Excision of an Unstable Pathogenicity Island in Salmonella enterica Serovar Enteritidis Is Induced during Infection of Phagocytic Cells

The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated “regions of difference” (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells

Servicios de atención clínica interdisciplinaria

Este proyecto tiene como objetivo principal favorecer el bienestar psicológico y nutricional de manera interdisciplinaria, brindando acompañamiento a personas que buscan alguno de estos servicios, así como, a personas que están pasando por algún proceso jurídico y requieren contención o acompañamiento psicológico. La metodología que se utilizó fue basada en el enfoque sistémico, enfoque psicoanalítico y enfoque nutricional, inicialmente se trabajaba con los usuarios de manera presencial, sin embargo, a causa de la pandemia por el Covid-19, se comenzó a trabajar con los usuarios de manera virtual o telefónica. El proyecto trajo consigo un gran aprendizaje en las alumnas, las ayudó a tener más claridad en cuanto a su ejercicio profesional, además de que fortaleció importantes herramientas tanto personales como profesionales. El proyecto tuvo como resultado un mayor autoconocimiento de los usuarios que fueron atendidos, así como una mejor habilidad para tener un control emocional. En conclusión, se pudo observar como el desarrollo del proyecto trajo consigo aprendizajes tanto para los practicantes como para las personas que recibieron el acompañamiento psicológico o nutricional. Así como el desarrollo de nuevas habilidades para atender las medidas sanitarias por la contingencia del Covid-19, sin descuidar o cancelar el servicio brindado a los pacientes.ITESO, A.C

Deletion of a prophage-like element causes attenuation of Salmonella enterica serovar Enteritidis and promotes protective immunity

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a wide host range serovar belonging to the S. enterica genus. Worldwide, it is one of the most frequent causes of food borne disease. Similar to S. Typhimurium, some virulence genes of S. Enteritidis are located in pathogenicity islands and prophages. In this study we have generated a mutant strain of S. Enteritidis lacking a prophage-like element, denominated φSE12. The resulting mutant strain was attenuated and promoted protective immunity in infected mice. Although S. Enteritidis strains lacking the complete prophage φSE12 remained capable of surviving inside phagocytic cells, they showed a significantly reduced capacity to colonize internal organs and failed to cause lethal disease in mice. Consistent with these data, infection with S. Enteritidis strains lacking prophage φSE12 promoted the production of anti-. Salmonella IgG antibodies and led to protection against a challenge with virulent strains of S. Enteritidis. The

ROD21 excision frequency increases when <i>S.</i> Enteritidis infects phagocytic cells.

<p>Bone marrow-derived DCs (<b>A</b>) and J774.3 macrophages (<b>B</b>) were infected with <i>S.</i> Enteritidis (MOI equal to 25). After 2, 18 and 24 h post infection (hpi) intracellular bacteria were recovered and the copy number of the <i>attB</i> sequence generated by type 1 excision was detected by quantitative PCR, using as template the genomic DNA obtained from intracellular bacteria. Frequency of excision is expressed as the ratio between the copy number of the <i>attB-1</i> sequence determined for intracellular and extracellular bacteria. The DNA amount was normalized by calculating the copy number of the <i>rpoD</i> gene. Data shown in graphs are average values of at least three independent experiments. The amount of intracellular bacteria recovered after 2, 18 and 24 hpi from DCs (<b>C</b>) and J774.3 (<b>D</b>) was determined by lysing either 1,000 DCs or 100 J774.3 cells with PBS-triton X100 (0.1%) and seeding the lysates in LB plates. Data shown are the average of at least 3 independent experiments.</p

Schematic representation of excisions type 1 and type 2 of ROD21 and the respective episomal elements generated.

<p>(<b>A</b>) Schematic representation of ROD21 and its surrounding region in the chromosome of the <i>S.</i> Enteritidis NCTC13349 strain. Light gray arrows indicate genes that are part of ROD21, black arrows indicate neighboring genes located outside ROD21 and dark gray arrows show neighboring genes specifically contained between the DRS limiting ROD21 and the <i>asnT-3</i> gene. Portions of the chromosome involved in type 1 and 2 excisions are shown by connecting the respective recombining DRS/tRNAs (dotted lines). Numbered arrows indicate the regions where the primers used in this study hybridize. (<b>B</b>) Schematic representation of the <i>attB-1</i> and <i>attP-1</i> sites formed after type 1 excision and the genes remaining in both the chromosome of <i>S.</i> Enteritidis and the episomal element. (<b>C</b>) Schematic representation of the <i>attB-2</i> and <i>attP-2</i> sites formed after type 2 excision, and the genes remaining in both the chromosome of <i>S.</i> Enteritidis and in the episomal element. Primer pairs used to detect the chromosomal excisions and episomal elements are indicated as black arrows.</p

ROD21 excision can be generated by means of two different recombination events.

<p>Amplification of <i>attB</i> and <i>attP</i> sequences generated after type 1 and type 2 excisions were detected by nested PCR in LK5, PT4, PT1 and PT21 strains of <i>S.</i> Enteritidis, using primer pairs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-t002" target="_blank">Table 2</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026031#pone-0026031-g002" target="_blank">Figure 2</a>. PCR products for <i>attB</i> (<b>A</b>) and <i>attP</i> (<b>B</b>) sequences for each type of excision were resolved in 1% agarose gels. The sequence of each PCR product was obtained (chromatograms in each Figure) and compared with the <i>attB</i> and <i>attP</i> sequences deduced for type 1 and type 2 excisions (labeled as theoretical). <i>attB</i> and <i>attP</i> sequences are highlighted in red in both alignments and chromatograms. Expected size for each PCR product: 591 bp for type 1 excision <i>attB</i>, 657 bp for type 2 excision <i>attB</i>, 995 bp for type 1 excision <i>attP</i> and 1050 bp for type 2 excision <i>attP</i>.</p

ROD21 is lost in bacteria undergoing type 2 excision.

<p>(<b>A</b>) Schematic representation of <i>tetRA</i> insertion in the ROD21::<i>tetRA</i> strain. This strain was used to isolate bacteria that have lost ROD21. The ROD21::<i>tetRA</i> strain was grown in the contraselection BM medium and the arising colonies were tested for the presence of ROD21 by PCR. The lines denominated 1 and 2 represent the expected PCR products that would be generated if ROD21 was inserted in the chromosome. (<b>B</b>) Detection of ROD21 by PCR analysis in WT <i>S.</i> Enteritidis and in one of the ΔROD21 strains isolated in a contraselection assay. This figure shows a representative agarose gel (1%) resolving the PCR products 1 and 2 (which denote each boundary of the integrated form of ROD21) obtained for WT and ΔROD21 <i>S.</i> Enteritidis strains. In addition, the <i>attB</i> sequences generated after type 1 (<i>attB-1</i>) and type 2 (<i>attB-2</i>) excisions were also detected by PCR. As a positive control, the <i>rpoD</i> gene was amplified by PCR. Data shown derive from one representative <i>S.</i> Enteritidis ΔROD21 strain selected out of 6 strains recovered in two independent experiments in which the <i>attB-2</i> sequence was detected. (<b>C</b>) C57BL/6 mice were orally infected with 1×10⁶ CFUs of either WT or ΔROD21 <i>S.</i> Enteritidis strains and the survival rate was measured daily. Uninfected mice were included as controls. Data shown are averages of two independent experiments, each including at least 4 mice per group. (<b>D and E</b>) Competitive infection assays, consisting of C57BL/6 mice infected either orally or intravenously with a mixture of the WT (Kn^R) and ΔROD21 (Cm^R) <i>S.</i> Enteritidis strains (input ratio equal to 1∶1). After 72 h, bacteria were recovered from spleens and livers of infected mice and the ratio of WT (Kn^R) over ΔROD21 (Cm^R) <i>S.</i> Enteritidis was calculated and compared to the input. Data shown in graphs are average values from two independent experiments for bacteria recovered from spleens and livers after intragastric gavage (<b>D</b>) or intravenous (<b>E</b>) infections of 3 mice per group.</p

Exposure to peroxide induces the excision of ROD21 from the <i>S.</i> Enteritidis chromosome.

<p><i>S.</i> Enteritidis strain PT1 was grown in LB medium and then approximately 3×10⁹ CFUs were transferred to fresh LB medium (LB) or to N-minimal medium (N), and incubated for 2 or 18 additional hours. Hydrogen peroxide was added at a final concentration equal to 0.25 mM during the last 30 min of incubation (N+H) and either genomic DNA or RNA was isolated. (<b>A</b>) The frequency of <i>attB-1</i> excision was quantified by qPCR using genomic DNA and was expressed as a relative value equal to the ratio between the copy number of <i>attB-1</i> over the copy number of <i>rpoD</i> gene. (<b>B</b>) <i>SEN1970</i> expression was determined by qPCR using cDNA and expressed as a relative value (the ratio between the copy number of <i>SEN1970</i> and the copy number of <i>rpoD</i>). The results are the average of three independent experiments. **; <0.01, one-way ANOVA and Tukey post test.</p

Primers used in this study.

<p>*Coordinates are those of the <i>S. enterica</i> serovar Enteritidis PT4 NTCT NCTC13349 sequence.</p><p>**Italics indicate the region that anneals to the 5′ or 3′ end of a mini <i>Tn</i>10 transposon.</p