Salmonella entérica serovar Enteritidis biofilm lifestyle induces lower pathogenicity and reduces inflammatory response in a murine model compared to planktonic bacteria / Salmonella entérica serovar Enteritidis en biopelícula induce una menor patogenicidad y respuesta inflamatoria en un modelo murino que su contraparte planctónica

Abstract Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most frequent serovar involved in human salmonellosis. It has been demonstrated that about 80% of infections are related to biofilm formation. There is scant information about the pathogenicity of S. Enteritidis and its relationship to biofilm production. In this regard, this study aimed to investigate the differential host response induced by S. Enteritidis biofilm and planktonic lifestyle. To this purpose, biofilm and planktonic bacteria were inoculated to BALB/c mice and epithelial cell culture. Survival studies revealed that biofilm is less virulent than planktonic cells. Reduced signs of intestinal inflammation and lower bacterial translocation were observed in animals inoculated with Salmonella biofilm compared to the planktonic group. Results showed that Salmonella biofilm was impaired for invasion of non-phagocytic cells and induces a lower inflammatory response in vivo and in vitro compared to that of planktonic bacteria. Taken together, the outcome of Salmonella-host interaction varies depending on the bacterial lifestyle.

Resumen Salmonella entérica serovar Enteritidis (S. Enteritidis) es la serovariedad más frecuentemente aislada en la salmonelosis humana. Se ha demostrado que alrededor del 80% de las infecciones están relacionadas con la formación de biopelículas. Sin embargo, la información disponible acerca de la patogenicidad de S. Enteritidis y su relación con la producción de biopelículas es escasa. Este trabajo tuvo como objetivo investigar la respuesta diferencial del huésped frente a S. Enteritidis en sus 2 estilos de vida: biopelícula y planctónico. Para ello, se inocularon bacterias en estado de biopelícula o planctónico en ratones BALB/c y cultivo de células epiteliales. Los estudios de supervivencia revelaron que Salmonella en biopelícula fue menos virulenta que su contraparte planctónica. Los animales inoculados con biopelículas presentaron una mayor conservación estructural del intestino y una menor translocación bacteriana que el grupo planctónico. Asimismo, Salmonella en biopelícula mostró una capacidad deficiente para invadir células no fagocíticas e indujo una menor respuesta inflamatoria in vivo e in vitro que las bacterias planctónicas. Se concluye que el resultado de la interacción Salmonella-huésped depende del estilo de vida bacteriano.

Salmonella enterica serovar Enteritidis biofilm lifestyle induces lower pathogenicity and reduces inflammatory response in a murine model compared to planktonic bacteria.

Salmonellaenterica serovar Enteritidis (S. Enteritidis) is the most frequent serovar involved in human salmonellosis. It has been demonstrated that about 80% of infections are related to biofilm formation. There is scant information about the pathogenicity of S. Enteritidis and its relationship to biofilm production. In this regard, this study aimed to investigate the differential host response induced by S. Enteritidis biofilm and planktonic lifestyle. To this purpose, biofilm and planktonic bacteria were inoculated to BALB/c mice and epithelial cell culture. Survival studies revealed that biofilm is less virulent than planktonic cells. Reduced signs of intestinal inflammation and lower bacterial translocation were observed in animals inoculated with Salmonella biofilm compared to the planktonic group. Results showed that Salmonella biofilm was impaired for invasion of non-phagocytic cells and induces a lower inflammatory response in vivo and in vitro compared to that of planktonic bacteria. Taken together, the outcome of Salmonella-host interaction varies depending on the bacterial lifestyle.

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.

Activation of iNKT Cells Prevents Salmonella-Enterocolitis and Salmonella-Induced Reactive Arthritis by Downregulating IL-17-Producing γδT Cells.

Reactive arthritis (ReA) is an inflammatory condition of the joints that arises following an infection. Salmonella enterocolitis is one of the most common infections leading to ReA. Although the pathogenesis remains unclear, it is known that IL-17 plays a pivotal role in the development of ReA. IL-17-producers cells are mainly Th17, iNKT, and γδT lymphocytes. It is known that iNKT cells regulate the development of Th17 lineage. Whether iNKT cells also regulate γδT lymphocytes differentiation is unknown. We found that iNKT cells play a protective role in ReA. BALB/c Jα18-/- mice suffered a severe Salmonella enterocolitis, a 3.5-fold increase in IL-17 expression and aggravated inflammation of the synovial membrane. On the other hand, activation of iNKT cells with α-GalCer abrogated IL-17 response to Salmonella enterocolitis and prevented intestinal and joint tissue damage. Moreover, the anti-inflammatory effect of α-GalCer was related to a drop in the proportion of IL-17-producing γδT lymphocytes (IL17-γδTcells) rather than to a decrease in Th17 cells. In summary, we here show that iNKT cells play a protective role against Salmonella-enterocolitis and Salmonella-induced ReA by downregulating IL17-γδTcells.

Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina.

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008. It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein. Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains. The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations. Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

Dam methylation regulates the expression of SPI-5-encoded sopB gene in Salmonella enterica serovar Typhimurium.

DNA adenine methylation is an essential factor in Salmonella virulence. Here, we investigate the involvement of DNA adenine methylase (Dam) in the expression and translocation of a SPI-5-encoded effector of S. Typhimurium. SopB expression and secretion were determined using SopB-FLAG-tagged wild type and dam strains of S. Typhimurium. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopB protein and sopB mRNA than the wild type strain under SPI-1 and SPI-2 inducing conditions in vitro. SopB secretion was also considerably impaired in the absence of dam. In agreement with in vitro experiments, SopB synthesis in dam mutants recovered from infected epithelial cells and from murine mesenteric lymph nodes was reduced by 40% respect to the wild type strain (p < 0.05). SopB translocation was neither detected in the cytosol of epithelial cells nor in the cytosol of cells isolated from mesenteric lymph nodes infected with the dam mutant. Taken together, our results demonstrate that, in S. Typhimurium, Dam methylation modulates the expression and translocation of SPI-5-encoded SopB effector.

AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice.

Salmonellosis is a major health problem worldwide. Salmonella enterica serovar Enteritidis (S. Enteritidis) has been a primary cause of Salmonella outbreaks in many countries. AvrA is an SPI-1 effector protein involved in the enteritis pathway, with critical roles in inhibiting inflammation and apoptosis. In this work, we constructed an AvrA-FLAG-tagged strain of S. Enteritidis to analyse the expression profile of AvrA in vitro, in cell culture and in vivo. AvrA expression and secretion were observed in vitro under culture conditions that mimicked intestinal and intracellular environments. In agreement, bacteria isolated from infected cell monolayers expressed and translocated AvrA for at least 24 h post-inoculation. For in vivo experiments, BALB/c mice were inoculated by the natural route of infection with the AvrA-FLAG strain. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes (MLN). Our results showed that AvrA continues to be synthesized in vivo up to day 8 post-inoculation. Moreover, AvrA translocation was detected in the cytosol of cells isolated from MLN 8 days after infection. Interestingly, we observed that AvrA is secreted by both type three secretion system (T3SS)-1 and T3SS-2. In summary, these findings indicate that AvrA expression is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously. The AvrA effector may participate also in systemic S. Enteritidis infection.

SopB effector protein of Salmonella Typhimurium is translocated in mesenteric lymph nodes during murine salmonellosis.

Salmonella Typhimurium harbors two Salmonella pathogenicity islands (SPIs), each encoding a type three secretion system for virulence proteins. Although there is increasing evidence of postinvasion roles for SPI-1, it has been generally accepted that SPI-1 genes are downregulated following the invasion process. Here, we analyzed the expression and translocation of SopB in vitro, in cell culture and in vivo. To this end, a sopB-FLAG-tagged strain of Salmonella Typhimurium was obtained by epitope tagging. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. SopB expression was observed in vitro under cultured conditions that mimic the intestinal niche and different intracellular environments. In agreement, bacteria isolated from infected monolayers expressed and translocated SopB for at least 24 h postinoculation. For in vivo experiments, BALB/c mice were inoculated intraperitoneally with the tagged strain of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes. Our results showed that SopB continues to be synthesized in vivo during 5 days after inoculation. Interestingly, translocation of SopB was detected in the cytosol of cells isolated from lymph nodes 1 day after infection. Altogether, these findings indicate that the expression and translocation of SopB during Salmonella infection is not constrained to the initial host-bacteria encounter in the intestinal environment as defined previously.

Dam and its role in pathogenicity of Salmonella enterica.

Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

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 (SEDeltadam) and the Salmonella serovar Enteritidis parental strain were examined by using electrophoresis and silver staining. Compared to the parental strain, SEDeltadam 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 SEDeltadam is threefold lower than the amount of Wzz produced by the parental strain. Concomitantly, the activity of the wzz promoter in SEDeltadam 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.

Impaired synthesis and secretion of SopA in Salmonella Typhimurium dam 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 degrees 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.

Sublethal infection with Salmonella Enteritidis by the natural route induces intestinal and joint inflammation in mice.

Reactive arthritis (ReA) is a sterile inflammation triggered by a distal mucosal infection, which suggests a contribution from bacterial products. Investigation on the pathogenesis of ReA is difficult because of the limited studies that can be performed in humans; therefore the availability of animal models is crucial. We hereby describe a murine model for studying the early stages of Salmonella-induced ReA. BALB/c mice infected by the natural route with a sublethal dose of S. Enteritidis showed long lasting gut inflammation, synovitis in the knee joint and a significant increase of CD4+ lymphocytes in the draining popliteal lymph nodes. S. Enteritidis infection induced histological changes in intact knees and exacerbated inflammation in previously damaged joints. Experiments performed with S. Enteritidis DeltainvG mutant suggest that the proinflammatory signalling mediated by Salmonella TTSS-1 in the gut is required for the induction of joint sequelae. Since this model is highly reproducible and easy to perform, it provides great potential for investigating both host and bacterial contributions to the early stages of ReA.

Host response to a dam mutant of Salmonella enterica serovar enteritidis with a temperature-sensitive phenotype.

The temperature-sensitive dam mutant strain of Salmonella enterica serovar Enteritidis SD1 is highly attenuated and induces innate and protective immunity in mice. SD1 activates NF-kappaB and induces gamma interferon secretion. Early interaction of the SD1 mutant with intestinal epithelial cells was associated with ruffling of enterocytes. Invading bacteria were found inside Peyer's patches after inoculation.

Involvement of intestinal inducible nitric oxide synthase (iNOS) in the early stages of murine salmonellosis.

Local induction of inducible nitric oxide synthase (iNOS) and apoptosis was examined in the intestine of mice infected with virulent Salmonella enterica serovar Enteritidis 5694 (S. enteritidis) and its attenuated derivative mutant E/1/3. Both, intestinal iNOS mRNA expression and iNOS activity showed a peak at 4 h only in animals receiving the virulent S. enteritidis. Aminoguanidine treatment abrogated intestinal epithelial damage produced by virulent S. enteritidis and diminished apoptosis at the tips of the villi. Unlike the virulent strain, mutant E/1/3 induced massive iNOS expression in Peyer's patches, these findings may be related to its protective capacity. Our results suggest that intestinal iNOS participates in the early response to intestinal infection and that the final effect depends on the nature of the insult.