate in three separate experiments, and the data represent the mean ± SD. In contrast, wild-type PA14 and all of the mutant strains derived from PA14 secreted ToxA into their culture supernatants, as detected by Western blotting (C). P. aeruginosa PA103 and PA103-29 were used as positive and negative controls, respectively, for the production of exotoxin A; ToxA Ag is the purified P. aeruginosa exotoxin A (1 μg) used as a positive control for Western blotting. Abs. 495, absorbance at 495 nm.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

FIG. 5.

Restoration of ExoU expression in the ΔnirS mutant by supplementation with NO donors. (A) All strains were grown for 6 h in LB broth supplemented with 0.5 M EGTA to chelate calcium and induce the expression of T3SS exoproducts (23). Cultures were supplemented with either a NaNO₂ or a NO donor (DETA-NO, GSNO, or SNP) 1 h prior to supernatant harvesting, as indicated. Culture supernatant proteins were precipitated and analyzed by Western blotting with a mouse MAb against ExoU. T3SS exoproducts were not detected in supernatants from the ΔnirS mutant supplemented with NaNO₂. In contrast, exoproducts were detected in the supernatant of the ΔnirS mutant supplemented with all of the NO donors. (B) NO consumption by PA14 and the ΔnirS mutant in the presence of 3 mM SNP. The steady-state level of NO release by SNP occurred within 150 s, with a plateau at 1.6 μM (black line). The wild-type PA14 NO level peaked at 1.3 μM and reached a steady state at 200 nM (dark gray line). The ΔnirS mutant NO level peaked at 300 nM and reached a steady state at 100 nM.

Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Infect Immun. Infect Immun;77(10):4446-4454.

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH Terms

Animals
Bacterial Proteins/genetics
Bacterial Proteins/physiology*
Cell Line
Cell Survival
Colony Count, Microbial
Gene Deletion
Gene Knockout Techniques
Humans
Membrane Transport Proteins/biosynthesis*
Microbial Viability
Monocytes/microbiology*
Mutation
Nitrate Reductase/genetics
Nitrate Reductase/physiology
Nitrite Reductases/genetics
Nitrite Reductases/physiology*
Pseudomonas aeruginosa/enzymology*
Pseudomonas aeruginosa/genetics
Pseudomonas aeruginosa/pathogenicity*
Virulence
Virulence Factors/genetics
Virulence Factors/physiology*

Substances

Bacterial Proteins
Membrane Transport Proteins
Virulence Factors
Nitrite Reductases
Nitrate Reductase

Grant Support

5 T32 AI007285/AI/NIAID NIH HHS/United States
R37AI33713/AI/NIAID NIH HHS/United States

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Nitrite reductase NirS is required for type III secretion system expression and virulence in the human monocyte cell line THP-1 by Pseudomonas aeruginosa.

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