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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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ompD expression in target organs from BALB/c mice infected with S. Typhimurium 14028 s.Bacteria were recovered from organs (liver and spleen) after three and five days post infection. The ompD (A) and ompW (B) transcript levels were measured by qRT-PCR and are indicated as relative transcript levels with respect to the levels of each gene in the bacterial inoculum (I) used for infecting mice (n = 3). Experiments were performed in biological and technical triplicate. Asterisks represent statistical differences between control and treated cells (* p≤0.05; *** p≤0.001). Values are mean ± SD.
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pone-0111062-g005: ompD expression in target organs from BALB/c mice infected with S. Typhimurium 14028 s.Bacteria were recovered from organs (liver and spleen) after three and five days post infection. The ompD (A) and ompW (B) transcript levels were measured by qRT-PCR and are indicated as relative transcript levels with respect to the levels of each gene in the bacterial inoculum (I) used for infecting mice (n = 3). Experiments were performed in biological and technical triplicate. Asterisks represent statistical differences between control and treated cells (* p≤0.05; *** p≤0.001). Values are mean ± SD.

Mentions: As an accurate measure of virulence, the colonization of target organs was analyzed by means of CI assays. Mixed infection assays were performed using mice co-infected with strains ΔompD, ΔompW or ΔssrB and 14028 s (1∶1 ratio), at total doses of 103 bacteria. The CI for the ΔompD/WT competitive mix in oral infection was 6.21 and 4.95-fold for the liver and spleen, respectively (Figure 4B). In intraperitoneal infections, the CI for these strains were 3.26 and 3.13-fold in the liver and spleen, respectively (Figure 4E). Thus, in both cases, the ΔompD strain resulted more virulent than the wild-type strain. In contrast, for the ΔompW/WT competitive mix, the CI values did not reflect differences in virulence, neither in the oral nor in the intraperitoneal infection (Figure 4C & F). As expected, the control mix ΔssrB/WT evidenced highly attenuated virulence for the ΔssrB strain (Figure 4D & G), as previously reported [37]. Taken together, these results confirm the deleterious effect of the OmpD porin in the survival of Salmonella during systemic infection. Hence, the possibility of a negative regulation of ompD expression when S. Typhimurium is colonizing target organs was evaluated by qRT-PCR in liver and spleen of mice infected with the wild-type strain, at 3 and 5 days post-infection. The ompD transcript levels decreased up to 80-fold in bacteria recovered from both liver and spleen, compared with its levels from the bacterial inoculum used for infecting the mice (Figure 5A). In contrast, the ompW transcript levels were similar or only increased slightly in bacteria recovered from organs with respect to those from the inoculum (Figure 5B). These results demonstrate that ompD expression is negatively regulated when Salmonella is in target organs during systemic infection.


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)

ompD expression in target organs from BALB/c mice infected with S. Typhimurium 14028 s.Bacteria were recovered from organs (liver and spleen) after three and five days post infection. The ompD (A) and ompW (B) transcript levels were measured by qRT-PCR and are indicated as relative transcript levels with respect to the levels of each gene in the bacterial inoculum (I) used for infecting mice (n = 3). Experiments were performed in biological and technical triplicate. Asterisks represent statistical differences between control and treated cells (* p≤0.05; *** p≤0.001). Values are mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111062-g005: ompD expression in target organs from BALB/c mice infected with S. Typhimurium 14028 s.Bacteria were recovered from organs (liver and spleen) after three and five days post infection. The ompD (A) and ompW (B) transcript levels were measured by qRT-PCR and are indicated as relative transcript levels with respect to the levels of each gene in the bacterial inoculum (I) used for infecting mice (n = 3). Experiments were performed in biological and technical triplicate. Asterisks represent statistical differences between control and treated cells (* p≤0.05; *** p≤0.001). Values are mean ± SD.
Mentions: As an accurate measure of virulence, the colonization of target organs was analyzed by means of CI assays. Mixed infection assays were performed using mice co-infected with strains ΔompD, ΔompW or ΔssrB and 14028 s (1∶1 ratio), at total doses of 103 bacteria. The CI for the ΔompD/WT competitive mix in oral infection was 6.21 and 4.95-fold for the liver and spleen, respectively (Figure 4B). In intraperitoneal infections, the CI for these strains were 3.26 and 3.13-fold in the liver and spleen, respectively (Figure 4E). Thus, in both cases, the ΔompD strain resulted more virulent than the wild-type strain. In contrast, for the ΔompW/WT competitive mix, the CI values did not reflect differences in virulence, neither in the oral nor in the intraperitoneal infection (Figure 4C & F). As expected, the control mix ΔssrB/WT evidenced highly attenuated virulence for the ΔssrB strain (Figure 4D & G), as previously reported [37]. Taken together, these results confirm the deleterious effect of the OmpD porin in the survival of Salmonella during systemic infection. Hence, the possibility of a negative regulation of ompD expression when S. Typhimurium is colonizing target organs was evaluated by qRT-PCR in liver and spleen of mice infected with the wild-type strain, at 3 and 5 days post-infection. The ompD transcript levels decreased up to 80-fold in bacteria recovered from both liver and spleen, compared with its levels from the bacterial inoculum used for infecting the mice (Figure 5A). In contrast, the ompW transcript levels were similar or only increased slightly in bacteria recovered from organs with respect to those from the inoculum (Figure 5B). These results demonstrate that ompD expression is negatively regulated when Salmonella is in target organs during systemic infection.

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