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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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Effect of OmpD in the proliferation of S. Typhimurium inside macrophages.Murine RAW 264.7 macrophages were infected (MOI of 100∶1) with S. Typhimurium 14028 s, and its ΔompD, ΔompW and ΔssrB derivative mutants, containing or not the pBAD vector, or the plasmids pBAD-ompD or pBAD-ompW that overexpress OmpD and OmpW, respectively. CFU of the different strains were determined after recovery from infected macrophages at the indicated time points. The relative percentage of invasion at 2 h post infection (A) and of proliferation at 6, 8 and 12 h post infection (B) was determined to analyze the effect of the absence of ompD, ompW or ssrB genes. The relative percentage of proliferation at 6 (C), 8 (D) and 12 h (E) post infection was determined to analyze the effect of the absence and overexpression of OmpD. The relative percentage of proliferation at 12 h (F) post infection was determined to analyze the effect of the absence and overexpression of OmpW. Asterisks represent significant statistical difference between 14028 s and mutants used in this study (* p≤0.05). Values are mean ± SD.
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pone-0111062-g001: Effect of OmpD in the proliferation of S. Typhimurium inside macrophages.Murine RAW 264.7 macrophages were infected (MOI of 100∶1) with S. Typhimurium 14028 s, and its ΔompD, ΔompW and ΔssrB derivative mutants, containing or not the pBAD vector, or the plasmids pBAD-ompD or pBAD-ompW that overexpress OmpD and OmpW, respectively. CFU of the different strains were determined after recovery from infected macrophages at the indicated time points. The relative percentage of invasion at 2 h post infection (A) and of proliferation at 6, 8 and 12 h post infection (B) was determined to analyze the effect of the absence of ompD, ompW or ssrB genes. The relative percentage of proliferation at 6 (C), 8 (D) and 12 h (E) post infection was determined to analyze the effect of the absence and overexpression of OmpD. The relative percentage of proliferation at 12 h (F) post infection was determined to analyze the effect of the absence and overexpression of OmpW. Asterisks represent significant statistical difference between 14028 s and mutants used in this study (* p≤0.05). Values are mean ± SD.

Mentions: In order to evaluate the participation of OmpD in the adhesion, invasion and proliferation of S. Typhimurium in host cells, infection assays with macrophages were performed using the 14028 s wild-type and its isogenic ΔompD strain. Since SsrB is a transcriptional regulator required for the expression of the SPI-2 genes, which are essential for the intracellular proliferation of Salmonella and thus for its systemic infection in mice [37], a ΔssrB strain was used in these assays as a negative control. On the other hand, to compare the effect of OmpD with that of another porin, the ΔompW strain was also tested. No significant differences in the adherence were observed between all the strains tested (Figure S1); however, the ΔompD strain showed an increase of about 20% and 35% in invasion and proliferation, respectively, in comparison to the wild-type strain (Figure 1A & B). In contrast, the ΔompW strain showed invasion and proliferation patterns comparable to the wild-type strain and, as expected, the ΔssrB strain showed a decreased proliferation phenotype (Figure 1A & B).


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)

Effect of OmpD in the proliferation of S. Typhimurium inside macrophages.Murine RAW 264.7 macrophages were infected (MOI of 100∶1) with S. Typhimurium 14028 s, and its ΔompD, ΔompW and ΔssrB derivative mutants, containing or not the pBAD vector, or the plasmids pBAD-ompD or pBAD-ompW that overexpress OmpD and OmpW, respectively. CFU of the different strains were determined after recovery from infected macrophages at the indicated time points. The relative percentage of invasion at 2 h post infection (A) and of proliferation at 6, 8 and 12 h post infection (B) was determined to analyze the effect of the absence of ompD, ompW or ssrB genes. The relative percentage of proliferation at 6 (C), 8 (D) and 12 h (E) post infection was determined to analyze the effect of the absence and overexpression of OmpD. The relative percentage of proliferation at 12 h (F) post infection was determined to analyze the effect of the absence and overexpression of OmpW. Asterisks represent significant statistical difference between 14028 s and mutants used in this study (* p≤0.05). Values are mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111062-g001: Effect of OmpD in the proliferation of S. Typhimurium inside macrophages.Murine RAW 264.7 macrophages were infected (MOI of 100∶1) with S. Typhimurium 14028 s, and its ΔompD, ΔompW and ΔssrB derivative mutants, containing or not the pBAD vector, or the plasmids pBAD-ompD or pBAD-ompW that overexpress OmpD and OmpW, respectively. CFU of the different strains were determined after recovery from infected macrophages at the indicated time points. The relative percentage of invasion at 2 h post infection (A) and of proliferation at 6, 8 and 12 h post infection (B) was determined to analyze the effect of the absence of ompD, ompW or ssrB genes. The relative percentage of proliferation at 6 (C), 8 (D) and 12 h (E) post infection was determined to analyze the effect of the absence and overexpression of OmpD. The relative percentage of proliferation at 12 h (F) post infection was determined to analyze the effect of the absence and overexpression of OmpW. Asterisks represent significant statistical difference between 14028 s and mutants used in this study (* p≤0.05). Values are mean ± SD.
Mentions: In order to evaluate the participation of OmpD in the adhesion, invasion and proliferation of S. Typhimurium in host cells, infection assays with macrophages were performed using the 14028 s wild-type and its isogenic ΔompD strain. Since SsrB is a transcriptional regulator required for the expression of the SPI-2 genes, which are essential for the intracellular proliferation of Salmonella and thus for its systemic infection in mice [37], a ΔssrB strain was used in these assays as a negative control. On the other hand, to compare the effect of OmpD with that of another porin, the ΔompW strain was also tested. No significant differences in the adherence were observed between all the strains tested (Figure S1); however, the ΔompD strain showed an increase of about 20% and 35% in invasion and proliferation, respectively, in comparison to the wild-type strain (Figure 1A & B). In contrast, the ΔompW strain showed invasion and proliferation patterns comparable to the wild-type strain and, as expected, the ΔssrB strain showed a decreased proliferation phenotype (Figure 1A & B).

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