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Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Ipinza F, Collao B, Monsalva D, Bustamante VH, Luraschi R, Alegría-Arcos M, Almonacid DE, Aguayo D, Calderón IL, Gil F, Santiviago CA, Morales EH, Calva E, Saavedra CP - PLoS ONE (2014)

Bottom Line: In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain.In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion.Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Microbiología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chile.

ABSTRACT
Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

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Intracellular levels of total ROS in RAW 264.7 macrophages infected and similarity between OmpD and other members of the OMP superfamily.(A) Total ROS were determined at 1 to 12 h post infection in macrophages infected with strains 14028 s and ΔompD using the oxidant-sensitive probe H2DCFDA (fluorescence adjusted per µg of total protein of the sample). Asterisks represent statistical significant differences (* p≤0.05). A.U.  =  arbitrary units. (B) Sequence similarity network of OmpD and its closest homologues in the Omp superfamily. Nodes represent protein sequences, and edges represent worst reciprocal blastp E-values that are higher than a given threshold. Visualization was performed using the organic layout in Cytoscape 2.8.3 [Cline et al. 2007]. Reviewed proteins from Uniprot that have evidence of existing at the protein level are shown in color. Squares correspond to proteins that have known crystal structures in the Protein Data Bank. Edges filtered to e-value <1e-14, median alignment length: 341 residues, median identity: 34.0%. (C) Electrostatic surface potential of the biological assemblies of OmpD and SLAM-recognized proteins OmpC and OmpF. c.1) Periplasmic loops are mapped into the molecular surface: L1 blue, L2 red, L3 violet, L4 orange, L5 yellow, L6 ochre, L7 green and L8 Pink; c.2) Calculated surface electrostatic potential of OmpF from Salmonella Typhi (PDB: 3nsg); OmpF from E. coli (PDB:2zfg); OmpC from Salmonella Typhi (PDB: 3uu2) and OmpC from E. coli (PDB:2j1n) contoured at +- 2.0 kT.
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pone-0111062-g003: Intracellular levels of total ROS in RAW 264.7 macrophages infected and similarity between OmpD and other members of the OMP superfamily.(A) Total ROS were determined at 1 to 12 h post infection in macrophages infected with strains 14028 s and ΔompD using the oxidant-sensitive probe H2DCFDA (fluorescence adjusted per µg of total protein of the sample). Asterisks represent statistical significant differences (* p≤0.05). A.U.  =  arbitrary units. (B) Sequence similarity network of OmpD and its closest homologues in the Omp superfamily. Nodes represent protein sequences, and edges represent worst reciprocal blastp E-values that are higher than a given threshold. Visualization was performed using the organic layout in Cytoscape 2.8.3 [Cline et al. 2007]. Reviewed proteins from Uniprot that have evidence of existing at the protein level are shown in color. Squares correspond to proteins that have known crystal structures in the Protein Data Bank. Edges filtered to e-value <1e-14, median alignment length: 341 residues, median identity: 34.0%. (C) Electrostatic surface potential of the biological assemblies of OmpD and SLAM-recognized proteins OmpC and OmpF. c.1) Periplasmic loops are mapped into the molecular surface: L1 blue, L2 red, L3 violet, L4 orange, L5 yellow, L6 ochre, L7 green and L8 Pink; c.2) Calculated surface electrostatic potential of OmpF from Salmonella Typhi (PDB: 3nsg); OmpF from E. coli (PDB:2zfg); OmpC from Salmonella Typhi (PDB: 3uu2) and OmpC from E. coli (PDB:2j1n) contoured at +- 2.0 kT.

Mentions: Previously, we reported that ompD expression is down-regulated by exposure of S. Typhimurium to H2O2[7]. As this down-regulation of ompD expression is also observed inside macrophages, we investigated whether the presence of this outer membrane protein influences the levels of ROS produced by macrophages during infection. Total ROS levels were determined in macrophages infected with the wild-type and ΔompD strains at 1 to 12 h post-infection. As shown in Figure 3A, the macrophages infected with the ΔompD strain evidenced significantly lower levels of total ROS than the macrophages infected with the wild-type strain. This difference was not observed between macrophages infected with ΔompW and the wild-type strain (data not shown). Despite this observation, a drastic decrease of total ROS at 7 to 9 h post-infection occurred (Figure 3A), probably explained by the sequential roles of oxidative and nitrosative species produced coordinately in a temporal relationship by an early NADPH oxidase and the late inducible nitric oxide synthase (iNOS), respectively [39], [40].


Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Ipinza F, Collao B, Monsalva D, Bustamante VH, Luraschi R, Alegría-Arcos M, Almonacid DE, Aguayo D, Calderón IL, Gil F, Santiviago CA, Morales EH, Calva E, Saavedra CP - PLoS ONE (2014)

Intracellular levels of total ROS in RAW 264.7 macrophages infected and similarity between OmpD and other members of the OMP superfamily.(A) Total ROS were determined at 1 to 12 h post infection in macrophages infected with strains 14028 s and ΔompD using the oxidant-sensitive probe H2DCFDA (fluorescence adjusted per µg of total protein of the sample). Asterisks represent statistical significant differences (* p≤0.05). A.U.  =  arbitrary units. (B) Sequence similarity network of OmpD and its closest homologues in the Omp superfamily. Nodes represent protein sequences, and edges represent worst reciprocal blastp E-values that are higher than a given threshold. Visualization was performed using the organic layout in Cytoscape 2.8.3 [Cline et al. 2007]. Reviewed proteins from Uniprot that have evidence of existing at the protein level are shown in color. Squares correspond to proteins that have known crystal structures in the Protein Data Bank. Edges filtered to e-value <1e-14, median alignment length: 341 residues, median identity: 34.0%. (C) Electrostatic surface potential of the biological assemblies of OmpD and SLAM-recognized proteins OmpC and OmpF. c.1) Periplasmic loops are mapped into the molecular surface: L1 blue, L2 red, L3 violet, L4 orange, L5 yellow, L6 ochre, L7 green and L8 Pink; c.2) Calculated surface electrostatic potential of OmpF from Salmonella Typhi (PDB: 3nsg); OmpF from E. coli (PDB:2zfg); OmpC from Salmonella Typhi (PDB: 3uu2) and OmpC from E. coli (PDB:2j1n) contoured at +- 2.0 kT.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215857&req=5

pone-0111062-g003: Intracellular levels of total ROS in RAW 264.7 macrophages infected and similarity between OmpD and other members of the OMP superfamily.(A) Total ROS were determined at 1 to 12 h post infection in macrophages infected with strains 14028 s and ΔompD using the oxidant-sensitive probe H2DCFDA (fluorescence adjusted per µg of total protein of the sample). Asterisks represent statistical significant differences (* p≤0.05). A.U.  =  arbitrary units. (B) Sequence similarity network of OmpD and its closest homologues in the Omp superfamily. Nodes represent protein sequences, and edges represent worst reciprocal blastp E-values that are higher than a given threshold. Visualization was performed using the organic layout in Cytoscape 2.8.3 [Cline et al. 2007]. Reviewed proteins from Uniprot that have evidence of existing at the protein level are shown in color. Squares correspond to proteins that have known crystal structures in the Protein Data Bank. Edges filtered to e-value <1e-14, median alignment length: 341 residues, median identity: 34.0%. (C) Electrostatic surface potential of the biological assemblies of OmpD and SLAM-recognized proteins OmpC and OmpF. c.1) Periplasmic loops are mapped into the molecular surface: L1 blue, L2 red, L3 violet, L4 orange, L5 yellow, L6 ochre, L7 green and L8 Pink; c.2) Calculated surface electrostatic potential of OmpF from Salmonella Typhi (PDB: 3nsg); OmpF from E. coli (PDB:2zfg); OmpC from Salmonella Typhi (PDB: 3uu2) and OmpC from E. coli (PDB:2j1n) contoured at +- 2.0 kT.
Mentions: Previously, we reported that ompD expression is down-regulated by exposure of S. Typhimurium to H2O2[7]. As this down-regulation of ompD expression is also observed inside macrophages, we investigated whether the presence of this outer membrane protein influences the levels of ROS produced by macrophages during infection. Total ROS levels were determined in macrophages infected with the wild-type and ΔompD strains at 1 to 12 h post-infection. As shown in Figure 3A, the macrophages infected with the ΔompD strain evidenced significantly lower levels of total ROS than the macrophages infected with the wild-type strain. This difference was not observed between macrophages infected with ΔompW and the wild-type strain (data not shown). Despite this observation, a drastic decrease of total ROS at 7 to 9 h post-infection occurred (Figure 3A), probably explained by the sequential roles of oxidative and nitrosative species produced coordinately in a temporal relationship by an early NADPH oxidase and the late inducible nitric oxide synthase (iNOS), respectively [39], [40].

Bottom Line: In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain.In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion.Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Microbiología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chile.

ABSTRACT
Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

Show MeSH
Related in: MedlinePlus