Dam Methylation Controls O-Antigen Chain Length in Salmonella enterica Serovar Enteritidis by Regulating the Expression of Wzz Protein▿

We reported previously that a Salmonella enterica serovar Enteritidis dam mutant expressing a truncated Dam protein does not agglutinate in the presence of specific antibodies against O9 polysaccharide. Here we investigate the participation of Dam in lipopolysaccharide (LPS) synthesis in Salmonella. The LPS O-antigen profiles of a dam null mutant (SEΔdam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEΔdam produced LPS with shorter O-antigen polysaccharide chains. Since Wzz is responsible for the chain length distribution of the O antigen, we investigated whether Dam methylation is involved in regulating wzz expression. Densitometry analysis showed that the amount of Wzz produced by SEΔdam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SEΔdam was reduced nearly 50% in logarithmic phase and 25% in stationary phase. These results were further confirmed by reverse transcription-PCR showing that wzz gene expression was threefold lower in the dam mutant than in the parental strain. Our results demonstrate that wzz gene expression is downregulated in a dam mutant, indicating that Dam methylation activates expression of this gene. This work indicates that wzz is a new target regulated by Dam methylation and demonstrates that DNA methylation not only affects the production of bacterial surface proteins but also the production of surface polysaccharides

Salmonella enterica serovar Enteritidis enterocolitis during late stages of gestation induces an adverse pregnancy outcome in the murine model.

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome

Impaired synthesis and secretion of SopA in <i>Salmonella</i> Typhimurium <i>dam</i> mutants

DNA adenine methylation regulates virulence gene expression in certain bacteria, including Salmonella Typhimurium. The aim of this study was to investigate the involvement of DNA adenine methylase (Dam) methylation in the expression and secretion of the SPI-1 effector protein SopA. For this purpose, SopA–FLAG-tagged wild-type and dam strains of Salmonella Typhimurium were constructed. The expression and secretion of SopA were determined in bacterial culture and in intracellular bacteria recovered from infected HEp-2 epithelial cells. Bacterial culture supernatants and pellets were used to investigate secreted proteins and cell-associated proteins, respectively. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopA than the wild-type strain. Interestingly, the strain lacking Dam synthesizes SopA under nonpermissive conditions (28 °C). In addition, SopA secretion was drastically impaired in the dam mutant. In vivo experiments showed that the intracellular Salmonella dam mutant synthesizes SopA although in lower amounts than the wild-type strain. Taken together, our results suggest that Dam methylation modulates the expression and secretion of SopA in Salmonella Typhimurium

Effect of LCFM on mesenteric cytokines after <i>Salmonella</i> enterocolitis.

<p>Cytokine expression was analyzed by qPCR 5 days after oral inoculation with 3-4 x 10³ UFC of the pathogen. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Seven animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments. </p

Salmonella Enteritidis foodborne infection induces altered placental morphometrics in the murine model

Introduction: Salmonella foodborne disease during pregnancy causes a significant fetal loss in domestic livestock and preterm birth, chorioamnionitis and miscarriage in humans. These complications could be associated with alterations in placental structure. This study was aimed to determine how a low dose of Salmonella Enteritidis during late gestation affects placental histomorphometric in mice. Methods: We used a self-limiting enterocolitis murine model. BALB/c pregnant animals received a low dose of Salmonella Enteritidis (3–4 x 102 CFU/mouse) on gestational day (GD) 15. At day 3 post infection bacterial loads, serum cytokines expression and placental histomorphometrics parameters were analyzed. Results: We found that a sub-lethal infection with Salmonella induced a significant drop in fetal weight -to-placental weight-ratio and an increase in the placental coefficient. After bacterial inoculation maternal organs were colonized, inducing placental morphometric alterations, including increased placental thickness, reduced surface area, and diminished major and minor diameters. Also, foci of necrosis accompanied by acute leukocyte infiltration in decidual zone, reduction of vascular spaces and vascular congestion in labyrinth zone, were also evident in placentas from infected females on GD 18. Our data shows that placentas from infected mothers are phenotypically different from control ones. Furthermore, expression of IFN-gamma and IL-6 was up regulated in response to Salmonella in maternal serum. Discussion: Our findings demonstrate that a low dose of Salmonella during late gestation alters the placental morphometry leading to negative consequences on pregnancy outcome such as significant reduction in fetal body weight.Fil: Betancourt, Diana M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Sarnacki, Sebastian Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Cerquetti, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Monzalve, Liliana Salazar. Universidad del Valle; ColombiaFil: Pustovrh, María C.. Universidad del Valle; ColombiaFil: Giacomodonato, Mónica Nancy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Effect of LCFM on intestinal IL-10 and TGF-β.

<p>Cytokines expression was analyzed by qPCR. <i>L. casei</i> only group: mice fed for a week with LCFM. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Four animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments.</p

Effect of LCFM consumption on intestinal pathology during <i>Salmonella</i> enterocolitis.

<p>A to C: Histology of the intestine 2 days after oral infection with the pathogen. D: Intestinal inflammation scores. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. (A) EC group: moderate enterocolitis, loss of normal villus architecture and height. (B) <i>L. casei</i> + EC group: regular display of intestinal villus, undistinguishable from Control group (C). HE; 40x. (NS): no significant differences. Data were collected from 3 independent experiments. </p

Effect of LCFM on intestinal permeability after <i>Salmonella</i> enterocolitis.

<p>A single dose of FITC–dextran was administered by gavage to the animals at day 2 post infection and the intensity of fluorescence was measured in serum 5 h later. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: uninfected mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. Seven animals per group were analyzed. (NS): no significant differences. Representative data from 3 independent experiments. </p

Effect of LCFM consumption on joint during <i>Salmonella</i> enterocolitis.

<p><b>A</b>. TNF-α was measured in joint homogenates by ELISA 5 days after oral infection with the pathogen. Five animals per group were analyzed. <b>B</b>. IL-17 expression was analyzed by qPCR at day 5 post infection in joint draining lymph nodes. Seven animals per group were analyzed. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. <i>L. casei</i> + Strep group: animals were fed with the probiotic for a week and then received streptomycin. Results are expressed as mean +/- SD. (NS): no significant differences. Data were collected from 3 independent experiments. </p

Effect of LCFM consumption on joint pathology during <i>Salmonella</i> enterocolitis.

<p>A to C: Histology of knee joints 5 days after oral inoculation with the pathogen. D: Synovial inflammation scores. EC group: mice received 20 mg of streptomycin 24 h before intragastric infection with 3-4 x 10³ CFU of <i>S</i>. Enteritidis. <i>L. casei</i> + EC group: mice received the probiotic for a week before enterocolitis onset. Control group: untreated mice. (A) EC group: moderate hyperplasia, with 3 to 5 layers of synoviocytes (arrows). (B) <i>L. casei</i> + EC group: normal synovial capsule (arrows), undistinguishable from Control group. (C) Control animals: normal synovial capsule (arrows). HE; 40x. Data were collected from 3 independent experiments. </p