María Cristina Cerquetti

Consumption of Lactobacillus casei fermented milk prevents Salmonella reactive arthritis by modulating IL-23/IL-17 expression.

Reactive arthritis is the development of sterile joint inflammation as a sequel to a remote infection, often in the gut. We have previously shown that a low dose of S. enteritidis inoculated to streptomycin-pretreated mice generates a self-limiting enterocolitis suitable for studying reactive arthritis. Here we show that consumption of Lactobacillus casei prior to infection abolishes intestinal and joint inflammation triggered by Salmonella. BALB/c mice were sacrificed after infection; intestinal and joint samples were analyzed for histological changes and expression of cytokines. TNF-α was measured by ELISA and the expression of IL-1β, IL-6, IL-10, IL-17, IL-23 and TGF-β was assessed by qPCR. L. casei consumption prevented Salmonella-induced synovitis, the increment of TNF-α in knees and the increase of IL-17 expression in popliteal and inguinal lymph nodes. At intestinal level consumption of L. casei drastically diminished S. enteritidis invasiveness and shortened splenic persistence of the pathogen. Bacterial loads recovered at days 2 and 5 from Peyer’s patches were 10-fold lower in mice fed with L. casei. In accordance, we found that the augment in gut permeability induced during enterocolitis was decreased in those animals. Consumption of L. casei prior to infection failed to increase anti- inflammatory molecules such as IL-10 and TGF-β in the intestine. On the other hand, consumption of L. casei abrogated the expression of TNF-α, IL-17, IL-23, IL-1β and IL-6 in cecum and mesenteric lymph nodes. These cytokines are needed for differentiation of immune cells involved in the development of reactive arthritis such as Th17 and γδ T cells. Trafficking of these inflammatory cells from the gut to the joints has been proposed as a mechanism of generation of reactive arthritis. Our results suggest that L. casei consumption prevents Salmonella-induced synovitis by altering the intestinal milieu necessary for differentiation of cells involved in the generation of joint inflammation.

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.

Nitric Oxide and Apoptosis Induced in Peyer's Patches by Attenuated Strains of Salmonella enterica Serovar Enteritidis

ABSTRACT Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyers patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 of S. enterica serovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 of S. enterica serovar Enteritidis at the time of immunization is necessary to generate a protective immune response.

Protective capacity of a temperature-sensitive mutant of Salmonella enteritidis after oral and intragastric inoculation in a murine model

Temperature-sensitive (ts) mutant E/1/3 of Salmonella enteritidis was selected to evaluate its capacity to induce protective responses after peroral (p.o.) or intragastric (i.g.) inoculation to mice. This ts mutant of coasting phenotype was detected in Peyers patches until day 4, and in spleen by days 3 and 4 after the mice were inoculated by the p.o. route with 10(10) colony forming units. Peroral immunization induced significant protection from oral challenge with 240 LD50 of the wild-type (wt) strain. Higher protection was achieved when the animals were boosted intraperitoneally after p.o. immunization. Intragastric inoculation with the same dose of the ts mutant increased both the level of protection, and colonization and persistence of the micro-organism in Peyers patches and spleen. Immunization with a single i.g. inoculation induced 70% protection from p.o. challenge of the animals with the wt S. enteritidis. Two i.g. immunizations with E/1/3 raised the level of protection to 90%. Specific IgG, IgM and IgA antibodies, measured in plasma using a micro-ELISA method, were detected after i.g. immunization with ts mutant E/1/3. In addition, specific antibody-secreting cells were detected by means of an ELISPOT assay in spleen and mesenteric nodes of mice immunized with the ts mutant.

Dam methylation is required for efficient biofilm production in Salmonella enterica serovar Enteritidis

The ecological success of Salmonella enterica to survive in different environments is due, in part, to the ability to form biofilms, something which is especially important for food industry. The aim of the current study was to evaluate the involvement of Dam methylation in biofilm production in S. Enteritidis strains. The ability to generate biofilms was analyzed in wild type and dam mutant strains. In S. Enteritidis, the absence of Dam affected the capacity to develop pellicles at the air-liquid interface and reduced the ability to form biofilm on polystyrene surfaces. Curli and cellulose production, determined by Congo red and calcofluor assays, were affected in dam mutant strains. Relative quantitative real-time PCR experiments showed that the expression of csgD and csgA genes is reduced in mutants lacking dam gene with respect to the wild type strains, whereas transcript levels of bcsA are not affected in the absence of Dam. To our knowledge, this is the first report on the participation of Dam methylation on biofilm production in Enteritidis or any other serovar of S. enterica. Results presented here suggest that changes in gene expression required for biofilm production are finely regulated by Dam methylation. Thus, Dam methylation could modulate csgD expression and upregulate the expression of factors related with biofilm production, including curli and cellulose. This study contributes to the understanding of biofilm regulation in Salmonella spp. and to the design of new strategies to prevent food contamination and humans and animals infections.

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.

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.

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.

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.

A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium

Abstract A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 °C but has a lower growth rate and forms filaments at 37 °C. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam“leaky” phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.